The Role of Selective Inhibition of the in Development and Progression of Colon

Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von Körner, Julia

an der

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie

Konstanz, 2018

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-yi88dwkvjay87

Tag der mündlichen Prüfung: 18. April 2018 1. Referent: Prof. Dr. Marcus Groettrup 2. Referent: Prof. Dr. Thomas Brunner

Chapter I

The Role of Selective Inhibition of the Immunoproteasome on Development and Progression of Colon Cancer

3

Abstract

Colorectal cancer (CRC) is the third most frequently diagnosed and lethal cancer type worldwide. Due to high therapy failure and mortality rates of patients treated with standard CRC therapy including surgical resection and chemotherapy, new treatment options and drug targets for colorectal carcinoma are a pressing medical need. CRC develops from adenomatous precursor lesions by a complex interaction of environmental carcinogens, the cumulative accumulation of independent genetic aberrations in or tumor suppressor and the host immune system. Besides several lifestyle associated factors, genetic predisposition and long-term chronic inflammatory conditions of the gastrointestinal mucosa as a hallmark of patients suffering from inflammatory bowel disease (IBD), including Ulcerative Colitis (UC) and Crohn’s Disease (CD), pose a significant risk factor for the development of colon cancer. CRC risk increases with severity and duration of the intestinal , demonstrating a tumor-promoting role of inflammation. While the majority of CRC patients have not experienced chronic inflammatory bowel disease before diagnosis, CRC typically exhibits immune/inflammatory infiltrates referred to as ‘tumor-elicited inflammation’. The clinical outcome and prognosis of CRC is strongly influenced by the type of immune response generated. Inflammatory mediators and produced by the pro-inflammatory T helper subsets Th1 and Th17, in particular IL-6, TNF, IL-17 and IL-23, directly promote colon tumor development and cause the attraction of lamina propria myeloid cells (macrophages, granulocytes) to the tumor site, which further accelerate tumor inflammation.

In recent years, a great number of studies report elevated expression of the immunoproteasome subunits LMP7 and LMP2 in inflamed tissue of IBD patients as well as in 70% of human CRC tumor specimen. The immunoproteasome is predominantly and constitutively expressed in immune cells, but stimulation of cells with pro-inflammatory cytokines such as IFN or TNF leads to replacement of the three constitutive catalytically active subunits 1, β, and 5 of the β6S proteasome with the inducible active subunits low molecular mass peptide β (LMPβ, 1i), multi-catalytic endopeptidase complex-like (MECL)-1 (βi), and LMP7 (5i). The immunoproteasome shapes the antigenic repertoire of MHC class I-restricted T cell epitopes at sites of inflammation resulting in more efficient activation of cytotoxic T Lymphocytes (CTLs). Apart from its pivotal role in antigen processing and presentation, the immunoproteasome was shown to have additional functions such as influencing production and T helper cell differentiation, as well as T cell survival.

Given that cancer is generally associated with increased proteasomal activity due to factors including rapid cell proliferation, increased oxidative stress and elevated cytokine levels, these findings led to the development of novel therapeutic options. So, subunit-selective inhibitors of the immunoproteasome have been designed to directly target cells with high immunoproteasome levels. Inhibition of the chymotrypsin-like activity of LMP7 with the cell-permeable, irreversible epoxyketone inhibitor ONX 0914 (formerly PR-957) has gained substantial interest in medical research. Treatment with the immunoproteasome selective inhibitor has already shown promising therapeutic effects in ameliorating clinical symptoms of various mouse models of autoimmune diseases and chronic inflammatory pathologies. Particularly, in DSS (dextran sodium sulfate)-induced experimental colitis, the mouse model for UC, ONX 0914 efficiently reduced disease severity. Inhibitor treatment as well as genetic ablation of LMP7 (and LMP2 or MECL) effectively inhibited inflammation-associated loss of body weight while improving other disease related symptoms. Treatment with ONX 0914 resulted in decreased secretion of pro-inflammatory cytokines and reduced intestinal inflammation and tissue destruction, as well as suppressed differentiation of CD4+ T cells into Th17 cells. These findings suggest

I

ONX 0914 treatment as a promising therapeutic intervention against colitis-associated pathologies such as inflammation-associated colon cancer.

Although proteasome inhibition showed profound anti-tumor activities against a variety of hematologic malignancies, considerably less is known about their effects on solid tumors. Since inflammation and cancer are intermitted and since the immunoproteasome promotes Th1 and Th17 differentiation and pro- inflammatory cytokine production, we were interested whether selective inhibition or genetic deficiency of the immunoproteasome would interfere with CRC development and exacerbation in either preventive or therapeutic settings of preclinical mouse models of colon cancer.

In this study, we report that inhibition of the immunoproteasome with ONX 0914 suppressed pathological symptoms of CRC in sporadic, hereditary as well as inflammation-driven development in vivo during the preceding inflammation phase and directly on the already established tumors. Treatment with the inhibitor decreased or completely blocked the emergence and progression of intestinal tumor formation in either chemically-induced (AOM/DSS) or spontaneously mediated colon carcinogenesis in response to AOM, as well as in transgenic ApcMin/+ mice, which resemble human familial adenomatous polyposis (FAP). Moreover, also genetic ablation of LMP7 markedly reduced tumor burden in AOM/DSS treated wild type mice and ApcMin/+ mice. ONX 0914 treatment strongly reduced DSS inflammation-induced loss of body weight, while survival rates were significantly enhanced. The anti-tumorigenic effect of ONX 0914 in colon cancer is characterized by combined action of a strong anti-inflammatory response in the tumor microenvironment (TME), as well as of anti- proliferative and pro-apoptotic signaling in pre-neoplastic and malignant cells. The LMP7 inhibitor curtailed both tumor associated inflammation and tumor growth by either mitigating or curtailing the production of pro-inflammatory cytokines, especially IL-6 and IL-17A, innate immune cell infiltration and inflammation-induced damages in the colon mucosa, which are critical drivers of pro-tumorigenic signaling in colon cancer. Furthermore, interference of the pleiotropic effects on homeostasis by ONX 0914 potently triggered apoptotic signaling and suppressed survival and proliferation of colon cancer cells. In addition, ONX 0914 might potentially decreases the activity of key oncogenic pathways and mutagenic changes in pre-malignant cells. Analysis of clinical CRC samples indicate that LMP7 and LMP2 subunits are upregulated in cancer tissue compared to non-malignant specimen, which verifies the immunoproteasome as a valid drug target in colorectal carcinoma.

In summary, these findings support an important functional involvement of the immunoproteasome as a pathogenic mechanism in development and progression of CRC. The data also provide the valuable approach that selective inhibition of the immunoproteasome may offer a therapeutic benefit for the treatment of CRC.

II

Zusammenfassung

Das Kolonkarzinom (Dickdarmkarzinom und Rektumkarzinom) zählt zu den dritthäufigsten malignen Tumorerkrankungen weltweit und ist dabei die zweithäufigste Krebserkrankung in Deutschland. Hinsichtlich der bestehenden hohen Mortalitätsraten sowie der geringer Heilungschancen nach konventionellen Therapie-Methoden (vollständigen operativen Entfernung des Primärtumors und neo- adjuvante Therapien, wie Chemotherapie oder Strahlentherapie) ist es dringend notwendig, neue onkologische Wirkstoffe und zielgerichtetere Therapieansätze zu entwickeln, um die Heilungschancen zu erhöhen. In den meisten Fällen geht Darmkrebs aus einem anfangs noch gutartigen Geschwulst der Darmschleimhaut hervor (Darmpolyp). Die weitere Entwicklung wird ausgelöst durch eine Anhäufung verschiedener Mutationen, wobei es sich entweder um die Inaktivierung von Tumor-Suppressions- Genen oder um die Aktivierung von Onkogenen handelt. Neben exogenen Umwelteinflüssen und dem individuellen Lebensstil, wie beispielsweise übermäßigem Nikotin- und Alkoholkonsum, gelten genetische Veranlagung, sowie chronisch entzündliche Darmerkrankungen (CED) als maßgebliche Prädispositionsparameter und Risikofaktoren für die Entstehung von Darmkrebs. Entzündliche Erkrankungen des Intestinal-Trakts, wie Colitis ulcerosa (CU) und Morbus Crohn (MC), die zu CEDs zusammengefasst werden, weisen darauf hin, dass wiederkehrende chronische Entzündungen des Darms eng mit der Entwicklung von Darmkrebs verbunden sind. Auch im sich spontan entwickelndem Darmkrebs, lässt sich eine erhöhte Anzahl entzündlicher Mediatoren und Immunzellen im Tumor- infiltrierendem Gewebe finden. Die Art der Immunantwort, welche sich im Rahmen der Krebsentstehung bildet, trägt maßgeblich zur Prognose bei. So ist die Ausbildung der Entzündungs- fördernden T Helfer 1 (Th1) und Th17 Antwort, sowie eine erhöhte Konzentration an Zytokinen den IL- 6, TNF, IL-17 und IL-23 dem Tumorwachstum förderlich. Es konnte gezeigt werden, dass sowohl CED-Patienten, als auch Patienten, die an Darmkrebs erkrankt sind, eine signifikant erhöhte Expression der Immunproteasom Untereinheiten LMP7 und LMP2 aufweisen. Das Immunproteasom, welches im Vergleich zum Standard-Proteasom unterschiedliche katalytisch aktive Untereinheiten (LMP2, LMP7, MECL-1) enthält, kommt in Immunzellen vor und ist in Geweben mit aktiven Entzündungsprozessen kurzzeitig durch Entzündungs-fördernde Zytokine (IFN () und/oder TNF (Tumor Nekrose Faktor Alpha)) induzierbar. Neben begünstigter Erweiterung des Antigen-Repertoires auf dem Haupt-Histokompatibilitäts-Komplex (MHC) der Klasse I, konnte gezeigt werden, dass das Immun-Proteasom eine Rolle bei der Zytokin- Produktion, der Differenzierung von T-Helfer Zellen und beim Überleben von T-Zellen spielt. Auf der Suche nach einem verbesserten molekularen Wirkstoff in der Krebstherapie, konnte gezeigt werden, dass in malignen Zellen eine erhöhte Proteasom-Aktivität infolge starker Expression von Immunproteasomen zu verzeichnen ist, welche die Zellzyklusphasen fördert und damit zur Tumorzell- Proliferation beiträgt. Dies führte zur Entwicklung spezifischer Inhibitoren von relevanten Proteasom- Untereinheiten. Durch selektive Hemmung der Chymotrypsin-ähnlichen Aktivität der Immunproteasom-Untereinheit LMP7 mit dem Wirkstoff ONX 0914 konnte die Entstehung und das Fortschreiten einiger Entzündungskrankheiten, sowie mehrerer Autoimmunkrankheiten im präklinischen Modell verhindert werden. Die Hemmung des Immunproteasoms weist im Vergleich zur Hemmung des konstitutiven Proteasoms weniger Nebenwirkungen auf und könnte somit einen neuen Therapieansatz für die Behandlung autoimmuner bzw. damit assoziierter Erkrankungen darstellen. Interessanterweise konnte die Behandlung mit ONX 0914 den Krankheitsverlauf von DSS (Dextran-Natriumsulfat)-induzierter Colitis, einem etablierten Tiermodell für CED, komplett unterdrücken.

III

Zurzeit finden Proteasom Inhibitoren klinische Anwendung nur in einer Reihe von hämatologischen Neoplasien, wie malignen Lymphomen und Leukämien. Jedoch bedingt durch die Tatsache, dass Krebs und Entzündung im engen Zusammenspiel zueinander stehen, sowie des erfolgreichen Therapieeinsatzes des Immunproteasom Inhibitors im Colitis-Mausmodell, wurde in dieser Dissertation der Effekt von ONX 0914 auf die Entstehung und Entwicklung von Darmkrebs in verschiedenen präklinischen Modellen getestet und beurteilt.

Die vorliegenden Ergebnisse zeigen, dass die Behandlung mit dem LMP7 Inhibitor die pathologischen Symptome der Darmkrebs-Erkrankung in spontanen und chemisch induzierten Mausmodellen, sowie in Mäusen mit Mutationen im APC (adenomatöse Polyposis des Colons)-Gen supprimiert. Die Tumor- Entstehung, sowie die progressive Fortschreitung der Tumorgenese wurden signifikant eingedämmt, sodass wir davon ausgehen können, dass die Inhibition des Immunproteasoms die physiologischen und pathophysiologischen Immun-Antworten bei der intestinalen Karzinogenese maßgeblich beeinflusst. Des Weiteren konnte der anti-neoplastische Effekt auch in LMP7-/- Mäusen, welche eine Defizienz der Immunproteasom Untereinheit LMP7 aufweisen, verifiziert werden. Es konnte gezeigt werden, dass ONX 0914 an der Eindämmung der intestinalen Entzündung beteiligt ist, sodass die immun- modulatorische Veränderung der Tumor-Mikroumgebung ein Tumorwachstum verhindert. Weiterhin wird durch die Beeinträchtigung der proteasomalen Abbaumechanismen mittels ONX 0914 eine Arretierung der Zellaktivität mit daraus folgender Apoptose in malignen Zellen induziert.

Zusammenfassend unterstreichen diese Ergebnisse den potentiell therapeutischen Einsatz von ONX 0914 als neuartige Strategie zur Behandlung des Kolonkarzinoms.

IV

Content

Abstract ...... I Zusammenfassung ...... III Introduction ...... 1 Characterization of the proteasome system (UPS) ...... 1 Structure and function of the 20S proteasome ...... 2 Catalytic Principle of the 20S Proteasome ...... 4 Different types of the 20S proteasome ...... 5 The immunoproteasome ...... 5 The thymoproteasome...... 6 Proteasome assembly ...... 6 The immunoproteasome in health and disease ...... 8 The proteasome as drug target in cancer ...... 10 Peptide aldehydes ...... 11 Peptide boronates ...... 11 -lactones ...... 11 Peptide vinyl sulfones ...... 12 Peptide epoxyketones ...... 12 Subunit-selective inhibitors ...... 12 5/LMP7-selective inhibitors (CT-L activity inhibitors) ...... 12 Immunoproteasome subunit-selective inhibitors ...... 13 LMP2-selective inhibitors ...... 13 LMP7-selective inhibitors ...... 13 Colorectal cancer (CRC) – epidemiology and etiology ...... 15 Hereditary colorectal cancer ...... 16 Sporadic colorectal carcinoma ...... 18 Inflammation-associated colon cancer ...... 18 Colon cancer therapy ...... 19 Mouse models of colon cancer ...... 19 Mouse models of spontaneously developing colon cancer ...... 19 Chemically-induced mouse models of colon cancer – the AOM/DSS m odel ...... 20 Tumor-promoting inflammation in CRC...... 22 The IL-6/STAT3 – TNF/NFκB axis in colon cancer ...... 23 Immune cells in colon cancer tissue ...... 25 Results ...... 26 Immunoproteasome inhibition leads to anti-proliferative activity and induction of in colon cancer cells in vitro ...... 26 Treatment with the LMP7 inhibitor ONX 0914 suppresses the formation of inflammation-driven colon carcinogenesis in the AOM/DSS mouse model ...... 28 Treatment with ONX 0914 abrogates colitis-associated colon carcinogenesis in a therapeutic setting of the AOM/DSS mouse model ...... 31 LMP7 inhibitor treatment suppresses further colorectal tumor growth in the absence of inflammation ...... 33 Treatment with ONX 0914 reduces intestinal tumorigenesis in the ApcMin/+ mouse model ...... 34 Inhibition of the immunoproteasome is also efficacious in therapeutic treatment of already established tumors in the ApcMin/+ mouse model ...... 37 Genetic deficiency of the LMP7 subunit ameliorates spontaneous formation of intestinal polyposis in ApcMin/+ mice ...... 38 The development of sporadic colon carcinogenesis is markedly suppressed by inhibition of the immunoproteasome with ONX 0914 ...... 40 Patient-derived CRC specimen showed elevated expression of immunoproteasome subunits...... 44 Discussion and future implications ...... 46 Material and Methods ...... 60 Cell lines and cell culture methods...... 60 Animals and ethics statement ...... 60 Genotyping of mice ...... 60 Human CRC samples ...... 61 Immunoproteasome inhibition in vitro and in vivo...... 61 Animal models of colon carcinogenesis ...... 62 The AOM/DSS tumor model ...... 62 ApcMin/+ mice and the ApcMin/+-DSS mouse model ...... 62 AOM-induced mouse model of sporadic colon carcinogenesis ...... 62 Histology ...... 63 Preparation of tissue sections ...... 63 Haematoxylin and eosin (H&E) staining ...... 63 Immunohistochemistry (IHC) ...... 63 Analysis of cytokine expression in serum or intestinal tissue ...... 64 Analysis of cell viability and cell proliferation ...... 64 The MTT assay ...... 64 CFSE labeling for assessment of cell proliferation ...... 64 Flow cytometric analysis...... 64 Annexin V/PI staining for flow cytometric analysis of apoptotic cell death ...... 64 Molecular Biological Methods - western blotting ...... 65 Protein extraction for western blotting ...... 65 Protein quantification using the BCA assay ...... 65 SDS- polyacrylamide gel electrophoresis (SDS-PAGE) ...... 65 Wet blotting procedure and protein detection ...... 66 Statistical analysis ...... 67 Supplementary Information ...... 68 Supplementary material and methods ...... 68 Tumor challenge and bioluminescence imaging (BLI) ...... 68 Intracellular staining of IL-17A in tumor-infiltrating lymphocytes (TILs) ...... 68 Analysis of cytokine expression in serum samples using multiplex immunoassay ...... 69 Supplementary Figures ...... 69 References ...... 72

1

Introduction

Characterization of the ubiquitin proteasome system (UPS) The ubiquitin proteasome system (UPS) and its underlying multicomponent molecular machinery is of utmost importance for maintaining intracellular protein homeostasis both at physiological conditions, as well as in the course of adaptive stress response by selective degradation of intracellular . This homeostatic process needs to be tightly controlled since proteasomal substrates not only include damaged and misfolded proteins but also mediators of many physiological and cellular processes.

Besides its fundamental role in protein quality control, the UPS regulates and orchestrates the abundance, intracellular localization as well as the enzymatic activity of many proteins. As the primary non-lysosomal ATP-dependent protein degradation machinery in eukaryotic cells (1, 2), the UPS is responsible for the degradation of short-lived poly-ubiquitin-tagged substrates. The UPS determines the availability of regulatory proteins and mediators of all kinds of cellular processes including progression, cell proliferation, signal transduction, differentiation and development, regulation, cell death, and inflammatory responses (3–5). Among them, prominent substrates of the UPS include cyclins, cdk (cyclin-dependent kinase) inhibitors, the factor NFκB (nuclear factor of kappa B kinase) as well as the tumor suppressors and Rb ( protein) (6–8). Cellular integrity is further preserved by preventing accumulation of irreversibly damaged and potentially toxic proteins (9). Additionally, the UPS confers crucial roles in antigen presentation. The resulting peptide fragments are presented on MHC class I molecules that are recognized by CD8+ cytotoxic T cell (2, 10– 12).

The attachment of an ubiquitin (Ub) moiety to a lysine residue is achieved via an ATP-dependent enzymatic cascade involving a sequential series of three classes of : an E1 ubiquitin-activating enzymes which catalyzes the C-terminal acyl-adenylation of Ub. After that, an activated thioester is transferred to an E2 ubiquitin-conjugating via transthioesterification. Eventually, an E3 ubiquitin ligase covalently attaches Ub to the substrate by forming an isopeptide bond between lysine side chains of the adjacent ubiquitin and the C-terminal glycine residue 76 (G76) of the Ub moiety (13– 15). Repeated series of these steps leads to linear or dendrimeric polyubiquitinylation to one of the seven internal modifiable lysine residues at position 48, also called K48 linked poly-ubiquitination. Noticeably, a Ub-polymer chain with a least four Lys48-linked Ub molecules appears to be the minimal recognition signal for proteasomal degradation by the 26S proteasome (16–19). Modifications with a single ubiquitin molecule (mono-ubiquitination) or with additional chains of other linkages (K6, K11, K27, K29, K33, K63, M1) are not degraded by the proteasome but are recognized as physiological target motifs in cellular homeostasis and mediate various signaling functions (15). For example, mono-ubiquitin plays a role in histone regulation and DNA repair, K11-linked poly-ubiquitin is involved in cell-cycle regulation, K33-linked poly-ubiquitin is responsible for kinase modification, while K63-linked poly-ubiquitin plays a role in endocytosis, DNA-damage responses and in signaling processes (15, 18, 20, 21). Linear poly-ubiquitin, linked via an N-terminal methionine (M1), is supposed to play a role in cell signaling (15).

The proteasome also catalyzes protein degradation in an ubiquitin-independent manner but therefore requires other specific factors bound to the substrate protein (22, 23).

1

Structure and function of the 20S proteasome The 26S proteasome is the multifunctional proteolytic key protease of the UPS located in both the cytoplasm as well as the nucleus. It is structurally and functionally divided into two multisub-complexes, namely the 20S proteasome core particle (720 kDa), where substrate occurs, and two associated 19S regulatory particles (RP) (900 kDa), which are responsible for recognition of ubiquitin- tagged substrates, unfolding, deubiquitination and translocation of the substrates into the core particle (24, 25) (see also Figure 1).

The 20S core particle is a barrel-shaped, cylindrical and multicatalytic complex composed of four axially stacked heptameric rings that encloses a central cavity by two outer α rings and two inner rings. Each ring is composed of seven distinct subunits displaying a α1-7 1-7 1-7 α1-7 stoichiometry. Each of these subunits occupies a fixed position within the 20S complex (26–28). The α-ring forms a toroidal entry gate into the inner cavity and provides contact points for interaction with the 19S regulatory particles, whereas the central compartment of subunits creates the proteolytic chamber (29). The catalytic activity of the proteasome is restricted to three subunits that expose threonine residues, which display N-terminal nucleophile (Ntn) hydrolase activity for peptide bound cleavage (30).

To prevent uncontrolled degradation of cellular proteins, the access to the active center of the 20S CP chamber is tightly regulated by an adjustable gate that is formed by the N-terminal tails of the α-subunits. First interaction of with the poly-ubiquitinated substrate proteins occurs via binding to subunits of the 19S regulatory cap that is associated with the 20S proteasome core (31, 32). Only upon direct binding of regulatory particles to the outer α rings, the conformational rearrangement of the N- terminal portions of the α subunits cause opening of the central chamber and facilitate access of proteins to the catalytic core (26, 29, 33). In the absence of a regulator particle, the free 20S proteasomes also shows detectable but low cleavage activity towards small proteins with intrinsically unstructured regions in the absence of ubiquitin and ATP (34).

The 19S regulatory particle (RP, PA 700, bleomycin-sensitive 10 cap, Blm10) ensures substrate unfolding in an ATP-dependent manner and translocation into the 20S core particle. The RP is formed by two sub-complexes, the lid and the base (25). The lid is composed of 13 non-ATPase subunits (Rpn1- 3, 5-13 and 15) which are responsible for recognizing and binding to ubiquitinated substrates by their Ub binding domain. After that, substrate proteins are then intrinsically deubiquitinated by the metalloisopeptidase Rpn11 and unfolded via the chaperone-like activity of the ATPases in order to enable entry in to the 20S core particle (35, 36). The base is composed of six paralogous AAA+-type ATPase subunits (Rpt1-Rpt6) that form a hexameric ring with two ubiquitin receptors, Rpn10 and Rpn13, associated to this ring. Additionally, two organizing subunits (Rpn1/2) serve as molecular scaffold. By its ATPase activity, the base sustains ATP-dependent unfolding and leads to gate-opening by direct interaction with the α-subunits (37–40).

Instead of the 19S cap, four alternative regulators, PAβ8α, PA28, PI31 and PA200, can associate to one or both ends with the 20S core particle. However, these alternative regulators lack ATPase activity and are unable to bind to Ub conjugates suggesting other Ub- and ATP-independent function (41).

The PA28 (11S cap, REG) proteasome regulator particle consists of two homologous IFN inducible SU (subunit) PAβ8α and PA28. These subunits form a hetero-heptameric ring of γα4 architecture which binds to the ends of the 20S proteasome in an ATP-independent manner. PAβ8α binding increases substrate affinity and facilitates α-ring opening for either access or release of proteins (4, 42, 43). Noticeably, PA28α has been shown to be involved in MHC class I antigen processing (42) as well

2 as in regulation of apoptosis (44). Although it is primarily associated with the immunoproteasome (IP), PAβ8α is not directly involved in broadening of the MHC class I restricted peptide variety (45). However, PAβ8α binding influences cleavage specificity and leads to increased formation of hybrid proteasomes of the 19S-20S-11S order (40). The homoheptamer PAβ8 (Ki antigen), a homolog of PAβ8α, forms homopolymeric complexes and is particularly associated with 20S proteasomes in the nucleus. In contrast to its structurally related family members, it is not inducible by IFN (46). Additionally, there are two other proteasome regulators, the monomeric PA200 which confers a possible role in DNA repair and the proteasome inhibitor (PI)31 which potentially interferes with MHC class I antigen presentation.

The formation of hybrid 26S proteasomes that are characterized by different regulators bound to each site of the core particle leads to diverse properties of these proteasome subtypes, which allows the cell to adapt in the needs of the respective physiological demand (4).

Figure 1. Major 20S proteasome subtypes and regulators. The 20S proteasome catalytic core is composed of four heptameric rings. Standard proteasomes are constitutively expressed and incorporate the catalytic subunits 1, β and 5. Pro- inflammatory cytokines (Interferon (IFN), (TNF)) up-regulate expression of the three inducible immunoproteasome subunits, low molecular mass protein (LMP)2, multi-catalytic endopeptidase complex-like (MECL)1, and LMP7. Additionally, the formation of mixed proteasomes is possible, containing both standard and immunoproteasome subunits. 20S proteasomes are activated by interaction with five regulatory particles which facilitate substrate entry: the preferentially assembled 19S regulator mediates ATP- and ubiquitin-dependent substrate degradation, the two heptameric regulators PAβ8α (IFN inducible with a proposed role in antigen presentation) and PAβ8 (only expressed in the nucleus, implicated in cell cycle regulation) as well as the two monomeric regulators PA200 and PI31. Regulators can bind to either one or both sides of 20S core particle. By binding of two different activators at each side hybrid proteasomes are formed. Depending on the type of the 20S proteasome, preferential association with regulators has been proposed, which is indicated by the line thickness. Figure adapted from (47).

3

Catalytic Principle of the 20S Proteasome The three catalytically active subunits of the constitutive 20S proteasome reside in the rings. Only 1c (PSMB6 (proteasome subunit beta), Y), βc (PSMB7, Z) and 5c (PSMB5, X) possess proteolytic activity to execute cleavage of protein substrates into peptide fragments of 3 to 22 amino acids by the peptidylglutamyl-peptide bond hydrolyzing (PGPH) activity, later termed as the caspase-like activity (C-L), the trypsin-like (T-L) activity and the chymotrypsin-like (CT-L) (48, 49). These activities were named after their preferential peptide bond cleavage specificity on the carboxyl-side of hydrophobic, basic and acidic amino acids, respectively (50–52). The caspase-like activity is assigned to 1c, the T- L activity to βc and the CT-L activity to 5c (29). The CT-L activity was shown to be the rate limiting step of proteasomal proteolysis (53). Interestingly, two other but minor proteolytic activities have been ascribed, which are identified as branched-chain amino acid activity (BrAAP) and small neutral amino acid activity (SNAAP) (54). The active site of the SNAAP activity could be assigned to the 7 subunit (26).

Each catalytically active subunit (SU) contains a threonine (Thr1) residue that is embedded around active site residues. Active sites are then formed at hydrogen bond-linked subunit interaction within the inner chamber of the 20S core (29). Hydrolysis of peptide bonds is accomplished by a nucleophilic attack onto the carbonyl carbon (-OH) of the peptide bond by the hydroxyl group of Thr1, whereas the amino group of Thr1 thereby serves as a proton acceptor (-NH2) (41, 55, 56) (see also Figure 2).

Figure 2. Catalytic mechanism of the 20S proteasome. Substrate in black; proteasome in cyan. Image from (57) This reaction creates an acyl-enzyme ester intermediate with the concomitant release of the N-terminal peptide fragment. Liberation of a water molecule after hydrolysis of the ester bond releases the C- terminal peptide fragment and restores the initial active site (58) (Figure 2). Most of these products are further hydrolyzed by cytosolic peptidases into single amino acids, whereas the majority of these peptide fragments enter the MHC class I presentation pathway (59, 60).

The mechanism of hydrolysis involving active site Thr1, Asp17 and Lys33 is identical for each subunit, while the substrate cleavage specificity is determined by distinct amino acids in the S1 binding pocket where substrates bind prior to cleavage. The Arg45 of the S1 pocket of 1 explains the preferential cleavage of peptide bonds after acidic amino acids. Gly45 together with Glu5γ of 2 renders a more spacious S1 pocket which prefers cleavage of peptide bonds after large, basic residues and Met45 of 5 substantially conduces the preference for cleavage after hydrophobic amino acids (29, 61).

4

Different types of the 20S proteasome The immunoproteasome Constitutive Proteasomes (CP) containing the catalytic subunits 1c, βc, 5c are ubiquitously expressed and represent the major proteasome type in almost every cell type – in both cytoplasm and nucleus (62, 63). However, studies of Monaco et al. (64) and Brown et al. (65) identified the incorporation of unique subsets into the 20S core complex in exchange of subunits common to constitutive proteasomes. This led to the hypothesis that proteasomes are able to change their specific functions in order to adapt to altered cellular demands. By replacing their constitutive subunit homologs, three inducible and alternative proteolytically active subunits, 1i (LMPβ, low molecular mass protein 2, PSMB9), βi (MECL-1, multicatalytic endopeptidase complex-like 1, PSMB10) and 5i (LMP7, PSMB8) can incorporate during nascent 20S proteasome biosynthesis to form a new 20S proteasome complex called immunoproteasome (IP) (66–68). The term results of its inducibility by IFN and the location of the LMP2 and LMP7 genes in close proximity to genes of the MHC II complex. Furthermore the IP is proposed to play significant roles in producing antigenic peptides for presentation on MHC I (66, 69, 70). Standard and immunoproteasome catalytic subunits show a high from 76% to 83% amino acid identities among the correlates (71). Murine LMP2 and LMP7 are encoded within the MHC class II region on 17 while MECL-1 is localized outside of the MHC in a cluster of unrelated genes on chromosome 8.

Cells of hematopoietic origin, such as T cells, B cells, monocytes, mTECs (medullary thymic epithelial cells) and especially antigen-presenting cells (DCs, macrophages) constitutively express high levels of immunoproteasomes and only low levels of constitutive proteasomes (3). Thus, the IP is highly abundant in lymphoid tissue such as thymus, spleen, lymph nodes and bone marrow in which it conveys specialized functions. As well, liver, and the intestine express a substantial amount of IP even under homeostatic conditions (71,12,72). The constitutive expression of immunoproteasome subunits is likely due to permanent activation of intracellular signaling mechanisms. It has been shown that unphosphorylated STAT1 binds to IRF1 (interferon regulatory factor 1) to form a complex which functions as a for constantly supporting LMP2 expression in hematopoietic cells (3, 74). However, IP expression can be contemporary induced in non-lymphoid tissue in response by various factors, such as pro-inflammatory cytokines (TNF, IFN) or environmental stressors like oxidative damage. Furthermore, the inducible expression of IP results in an accelerated proteasome assembly which allows rapid adaption to environmental changes and ongoing immune responses (75).

Incorporation of immunoproteasome subunits alters the catalytic activities of the proteolytically relevant subunits, which leads to different substrate cleavage patterns. These alterations between the CP and IP can be explained by structural differences and amino acid substitutions in the substrate binding pockets. Particularly, chymotrypsin-like and trypsin-like activities are increased, whereas C-L activities are almost completely eliminated. Thus, cleavage after acidic residues is nearly abolished in the IP (76). LMP7 and MECL-1 display the same cleavage specificity as their homologous standard subunits. However, in contrast to 1c, the LMP2 subunit now provides chymotrypsin-like activity, which possibly promotes the generation of MHC class I restricted peptides that are characterized by hydrophobic C- terminal anchor residue (10, 77, 78). Compared to 1, the substrate specificity pocket S1 of LMP2 contains two prominent amino acid substitutions. Thr21 is replaced by Val and Arg45 is replaced by Leu. These substitutions entail a reduced size of the S1 pocket in LMP2 and an altered polarity from positive to neutral. Additionally, crystallographic analyses reveal conformational changes of Met45 that results in a larger S1 pocket in LMP7 compared to 5c which is responsible for enhancing the CT-L activity of LMP7 (27). Although, both subunits possess CT-L activity, LMP7 has a stronger preference for cleavage after bulky, hydrophobic residues compared preferential cleavage after small, hydrophobic

5 amino acids such as alanine by 5c. No major structural varieties were observed between the 2c and MECL-1 binding pockets except for Asp53 replacement by Glu in MECL-1 (26, 79). Collectively, the altered cleavage preferences increase the quantity of peptide fragments with basic and hydrophobic C- termini which efficiently fit into the MHC I binding groove. Therefore, subunit replacement in the IP generates shapes subsequent CTL responses.

The thymoproteasome The group of Tanaka (80) has previously identified a third homolog of the 5 subunit, which incorporates into otherwise immunoproteasomes containing LMP2 and MECL-1. This results in the proteasome complex named ‘thymoproteasome’ due to the exclusive expression of the 5t (PSMB11) subunit in cortical thymic epithelial cells (cTECs) in the thymus. In contrast to 5c and LMP7, the S1 pocket of 5t includes Thr45 and is predominantly composed of hydrophilic amino acids, resulting in 60-70% decreased CT-L activity. Thus, the contingent of peptides with high affinity to MHC class I is reduced in this proteasome subtype. However, these low-affinity MHC class I binding peptides appear to play an important role for positive selection of CD8+ T cells as well as for cytokine release (80–82). During positive selection, self-peptides in complex with self MHC molecules are presented on the surface of cTECs to immature thymocytes while they move through the thymic cortex. Those thymocytes bearing T cell receptors capable of appropriately interacting with these complexes are positively selected for further development, while the remaining thymocytes undergo programmed cell death. Additionally, it has been proposed that the presentation of these thymoproteasome-specific peptides during positive selection may protect the development of autoimmune responses against these same peptides in the periphery (83).

Proteasome assembly The chaperone-assisted and regulated assembly of 20S proteasomes is quite similar for all proteasome subtypes and starts with the formation of the α-rings. This process is assisted by two heterodimeric chaperone complexes PAC1-PAC2 (proteasome assembling chaperones) and PAC3-PAC4. The PAC1- 2 chaperones function as a scaffold in order to prevent α-subunit association with other nascent α-rings. PAC3-4 facilitates the formation of a closed heptameric α-ring, thus preventing improper α-subunit aggregation (84). Fully and proper assembled α-rings then serve as a scaffold for subsequent -ring assembly. In case of the standard proteasome the -ring formation begins with the incorporation of 2c which is recruited and correctly positioned by the help of PAC3-4 and POMP (proteasome maturation protein, proteasemblin) (85). Next, 3 is incorporated, while the PAC3-PAC4 complex is concomitantly released (86). The remaining -subunits are incorporated into the nascent complex in a defined order (4, 5c, 6, 1, 7) eventually forming half proteasomes with the assembled α-ring (87). Each catalytic subunit is synthesized as a precursor with an N-terminal pro-peptides ranging from 2 to 7 kDa in size that serves as an intramolecular chaperone. Only the removal of these pro-peptides via autocatalytic mechanism within the fully assembled proteasome leads to maturation of the 20S core and finally to the liberation of the catalytic active threonine residues at the newly formed N-termini (30, 88, 89). In this way the proteasome avoids random protein degradation prior to proper subunit incorporation (90, 91). Finally, two half proteasome core particles are combined by dimerization with the help of POMP and the C-terminal tail of 7 (92, 93). Formation of the complete 20S core initiates autocatalytic cleavage of the N-terminal pro-peptides from the three pairs of catalytic -subunits. This leads to the first proteolytic degradation of the POMP and PAC1-PAC2 chaperones by the fully-assembled and mature 20S proteasomes (94). Trimming of the pro-peptides upon completion of the assembly severs as another protective mechanism to avoid unregulated cleavage prior to proper incorporation into the complex (88).

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The assembly of IPs is somewhat different. Unlike the order of subunit incorporation in CPs, co- incorporation of LMP2 and MECL-1 is the initial step in assembly of the -rings in IPs (87). After that, 3 is incorporated, which is immediately followed by either LMP7 or 4 and the leftover subunits to complete the assembly of the ring. Proteasome assembly is biased to promote immunoproteasome formation in presence of all catalytic subunits in the same cell (95). This preferential IP assembly is partly due to differences between the LMP7 and 5c pro-peptides, since POMP binds to the LMP7 pro- peptide with a higher affinity than to 5c (96). Additionally, LMP7 is preferentially assembled prior to 4. Furthermore, favored LMP2 incorporation facilitates (or even directs) MECL-1 incorporation (75, 97). Finally, LMP7 facilitates maturation of the nascent 20S core by aiding cleavage of pro-peptides from LMP2 and MECL-1 (97).

Under certain circumstances, asymmetric mixed proteasome subtypes comprised of different CP and IP catalytic SU composition may assemble. Albeit, only specific combinations of CP and IP subunits can be simultaneously incorporated (98). LMP7 is the only immuno-subunit that can be incorporated into proteasomes independent of the other subunits (88). Thus, intermediate proteasomes either contain only one (LMP7) or two (LMP7 and LMP2) inducible IP subunits (99, 100). Interestingly, these preferential subunit combinations preclude proteasome subtypes comprising MECL-1 together with constitutive SU or 5c together with either of one of the immunoproteasome subunits (95). Although it was previously reported that LMP2 is not incorporated into proteasomes independently of MECL-1 (which would lead to the fact that all MECL-1 containing proteasome complexes also must contain LMP2) (97), there are also controversial findings. Since MECL-1-/- mice still exhibit LMP2 expression, this subunit must be able to incorporate into proteasome without co-incorporation of MECL-1. Therefore, LMP7 is required for proper maturation of LMP2 and MECL-1-containing proteasomes, indicating that LMP7 is incorporated into all subtypes of 20S proteasomes that contain any of the IP subunits. LMP7 is additionally able to incorporate into proteasome complexes that contain constitutive subunits (75).

The above mentioned findings suggest the formation of four different subtypes of 20S proteasomes: CP, IP and two intermediate forms composed of 1c-2c-LMP7 or LMP2-2c-LMP7. Dahlmann (100) and others described the presence of intermediate proteasomes in various non-diseased tissues and cancer cells from both murine and human samples (99, 101). Healthy human tissue such as kidney and liver possess considerable amounts of intermediate proteasomes between 30% and 50% of the total proteasome pool, respectively, with the balance making up mainly CPs (102). Moreover, the small and large intestines only contain intermediate proteasomes and immunoproteasomes. Noticeably, these intermediate proteasomes are highly abundant in pAPCs such as macrophages and DCs, for example in mouse spleen. Also, cancer cells such as shown in melanoma, NSCLC or myeloma cell lines constitute a quantity of about 15-20% of intermediate proteasomes (103–106). Mixed proteasomes enhance the antigenic peptide repertoire presented on MHC class I including tumor-associated antigens (103, 107, 108). Collectively, intermediate proteasome represent a significant portion of the total proteasome pool in cells. However, these diverse proteasome populations and their distinct distribution pattern are highly tissue-specific, suggesting that these proteasomes confer specialized cellular function due to varying activity profiles (100, 109, 110).

An additional layer of diversity is given by the ability to form hybrid proteasomes with different combinations of proteasome regulators, for examples PA28-20S-19S (111). Including the different RPs which are able to associate with each end of the 20S core, it is possible to generate 36 proteasome subtypes – each of them different in their proteolytic capacity and specificity. Therefore, different

7 proteasome subtypes allow the cell to adapt to changing environmental conditions by adjusting proteolytic capacity and the substrate repertoire to each new encounter.

The immunoproteasome in health and disease Undeniably, the immunoproteasome plays important roles in the adaptive immune system. The altered cleavage preferences improve the versatility of antigenic and immunogenic peptides that contribute to mount efficient anti-bacterial and anti-viral CTL responses (3, 12, 78, 112). Functions of immunoproteasomes in pathogenic infection models have been analyzed using immunoproteasome deficient mice (3, 78, 88, 113). Indeed, IPs produce immunodominant epitopes derived from ovalbumin or LCMV more efficiently than CPs (49, 114). LMP7-deficient mice (115) displayed 25-50% reduction in surface expression of MHC class I and ineffectively presented endogenous peptides. This suggest inferior production of antigenic peptides in the absence of IP subunits, emphasizing the outstanding role of LMP7 in enhancing the generation of MHC class I ligands for antigen presentation. LMP7 deficiency leads to inefficient presentation of the endogenous, male specific minor histocompatibility antigen HY, the LCMV epitope GP33-41 and epitopes of the multiple murine cytomegalovirus (MCMV). Furthermore, LMP7-/- mice exhibit an impaired generation of an epitope of the intracellular bacterium Listeria monocytogenes (116). Simultaneously, these mice show normal parasite-specific CD8+ T cells frequencies and could yield pathogen clearance in spleen, but not in liver. Additionally, LMP7-deficient mice succumb to protozoan Toxoplasma gondii infection, due to decreased IFN response of CD8+ T cells which is in line with the proposed defect in antigen processing (117). Moreover, LMP7-/- mice failed to clear protein aggregates that accumulated in cardiomyocytes following Coxsackie virus B3 (CVB3) infection. Subsequently, LMP7 KO mice suffered from aggravated myocarditis due to impaired protection against virus-mediated protein damage (118). Furthermore, van Kaer et al. (119) demonstrated that CD8+ T cell responses against Influenza were inferior in LMP2-deficient mice resulting from less efficient presentation of a nucleoprotein epitope of influenza A virus in these mice. LMP2 or MECL-1 deficiency alters the amount of CD8+ T cells compared to LMP7 deficient mice (119, 120). Although, MECL-1 deficiency did not alter MHC class I surface expression in contrast to LMP7 KO mice, these mice exhibit reduced frequency of influenza NP-specific CTLs and a decreased response to two class I restricted epitopes of LCMV (lymphocytic choriomeningitis virus) (GP276-286 and NP205-212) (120), but normal response to Sendai virus (121). On the other hand, LMP2-/- mice show reduced NP366-374-specific T cells. Additionally, LMP2, LMP7, MECL triple KO mice show severe defects in MHC antigen presentation and accompanied 50% reduction of surface expression of MHC I molecules. These KO mice poorly presented LMCV epitopes except for GP276-286, which was increased. Thus, different effects on CTL generation are attributable to defects in antigen presentation in the periphery and an altered T cell repertoire or reduced CTL precursor frequency in these IP deficient mice (120, 122, 123).

Immunoproteasomes hold regulatory roles in T cell survival and proliferation (120, 122, 124, 125), as well as T helper cell differentiation (126) and the production of pro-inflammatory cytokines (127, 128). Additionally, IP formation appears to be essential for protein quality control and for preserving protein homeostasis in response to oxidative stress. Induction of immunoproteasomes was shown to be involved in enhanced elimination of damaged proteins and a superior degradation of DRiPs (defective, ribosomal products) which are misfolded, oxidized proteins (129). In relation to that, immunoproteasomes preserve cell viability and protect cells against harmful inflammation-induced accumulation of protein aggregates but also increase cytokine-mediated protein synthesis during inflammatory responses (9, 130). Moreover, the immunoproteasome was shown to contribute to the regulation of mass

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(131). An alleged role of IP in cell signaling by activation of NFκB was revised by findings in our laboratory and elsewhere (132, 133). They could show that the catalytic activity of LMP2 and LMP7 is not required for canonical NFκB activation.

Besides its tremendous role in antigen presentation and pathogenic infections, immunoproteasomes have been implicated in several other biological and pathological processes resulting in the development and aggravation of many diseases, including neurodegenerative diseases, inflammatory and autoimmune diseases (88). Consistent with that, the expression of immunoproteasome subunits in non-lymphoid tissues is strongly upregulated upon immunological and/or pathological challenge (3). Under healthy conditions immunoproteasomes are absent in neurons, but IP formation is heavily induced in animal models of neurodegenerative diseases such as Alzheimer´s disease (134), Huntington´s disease (135), and amyotrophic lateral sclerosis (ALS) (136). Elevated immunoproteasome levels have also been associated with inflammation and the development and progression of a number of autoimmune and inflammatory diseases, such as Crohn´s disease (137, 138), ulcerative colitis (139) and hepatitis (140). A pronounced immunoproteasome expression was found in liver of patients with cirrhosis (141) and in clinical specimen of multiple sclerosis (141), as well as in intestinal tissues of IBD patients, particularly of LMP2 and MECL-1 (142, 143). Also, LMP2 was found to be upregulated in DSS-induced colitis model, while transgenic mice lacking the LMP2 subunit did not develop colitis which is accompanied by mitigating damages to colon mucosa of experimental colitis due to low levels of intestinal inflammation (139, 144). Of particular note, elevated expression of LMP2 and LMP7 has been detected in approximately 70% of human CRC (colorectal cancer) tumor samples (145, 146). Moreover, immunoproteasome formation was upregulated in microglia cells in response to infection with LCMV (147), as well as in liver cells upon infection with Listeria monocytogenes (148). The involvement of immunoproteasomes in inflammatory disorders was complemented by descriptions of diseases in humans linked to mutations or single-nucleotide polymorphisms in genes encoding immunoproteasome subunits (149). Notably, missense or nonsense mutations in the PSMB8 gene encoding LMP7 cause decreased chymotrypsin-like activity. Patients carrying these point mutations suffer from symptoms displaying recurrent fever, elevated levels of acute phase molecules, and high levels of IL-6. These symptoms probably occur due to aberrant accumulation of poly-ubiquitinated and oxidized proteins. The underlying auto-immune disorders include the autosomal recessive autoinflammatory JMP syndrome (joint contractures, muscle and -induced lipodystrophy syndrome), the CANDLE syndrome (chronic atrophical neutrophilic dermatosis with lipodystrophy), the JASL syndrome (Japanese auto-inflammatory syndrome with lipodystrophy) which causes increased p38 phosphorylation and IL-6 production, and finally the Nakajo-Nishimura syndrome (NNS) (150–153). However, it is still controversial whether IP forces inflammatory diseases or whether its induction results as a consequence of excessive cytokine release and acquired cell stress. Whether immunoproteasome deficiency exerts a protective effect or exacerbates disease progression depends on the underlying pathology and the contributing inflammatory environment (39, 88). Using immunoproteasome-subunit KO mice it was concluded, that the immunoproteasome has a rather protective role and efficiently suppresses autoimmunity (154) due to increased intrinsic catalytic activity and the prevention of accumulation of harmful aggregated proteasome substrates during inflammation (9). However, it was also reported that immunoproteasomes are not superior in substrate degradation compared to the constitutive counterpart (155). Nonetheless, the use of selective immunoproteasome inhibitors helped to identify the involvement of the immunoproteasome in a variety of immunological and physiological

9 processes which are further described below and which are reviewed in Kisselev and Groettrup et al. as well as in Basler et al. (156, 157).

The proteasome as drug target in cancer Soon after its discovery, it became evident that the UPS mediates the degradation of regulatory proteins of almost all fundamental cellular mechanism and pathways like cell cycle progression, signal transduction, survival and differentiation, cell proliferation, immune response, antigen presentation or apoptosis (158–161). Dysfunction or defects of the UPS has been implicated with several pathological conditions including cardiac and neurodegenerative diseases, such as Alzheimer’s disease (AD) and Huntington’s disease (HD), as well as neoplastic disorders (5). Rapidly proliferating neoplastic cells with increased levels of proteasomes are majorly relying on the integrity of the UPS due to their expedited cellular metabolism and constantly increased proteotoxic stress levels in response to excessive activation of oncogenic signaling cascades (162). This is illustrated by prominent targets of the UPS in cancer which are the tumor suppressor proteins p53 and Rb (163, 164), the cdk inhibitors 1B (p27Kip1) and p21WAF/Cip1 (165–167), members of the Bcl-2 family, oncogenes such as c-, c-Fos , c-Jun, the transcription factors E2A, E2Fa, as well as factors of the NFκB pathway, namely IκB (inhibitor of kappa b) and c-Rel (168, 169).

Originally developed to further characterize the catalytic activities and cellular functions of the proteasome, small molecules selectively inhibiting the activity of proteasome were discovered to be cytotoxic to rapidly proliferating malignant cells with high rates of protein turnover while omitting healthy ones (170, 171). After that, proteasome inhibitors (PI) have largely been developed as anti-tumor agents. The anti-neoplastic activity of proteasome inhibition is attributed to multifactorial effects including accumulation of misfolded, poly-ubiquitinated and potentially toxic proteins, while simultaneously preventing clearance of misfolded proteins by the ERAD (ER-associated protein degradation) pathway. Furthermore, inhibition of the NFκB signaling pathway, the overproduction of RONS (reactive oxygen and nitrogen species), the initiation of the unfolded protein response and a defective balance of pro- and anti-apoptotic mediators finally results in upregulated ER stress, cell cycle arrest and cell death (172–177). Currently, proteasome inhibition is the first line treatment of human malignancies of the hematopoietic system such as mantle cell (MCL), non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, multiple myeloma (MM), acute and chronic lymphocytic (ALL and CLL). On the other hand, PIs only show limited success in the treatment of solid (178). In particular, plasma cell derived - multiple myeloma cells provide the optimal basis for PI treatment since they constitutively express elevated levels of proteasomes and exhibit increased protein biosynthesis triggered by immunoglobulin production (179).

While some PIs interact with the α-subunits of the proteasome to inhibit access to the catalytic core, the majority of proteasome inhibitors are active-site directed peptide-based small molecules that block catalytic activities of -subunits. Most of the inhibitors are composed of a short, characteristic peptide sequence followed by a C-terminal pharmacore that either reversibly or covalently interact with the nucleophilic hydroxyl side chain of the active-site Thr1 residue of the catalytic subunits, thus imitating natural proteasome substrates. The individual subunit binding preference is determined by side chains (P sites) of the respective compound as well as by the active site of the proteasome subunit itself. Consequently, the amino acid sequence of the inhibitor specifies its either broad-spectrum or subunit specific activity. The overall effect of a

10 proteasome inhibitor is determined by its bioavailability, its site of interaction, binding kinetics, as well factors that rely on target cell such as proteasome subtype expression, proliferative activity and protein turnover rate. Most of the PIs preferentially target the CT-L activity of the 5 subunit. According to their chemical and crystallographic structure, there are currently seven main subclasses of active-site directed proteasome inhibitors available, including peptide aldehydes, peptide boronates, - lactones, peptide vinyl sulfones, peptide α’’epoxyketones, vinylamides (syrbactins) and α- ketoaldehydes (glyoxals) (180–182). Peptide aldehydes Peptide aldehydes were the first synthetic proteasome inhibitors (183). The most well-known representative of this class of inhibitors is the tripeptide aldehyde MG132. Proteasomal inhibition is achieved by formation of rapidly reversible hemiacetal adducts between the aldehyhde pharmacore and the catalytic threonine residues (29). A potential anti-cancer effect was verified by the induction of apoptosis in cancer cells (184–186). Peptide boronates Boronic acid peptides are structural analogs to peptide aldehydes and are reversibly binding to the proteasome, thereby displaying increased potency compared to the aldehyde analogs (187). The boronate pharmacore forms a slowly reversible tetrahedral conjugate with the catalytic threonine residue Thr1, which is stabilized by the formation of a hydrogen bonds between any of the boronic acid hydroxyl group and the N-terminal amino group of the threonine (188, 189). The most notable inhibitor of this class is the dipeptidyl boronic acid bortezomib (BZ), which was the first proteasome inhibitor to receive FDA approval in 2003 for treatment of relapsed or refractory MM and MCL. BZ (formerly known as PS-341, now Velcade®, Millenium (Takeda Oncology Company)) potently inhibits proteasomal CT-L and C-L activities, with minimal impact on the T-L activity (190). However, using subunit-specific fluorogenic subtrates, similar potencies in inhibiting LMP2, 5 and LMP7 with low nanomolar IC50 (maximal inhibitory concentration) values of 7 nM and 4 nM have been revealed (170). BZ was shown to reduce tumor growth of mouse xenografts of prostate cancer. BZ-mediated proteasome inhibition induces dysregulation of the cell cycle by elevated levels of the cdk-inhibitor p21 which eventually leads to caspase-4, 8 and 9 dependent apoptotic cell death. Additionally, stabilization of IκB resulted in reduction of NFκB activity (191). Despite its clinical success, BZ-treated patients experience several side-effects including peripheral sensory neuropathy (PN), fatigue, modest thrombocytopenia, gastrointestinal disorders as well as the development of inherited or acquired drug resistance. In order to achieve preferential specificity and more favorable toxicity profile, the first orally available PI ixazomib was developed. Ixazomib (previously MLN2238, Millenium Pharmaceuticals Inc., now NINLARO®, Takeda) is administered as a boronic ester prodrug (ixazomib acid, MLN9708) which rapidly hydrolyzes into its active form in aqueous solution or plasma. Ixazomib is the third PI approved by the FDA in 2015 for combination therapies in MM patients. Ixazomib is an N-capped dipeptide boronic acid preferentially targeting 5 CT-L activity. Increased anti-tumor response of xenograft models of solid cancers have been additionally identified (192). The superior tissue distribution in comparison to BZ is attributable to a shorter proteasome dissociation half-life. The major molecular mechanism of this proteasome inhibitor include induction of UPR responses and ER stress. Another reversibly binding boronate-based PI is delanzomib, but its anti-tumor activity remains to be determined.

β-lactones Inhibitors of this class are natural products, for example the Streptomyces lactacystinaeus metabolite lactacystin. These inhibitors inactivate the catalytic threonine by esterifying the hydroxyl group and formation of an acyl-ester adduct. Lactacystin was shown to block cell cycle progression and to

11 preferentially induce apoptosis in chronic lymphocytic leukemia (B-CLL) by inhibiting the CT- L activity after covalently binding to 5c in malignant cells (193). To overcome the general issue of instability in aqueous solutions, another representative of the -lactone inhibitors is currently under advanced clinical investigation, which is the orally available, irreversible inhibitor Marizomib (MZ, Salinosporamide A (NPI-0052)), that is produced by the marine gram-positive actinomycete Salinospora tropica. Its subunit selectivity profile shows rapid and prolonged inhibition of the CT-L and T-L enzymatic activities, while displaying increased cytotoxicity compared to BZ. Peptide vinyl sulfones These synthetic, cell permeable inhibitors irreversibly bind to the proteasome by forming a covalent bond between the hydroxyl side chain of the threonine and the vinyl sulfone pharmacore preferentially targeting the CT-L activity (194). However, like peptide aldehyde inhibitors, vinyl sulfones also lack specificity due to off-target effects with other cellular proteases. Peptide epoxyketones The natural compounds eponemycin and epoxomycin, originally isolated from Streptomyces or Actinomycete, respectively, were initially discovered as broad-spectrum inhibitors and proofed anti- tumor activity against B16 melanoma (195). Epoxyketones potently inhibit catalytic activities upon binding of the α,-epoxyketone moiety to both the hydroxyl group and the free α-amino group of threonine residue, thereby forming an irreversible, six-membered morpholine adduct (196). Differential subunit binding preferences specify eponemycin as immunoproteasome selective with LMP2 being the primary target, while epoxomycin targets 5, LMP7, 2 and MECL-1 (197). Due to their specificity and the lack of off-target effects, a variety of synthetic peptide epoxyketones have been designed, ultimately leading to the development of potential therapeutic agents. The second-generation peptide epoxyketone Carfilzomib (CFZ, formerly PR-171, now Kyprolis®, Onyx Pharmaceuticals (Amgen), Proteolytix Inc.) was approved by the FDA in 2012 for treatment of relapsed or refractory MM in BZ-resistant patients. Compared to BZ, CFZ displays a more selective CT-L inhibitory profile and increased unparalleled binding specificity for the 20S subunits, together with lower in vivo toxicity. The orally available CFZ analog oprozomib (formerly PR-047, ONX 0912, Onyx Pharmaceuticals) selectively inhibits 5 and LMP7 and is currently evaluated in clinical trials for treatment of hematological malignancies and some solid cancers.

Nevertheless, the clinical activity of bortezomib and carfilzomib is often limited by unwanted off-target effects. As well, the absence of validated, predictive biomarkers of therapeutic outcome and the generally lack of therapeutic activity against solid tumors has led to a novel approach of designing more selective and potentially more potent proteasome inhibitors (27). Subunit-selective inhibitors β5/LMP7-selective inhibitors (CT-L activity inhibitors) The tetrapeptide epoxyketone YU-101 (which later yielded its derivate Carfilzomib by N-terminal modifications), was developed as the first proteasome-specific inhibitor for selective targeting of the CT-L activity without affecting the C-L or T-L activities. While exceeding other classical proteasome inhibitors in potency and selectivity, YU-101 showed anti-inflammatory and anti-proliferative effects. In relation to that, other peptide epoxyketone inhibitor were designed, including NC-005, which exhibits similar potency to YU-101 while being more selective in targeting the CT-L activity. However, these inhibitors did not yield antitumor activity.

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Immunoproteasome subunit-selective inhibitors Since several reports declared important roles of the immunoproteasome in diverse pathologic mechanisms, considerable effort has been spent on the development of immunoproteasome subunit selective inhibitors during recent years. LMP2-selective inhibitors The first immunoproteasome subunit selective inhibitor, UK-101, a peptide epoxyketone, targets LMP2 (113). It was shown to have anti-proliferative effect and significantly inhibited tumor growth in a xenograft of prostate cancer (132, 198, 199). Another advanced LMP2 selective inhibitor is IPSI-001 (Calpeptin) which also showed pro-apoptotic activity, ubiquitin conjugate accumulation and caspase- mediated apoptosis in myeloma-cell lines. IPSI-001 represents a 100-fold selectivity towards the CT-L activity of the IP over the CP. However, due to well-known off-target inhibition and cross-reactivity of the aldehyde pharmacore with other cellular serine and cysteine proteases such as cathepsin A and G and calapains, these inhibitors were not suitable for further assessment of clinical use (200). LMP7-selective inhibitors The first 5i-selective inhibitor was the tripeptide epoxyketone PR-957 (later ONX 0914, Onyx Pharmaceuticals (Amgen)) (Figure 3). Its selectivity and subunit binding preferences was identified by quantifying occupied proteasome active sites in the human leukemia cell line Molt-4 expressing both constitutive and IP subunits via subunit-specific ELISA (127). ONX 0914 displayed 20 to 40-fold increased selectivity for LMP7 over its secondary sensitive targets 5 and LMP2, respectively. Furthermore, ONX 0914 displayed a significantly low toxicity profile. ONX 0914 also inhibits the remaining catalytic subunits MECL-1, βc and 1c – but only at high concentrations. Proteasome selectivity is enabled by binding of ONX 0914 to the more spacious S1 pocket of LMP7. The selectivity was further verified by using knock-out mice lacking LMP7. CT-L substrate hydrolysis and peptide presentation on MHC I was inhibited in wild type animals but not in LMP7-/-. Crystal structures of the murine IP in complex with ONX 0914 highlighted that the N-terminal morpholinyl acetyl cap of the inhibitor is apparently not in direct contact with the LMP7 subunit, but rather serves to improve solubility and pharmacokinetic properties of ONX 0914 (79). Noticeably, in predominantly IP expressing PBMCs, ONX 0914 treatment inhibited LMP7 without considerable inhibition of LMP2 or MECL-1.

Figure 3. Top. Chemical structure of α’,’-epoxyketone ONX 0914. The pharmacore is shown in red. The S1 specificity pocket is illustrated as a blue curved line. Bottom. Mechanism of LMP7 inhibition © Sarah Mundt.

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LMP7-selective inhibition by ONX 0914 was shown to suppress the production of several pro- inflammatory cytokines including IL-6, IL-23 and TNF in human PBMCs. Furthermore, inhibitor treatment led to reduced expression of MHC I molecules on the cell surface. ONX 0914 treatment suppressed T helper cell differentiation of naïve CD4+ T cells into Th1 and Th17 and instead promoted the generation of Tregs most likely by inhibition of STAT3 phosphorylation (126). However, Th2 cells remained unaffected. Since both, Th1 and Th17 are intimately associated with the pathogenesis of several autoimmune diseases, these findings mark LMP7 as a potential therapeutic target for the treatment of chronic inflammatory diseases and autoimmune disorders.

Treatment with the LMP7-specific inhibitor ONX 0914 protected mice in several different preclinical models of inflammatory disorders and autoimmune diseases by diminishing pathological signs, disease severity and inflammation or even by suppression of disease progression. The anti-inflammatory effect of ONX 0914 was shown in mouse models of experimental DSS-colitis (139, 144), systemic erythematosus (SLE) (201), multiple sclerosis (MS) (202), type 1 diabetes and (RA) (127), as well as Hashimoto’s thyroiditis (203), myasthenia gravis (204) and autoimmune neuritis (205). These findings further highlight the therapeutic benefit of LMP7 inhibitors in the treatment of inflammatory disorders of diverse pathophysiological syndromes. By multiple measures, ONX 0914 proved to be more effective in inhibiting development or progression of experimental rheumatoid arthritis, in contrast to the clinically relevant etanercept as well as the 5-selective inhibitor PR-825 or the 5/LMP7-selective inhibitor Carfilzomib which either lacked efficacy or were only moderately efficient in amelioration of disease symptoms. Importantly, the therapeutic window of ONX 0914 of doses much lower than the maximum tolerated dose (MTD) of 30 mg/kg would not be achieved by broad spectrum proteasome inhibitors. In contrast, ONX 0914 had considerable therapeutic effects far below the MTD, at a dose level of 2 mg/kg. ONX 0914 treatment attenuated progression of arthritis by blocking IL-23 and TNF production in activated monocytes and by suppressing IFN and IL-2 secretion by T cells, thereby reducing immune infiltration in arthritic lesions and autoantibody levels (127). In mouse models of colitis, ONX 0914 treatment resulted in suppressed pro-inflammatory cytokine production, less inflammation and tissue destruction, as well as reduced differentiation of CD4+ T cells into Th17 cells (139, 144, 145). Interestingly, LMP7 inhibition did not always provoke the same effect on disease development as genetic LMP7 deficiency. For example, ONX 0914 treatment protected mice from disease progression in an experimental EAE model in contrast to LMP7-/- mice (202). Only recently, Li et al. could show that inhibition of the immunoproteasome by ONX 0914 treatment prevent chronic antibody-mediated allograft rejection in renal transplantation in rats (206).

Subsequently, a second LMP7-selective peptide epoxyketone inhibitor was developed with structural similarities to ONX 0914. PR-924 (IPSI) exhibits 130-fold selectivity towards LMP7 compared to 5c with little to no inhibition for other catalytic subunits. At higher concentration, it also inhibited LMP2. PR-924 efficiently induced apoptosis and suppressed further growth of multiple myeloma cells without affecting healthy PBMCs. As well, antitumor activity and prolonged survival was shown in mouse xenograft models with PR-924 being well-tolerated, which created the rational to further develop PR- 924 for clinical use in anti-myeloma therapy (207). Moreover, a lot of other LMP7-selective inhibitors have been developed lately (208).

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Colorectal cancer (CRC) – epidemiology and etiology With an annual incidence of more than one million new cases worldwide, colorectal cancer is the third most prevalent malignancy and the fourth leading cause of cancer morbidity and mortality with a considerable geographic variation and high incidences in industrialized countries (209–213). Almost every second human being will develop at least one benign polyp during lifetime but only less than 3% of these neoplasms will eventually evolve to colorectal cancer. Since disease-related symptoms are rare until very late stages of the disease, most cases remain undetected (211). The primary cause of cancer- related deaths in advanced stage CRC is the global distribution of metastases to lymph nodes, liver and lung (214). Although both men and women are similarly affected, men seem to have earlier disease onset and a higher incidence of CRC.

The etiology of CRC is still imprecise and not fully understood. The major causes of this pathology are heterogeneous and compose of complex interaction of environmental carcinogens, the accumulation of independent genetic aberrations and the host immune system. However, many different factors contribute to colon cancer development including diets rich in unsaturated fats and high consumption of red meat, excessive alcohol abuse, cigarette smoking. Furthermore, obesity and low levels of physical exercise have convincingly been associated with increased risk of CRC development. One of the main contributor of CRC development is chronic intestinal inflammation, as seen in inflammatory bowel diseases (IBD) (215–220). Although most cases of colon cancer can be attributed to sporadic CRC (85-95%), there are also hereditary syndromes (HNPCC, FAP, see below) and inflammation – mediated tumors (2-3%) (221). Despite distinct sets of tumor promoters and passenger mutations in every human colon cancer type, the most commonly genetic alterations in colon tumor lesions are Apc (81%), p53 (60%), K-Ras (43%) and Smad4 (9%).

CRC carcinogenesis is a multistep process of successive accumulation of mutations in tumor suppressor genes and oncogenes important for tumor initiation, while CRC progression develops due to genomic instability. Independent of the underlying CRC etiology, the essential stages of CRC development are: adenomatous, dysplastic precursor lesions (aberrant crypt foci, ACF) formation - adenoma – carcinoma. Although different pathogenic sequences have been proposed for colitis-associated CRC (CAC) following: inflammation – injury – dysplasia – carcinoma (222). The order and timing of mutations in this multistep carcinogenesis depends on the colon cancer type and differs among hereditary, sporadic and inflammation-mediated colon cancer (223) as illustrated in Figure 4.

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Figure 4. Molecular changes associated with heterogeneous colorectal cancer development. Top. In sporadic colon cancer of hereditary origin (familial adenomatous polyposis (FAP)), an early event is nonsense mutation in Apc which is accompanied by loss of cell polarity and tight junctions. This enables the invasion of bacteria and increased the production of the pro- inflammatory cytokines IL-23 and IL-17. Apc mutation is followed by mutations in the KRAS and the gene encoding p53 (TP53), which promotes progression from adenoma to carcinoma. Bottom. In colitis associated cancer, tumors develop from dysplastic lesions due to overactive colonic inflammation. Early mutations of the gene encoding p53 result in prolonged activation of NFκB and enhanced inflammation. The inflammatory response is further enhanced by barrier dysfunction and invasion of bacteria. Additionally, the inflammatory environment generates reactive oxygen species (ROS), which can drive DNA damage, that finally results in mutations of the Apc gene and the development of carcinomas. Images, modified from (214, 224, 225).

Hereditary colorectal cancer Inherited CRC manifestations are rare. Only about 5% of CRC arises in patients with a characterized germline mutation of autosomal and dominant manner. However, the causative mutations of HNPCC (hereditary non-polyposis colon cancer) and of FAP (familial adenomatous polyposis) are present in 100% of all sporadic CRCs. Of that 85% of all CRC types exhibit somatically mutant Apc and 15% harbor at least one of the typically mutated genes in HNPCC (226). These hereditary syndromes are characterized by an early age of disease onset at approximately 45 years of age with predominant involvement of the right-side colon (227–229).

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Hereditary non-polyposis colon cancer (HNPCC) HNPCC, also known as the Lynch Syndrome, is an autosomal dominant disorder caused by germline mutation in DNA mismatch repair (MMR) genes and results in adenoma formation in the ascending colon (230–233). The DNA repair genes that are predominantly affected are MSH2 or MLH1 (90%) as well as MSH6, PMS2 or MLH3 (234). Inactivation of the MMR is associated with microsatellite instability (MSI) which eventually promotes frame shift mutations in other tumor-promoting genes including CTNNB1 (the gene for -catenin), BRAF, as well as type II TGF and BAX (227). MSI is characterized by a defective DNA mismatch repair (MMR) system results and is further defined as insertions or deletions in DNA microsatellite repeat sequences (235). Malignant growth is thus initiated by mutations in microsatellite containing tumor suppressor genes such TGFRβ (transforming growth factor beta receptor 2), IGFIIR (insulin-like growth factor II receptor) or Bax (Bcl-2 (B cell lymphoma 2) – associated X protein) (236). MSI-mediated tumors are of the exophytic or sessile polyp type and characterized by excessive mucus production, poor differentiation and aberrant lymphocytic infiltration (237–239). Familial adenomatous polyposis (FAP) Hereditary FAP is mediated by nonsense mutations of the Apc (adenomatous polyposis coli) tumor suppressor gene at chromosome 5q21 and accounts for 1% of all CRC cases (240–242). This mutation generates a premature and inactivated protein due to insertion of an early stop codon. Apc mutation leads to chromosomal instability (CIN) and CRC development along the CIN pathway (243). CIN is characterized by abnormal chromosomal segregation and defects in chromosome cohesion and mitotic checkpoints which cause imbalance in chromosome numbers. Aneuploidy further causes inactivation of a functioning allele of tumor suppressor genes (loss of heterozygosity (LOH)) such as on chromosome 5q for Apc (244). FAP patients display several hundred pedunculated adenomatous polyps throughout the colon and rectum. If untreated, these precancerous benign lesions have a 100% prevalence for developing CRC. Loss of APC function leads to -catenin accumulation which stimulates cell proliferation and delays differentiation. Besides the formation of polyposis, FAP patients are further encountered with extracolonic manifestations including pancreatic mucinous adenocarcinoma, hepatoblastoma and desmoid tumors as well as congenital hypertrophy of the retinal pigment epithelium (CHRPE) (245, 246). The APC mutation is already present in early lesions. Transition of ACF to adenomas in the Apc-mediated tumorigenesis involves activating mutations of the MAPK pathway, predominantly in the K-ras proto-oncogene. Polyp progression to invasive and metastatic cancer is further driven by sequential accumulation of mutations in TGF and DNA repair pathways with LOH of the tumor suppressors Smad4 and p53. Apc in CRC and its functions in the Wnt signaling pathway Apc is a multidomain-containing protein composed of 2843 amino acids. It regulates cell migration, cell-cell interaction, chromosomal segregation and apoptosis in colonic crypts by multiple binding sites for various proteins. However, the most prominent role of Apc is its function as a canonical tumor suppressor by down-regulation of Wnt signaling cascade. Thus Apc prevents continuous activation of the oncogene -catenin. In the absence of Wnt activating ligands, Apc forms a complex with Axin, GSKγ (glycogen synthase kinase 3 beta), EB1 (end-binding protein 1) and several other proteins that bind -catenin and prevent nuclear translocation of the latter. Instead, complex formation promotes phosphorylation, ubiquitination and subsequent proteasomal degradation of -catenin. Loss of Apc function results in -catenin accumulation and nuclear translocation. Binding of -catenin to the Tcf/Lef (T cell factor/lymphoid enhancer factor) transcription factor complex induces transcription of a wide variety of -catenin target genes such as the proto-oncogenes cyclin D1 or c-myc, the transcription factor AP-1 (activator protein 1) as well as MMP-7 (matrix metalloproteinase 7), PPAR (peroxisome

17 proliferator-activated receptor gamma), c-jun or Akt kinase. Thus, the tumorigenic action of unregulated Apc/-catenin/Wnt signaling confers to direct stimulation of cell proliferation and the alteration of different underlying signaling cascades (247).

Inactivating somatic mutations of the Apc gene are found in 85% of human sporadic colorectal tumors which confirms the pivotal role of the Apc protein in colorectal tumorigenesis. Although, Apc mutations are sufficient to initiate adenoma formation, additional genetic events are required for tumor progression. Sporadic colorectal carcinoma The majority of sporadic CRC arise from benign dysplastic precursors in the descending colon and rectum which are usually polypoid, flat or depressed adenomatous polyps that progress over several decades to carcinoma (248, 249). At least two major molecular events contribute to further progression of sporadic CRC, which are CIN to 85% of all CRC cases, while MSI accounts for 15%. Since these two pathways can be already detected in precursor lesions, it indicates that genomic instability is already an early step in CRC development (250).

One of the earliest molecular events in sporadic CRC is loss of Apc function. Subsequent accumulation of activating mutations such as in the K-ras oncogene result in adenoma growth and progression. The final transition to adenocarcinoma is probably mediated by inactivation or mutation of p53. So, loss of p53 tumor suppressor function occurs rather late in sporadic CRC tumor development and was identified with a prevalence of 50% in aggressive lesions (251–254). Inflammation-associated colon cancer Colitis-associated cancer (CAC) is characterized by chronic inflammation and is associated with increased levels of pro-inflammatory cytokines, chemokines, growth factors and oxidative damage which in its entirety leads to the inception of colon cancer formation. The prominent hallmark of CAC is inflammation-associated oxidative and nitrosative stress due to an overproduction of reactive oxygen and nitrogen species (RONS) and depletion of antioxidant molecules. Elevated oxidative stress are probably induced via permanent activation of iNOS (inducible nitric oxide synthase) in the colon mucosa leading to cytotoxic concentrations of NO (nitric oxide). The associated massive increase in ROS level during intestinal inflammation results in DNA damage due to compensatory epithelial proliferation (“wound-healing”) in addition to the accumulation of genetic alterations in these epithelial cells. Inflammation is further augmented by aberrant DNA methylation as well as excessive cytokine production of infiltrating inflammatory cells. Furthermore, a considerable amount of genotoxin- producing bacteria which enter the intestinal lumen due to disrupted epithelial barriers also contributes to neoplastic transformation in the inflamed colon tissue.

While sporadic CRC predominantly progresses from polypoid lesions, IBD-associated cancers typically evolve from single, non-polypoid or flat, multifocal dysplastic mucosa (223, 251, 255, 256). Mutations or allelic loss of function of tumor suppressor genes p53 and DCC (deleted in colorectal cancer) is readily detected at early stages of CAC in inflamed, non-dysplastic mucosa and flat dysplasia. On the other hand, tumor progression is associated with mutations in Apc and proto-oncogenes like K-ras or Bcl-2, which predominantly occurs in high-grade dysplasia (HGD) and carcinomas (257).

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Colon cancer therapy For Stage 0 CRC (Carcinoma in situ) that is limited to the intestinal mucosa, local excision is the most favorable method of treatment for these patients with curable CRC. Treatment options of all other CRC stages include primary surgical resection, as well as adjuvant chemotherapy and neoadjuvant radiation therapy or chemoradiotherapy in order to reduce mortality in surgically treated patients or to reduce the risk of recurrence. Nevertheless, the 5-year survival probability strongly depends on the CRC tumor stage and decreases from 93% for stage I CRC to less than 5% for fully advanced stage IV CRC tumors. Thus, every second patient fails therapy and succumbs from CRC. There are various screening methods for early detection of colorectal tumors available, however, their diagnostic value is limited regarding sensitivity (fecal occult blood test), costs and inconvenience (colonoscopy). Recently, also non-steroidal anti-inflammatory drugs (NSAIDs) such as sulindac or aspirin, which inhibit COX-β and NFκB activity, have been shown to reduce polyp development in CAC and even in FAP patients (258, 259). In addition to standard treatment option (a combination of the cytotoxic drugs oxaliplating and 5- (5- FU), the introduction of monoclonal antibody therapy against EGFR (epidermal growth factor receptor) or VEGFR (vascular endothelial growth factor receptor) (Cetuximab, Bevacizumab and Panitumumab) has led to increased efficacy of metastatic CRC treatment, since aberrant expression of EGFR is found in 80% of colon cancers (260, 261). Novel therapies include mTOR (mammalian target of rapamycin inhibitors), PKC (protein kinase C) or Src (sarcoma family) kinase inhibitors. (262).

Mouse models of colon cancer Mouse models of spontaneously developing colon cancer ApcMin/+ transgenic mice as a mouse model for human FAP The ApcMin/+ (adenomatous polyposis coli / multiple intestinal neoplasia) mouse was identified in a random ENU (N-ethyl-N-nitrosurea) mutagenesis screen of C57BL/6J (B6) mice. The underlying phenotype is introduced by a missense mutation at position 850 on one allele of the Apc tumor suppressor gene which results in the 2549 amino acid long truncated Apc protein (263, 264). Due to its critical role in Wnt signaling and the regulation of development, homozygous mutation of germline Apc causes embryonic lethality. Thus, genetically modified ApcMin/+ mice are heterozygous. Although, the heterozygous phenotype is the preliminary step for intestinal tumor formation, initiation of tumorigenesis only starts after somatic loss of the remaining wild type allele via loss of heterozygosity (LOH) by somatic recombination (245, 265). This leads to accumulation and nuclear translocation of - catenin. The aberrantly activated Wnt signaling pathway subsequently drives neoplastic transformation within the crypt axis which results in the development of multiple polyps throughout the whole intestinal tract (266, 267). The Wnt-driven ApcMin/+ mouse model quickly recapitulates both genetic and phenotypic characteristics of human sporadic intestinal tumorigenesis by exhibiting polyposis following loss of APC within 16 to 20 weeks of age (263, 268). Homologous to murine Apc, deregulation of Wnt/-catenin signaling following inactivating mutation in the wild type allele of this gene is the major initiating factor in the etiology and pathogenesis of FAP. Moreover, Apc mutations are frequently found in more than 80% of sporadic CRC in human patients (269).

Premalignant lesions are visible as early as 4 weeks after birth. Due to rapid adenoma formation, these mice spontaneously develop approximately 24-30 both pedunculated and flat lesions, as well as multiple microadenomas throughout the whole intestinal tract (264, 270). While the small intestine is the primary site of polyp formation, tumor lesions in human FAP patients mainly occur in the colon. This discrepancy of tumor location and favorable expression in small bowel is still not explained. In contrast to human FAP that is predominantly characterized by colon adenomas which routinely progress to

19 invasive adenocarcinomas, ApcMin/+ mice rarely exhibit submucosal invasion or adenocarcinoma development. Moreover, there are no incidences of formation in these mice (271, 272). However, similar to human patients, ApcMin/+ mice develop extra-colonic manifestations including desmoid tumors, and a predisposition to mammary adenoma formation at later ages (254, 267). The average life expectation is rarely beyond a maximum of 120 days due to excessive intestinal tumor formation. As well, anemia is a frequent complication due to rectal bleeding of hemorrhagic tumors, thus negatively affecting general health status of the mice (273). The ApcMin/+ mutation is characterized by chromosomal instability and MSI associated mutations which cause mitotic defects in intestinal crypts of the small intestine. This initiates the generation of tetraploid cells, misoriented spindles and misaligned and leads to cell cycle defects, alterations in cell proliferation and apoptosis. As well, a reduced crypt to villus migration was observed in these mice (274).

Several genetically engineered Apc mutant strains with targeted disruptions of Apc have been developed in order to more accurately reflect FAP and sporadic CRC (224). Besides similar histopathological features of small intestinal polyposis, these mutant ApcMin/+ mice display Apc mutation-independent phenotypic characteristics such as altered tumor numbers and location, as well as disease onset and severity. For example, APC1322T, APCΔSAMP, ApcΔ14, APC5802 and ApcΔ580 mice have a more severe phenotype, APCΔ15 mice exhibit an intensively high intestinal tumor burden, while APC1572T mice display extracolonic manifestations rather than intestinal cancers. Other sporadic colon cancer mouse models IL-10 -/- mice spontaneously develop severe colitis and CAC with an aberrant Th1 phenotype (275). The development of invasive carcinomas in these mice is probably associated to colonic bacterial infection, as indicated by an accelerated AOM (azoxymethane)-induced tumorigenesis after H.pylori infection. Although IL-10 mainly acts as an anti-inflammatory cytokine, together with TGF it contributes to immunosuppression and might favor tumor progression by controlling functions of Tregs (regulatory T cells) or tumor-specific CD8+ T cells (276). Moreover, Gai2-/- (Guanine nucleotide binding (G)-proteins) mice develop UC like IBD eventually forming adenocarcinomas in the colon (277).

Chemically-induced mouse models of colon cancer – the AOM/DSS m odel Azoxymethane (AOM) Parenteral injections of the pro-carcinogen AOM and its derivates DMH (1,2 dimethylhydrazine) and MAM (methyl azoxy methane acetate) leads to colorectal cancer by DNA alkylation preferentially in the distal colon (278, 279). Compared to DMH, AOM exhibits higher potency and enhanced stability in dosing solutions (280). After application into the peritoneum, AOM requires further metabolic activation to exert its mutagenicity in the colon. Metabolism of these compounds in the liver involves several N-oxidation and hydroxylation steps. Consequent secretion of methylazoxymethanol-glucuronic acid (MAM) conjugates are further dissociated by the intestinal flora via bacterial -glucuronidase in the distal colon and rectum to eventually release active MAM (shown in Figure 5).

The first step is hydroxylation of the methyl group of AOM to generate MAM. The unstable MAM undergos further biotransformation or is spontaneously decomposed to formaldehyde and mehtyldiazonium, a highly alkylating species in the liver and colon (282). The released methylazoxymethanol is the main genotoxic compound causing DNA alkylation and the formation of DNA adducts (281, 283). The inducible cytochrome P450 isoform CYP2E1 is directly involved in activation of AOM and MAM. The active metabolites are further transported via bile or the bloodstream. Additional activation of these metabolites is proceeded via P450-independent mechanisms including oxidation of MAM by alcohol dehydrogenases directly in the colon. The bioactivation of MAM by unique colon-specific P450 isoforms further contributes to organ-specific induction of distal colon

20 tumors. Inhibition of CYP2E1 or genetic ablation of CYP2E1 decreased tumor numbers in the AOM model (284).

Figure 5: Metabolic pathway of azoxymethane (AOM) activation. AOM is hydroxylated to MAM by the P450 enzyme CYP2E1 in the liver. MAM is oxidized to methylazoxyformaldehyd in reactions catalyzed by CYP2E1 in the liver or the colon, but it can also spontaneously decay. Gut microbial factors lead to release of formic acid yielding the methyldiazonium ion, which methylates DNA bases in the colon. AOM, azoxymethane; MAM methylazoxymethanol. Image from (281) with minor adaptations. AOM-induced tumors show histopathological and molecular resemblances to human colorectal neoplasia with tumors of polypoid growth frequently located in the distal part of the colon. However, human tumors arise from adenomatous polyps, while AOM-induced adenocarcinoma develops from flat, non-polypoid mucosa. While in humans up to 50% of all patients develop metastasis in regional lymph nodes, AOM-mediated carcinogenesis lacks mucosal invasiveness and metastasis formation (271, 285).

AOM activates several molecular pathways including K-ras, -catenin, PI3K (phosphoinositide-3- kinase)/Akt and TGF (286) and thus further activates downstream targets for cell survival (NFκB, Bcl- XL (B cell lymphoma extra large)) and increases cell proliferation by promoting cyclin D1 and c-myc expression. Secondly, MAPK (mitogen activated protein kinase) together with ERK (extracellular signal regulated kinase) promote AOM-mediated tumorigenesis by targeting proteins such as c-myc or Rb. On the molecular level, AOM-induced dysplasia and neoplasms display aberrant expression of cell cycle promoters cyclin D1 and cdk4 which contribute to early phases in AOM-induced carcinogenesis in the mouse colon (280, 287). Similar to human CRC, AOM directly induces K-ras mutations (288, 289) and leads to altered cellular localization of -catenin (290). Furthermore, augmented expression of COX-2 (cyclooxygenase 2) and iNOS is induced in colon tumors (291). The AOM/DSS mouse model The two stage AOM/DSS tumor model is based on a single injection of AOM followed by repeated administration of the pro-inflammatory agent dextran sodium sulfate (DSS) which results in development of multiple large colonic tumors already after 8-10 weeks (279, 292–294). This model highlightes the tumor-promoting activity of chronic inflammation in development of AOM-induced tumors. Tumor lesions are mainly localized in the distal part of the colon, thus resembling the predominant site of UC-mediated neoplasia in humans. Also, the clinical course is similar to human UC which is characterized by spontaneous onset of active inflammation with intermittent periods of disease inactivity. Pathological progression from ACF (aberrant crypt foci) with crypt fission propagate DNA damage to neighboring crypts which eventually leads to the emergence of microadenomas (292, 295).

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AOM/DSS induced tumorigenesis is associated with increased infiltration of inflammatory cells, mainly macrophages and lymphocytes. Moreover, increased activity of the JAK/STAT3 (Janus kinase/signal transducer and activators of transcription 3) pathway that is activated in response to increased levels of pro-inflammatory IL-6, facilitates transition to cancer (282, 296–298). Additionally, AOM/DSS causes alterations in the intestinal microbiome, leading to dysbiosis due to impaired epithelial integrity (299, 300).

Tumor-promoting inflammation in CRC Inflammation is a key component of the tumor microenvironment and affects every facet of cancer development, leading to the fact that inflammation is the seventh inherent hallmark of cancer (301). The intricate connection of inflammation and cancer is not restricted to a subset of cancer, but is rather present in all neoplastic tissues, including those that are causatively unrelated to inflammatory processes. At least 20% of all cancer types are associated with persistent infection or chronic inflammation including gastric ulcer (Helicobacter pylori), liver cancer (Hepatitis Virus B/C), cervix carcinoma (human papilloma virus), autoimmune diseases or overt inflammatory conditions (e.g. prostatitis for prostate cancer) (301–303). Even cancer tissue that did not develop due to inflammation exhibits extensive inflammatory infiltrates and high levels of pro-inflammatory cytokines, referred to as ‘tumor- elicited inflammation’ (304–307). Cancer and inflammation are connected by each other schematically via two pathways. The intrinsic oncogenic-driven pathway is mediated by cancer-related changes, which guide the construction of the inflammatory milieu. The extrinsic pathway is activated by tumor-unrelated, inflammatory conditions such as pathogens or pro-inflammatory cytokines which directly promote cancer development (308, 309). Microbiota are one of these exogenous mediators associated with CRC development. A lot of human and animal studies demonstrate the role of particular bacteria as possible causative agents of colorectal cancer. For example, Helicobacter hepaticus and the enterotoxigenic Bacterioides fragilis enhance colorectal cancer formation in ApcMin/+ mice (310), Streptococcus bovis augments AOM- induced cancer in rats and Fusobacteria are increased in human CRC tissue (311–313). CAC represents the classical inflammation-mediated cancer model, where a single application of the pro-carcinogenic agent AOM only gives rise to colon tumors in combination to the induction of chronic colitis (278, 279, 314). Already described in 1925, Crohn and Rosenberg first reported the development of colitis-associated neoplasms. Long-term inflammatory conditions of the gastrointestinal mucosa as a hallmark of patients suffering from IBD pose a significant risk factor for the development of colon cancer as shown both in clinical studies as well as in mouse models of chronic inflammation (219, 251, 256). Although representing only 1% of all CRC cases, the overall risk for CAC development in long standing IBD (especially in UC) is 5 fold higher compared to healthy individuals (315). The risk factor increases with prolonged duration and severity of inflammation further demonstrating a tumor- promoting role of inflammation (256, 297, 316–318). The relationship of inflammation and colon cancer is moreover illustrated by successful anti-tumor efficacy using non-steroidal anti-inflammatory agents (NSAIDs) or blocking agents against TNF that resulted in decreased infiltration of neutrophils and macrophages into the colonic mucosa (259, 317, 319–322). Chronic inflammation leads to extensive accumulation of activated immune cells and their mediators (chemokines, cytokines), epithelial cell damage and the release of large amounts of reactive oxygen and nitrogen species (RONS) by activated macrophages and neutrophils (323). By causing oxidative and nitrosative stress, the highly mutagenic entities ROS (reactive oxygen species) and RNI (reactive

22 nitrogen intermediate) potentiate neoplastic transformation and tumor promotion (323–325). Recurrent cycles of mutagenic assault, tissue renewal and persistent DNA damage eventually leads to oncogene activation or inactivation of tumor suppressor genes. Genomic instability is constantly maintained by released factors of the pro-inflammatory environment that support carcinogenesis. This smoldering inflammation additionally potentiates epigenetic modification such as CpG (cytosine-phosphate-guanin) island methylation, histone modification or the activity of miRNAs and affects proliferation and survival of neoplastic cells, angiogenesis and metastasis (326).

The IL-6/STAT3 – TNF/NFκB axis in colon cancer A plethora of immune cells and cytokines contribute to tumorigenesis. IL-6 and TNF are the key tumor- promoting molecules in human and mouse colitis and colorectal cancer and are associated with activation of the intracellular signaling pathways STATγ and NFκB, respectively. A third important contributor of cancer-promoting inflammation is considered to be the activation of AP-1 by IL-1 (327, 328). Cytokine-mediated tumor promotion was verified in several CAC and CRC mouse models displaying high upregulation of IL-6, TNF and IL-1 (256, 317, 329). As well, STATγ and NFκB are aberrantly activated in 50% of CRC and CAC tumors thereby acting as non-classical oncogenes (304, 330–334). Chronically increased levels of IL-6 and TNF were shown to promote transition of colitis to CAC (316, 335). Elevated systemic and intestinal expression of both cytokines, as well as sIL-6Rα (soluble IL-6 receptor) in serum samples from IBD patients was directly correlated with increased risk for CAC- mediated tumors. As well, circulating IL-6 levels are highly elevated during colon tumorigenesis in CRC and correlate with advanced pathological tumor stage and poor prognosis in human CRC samples and liver metastasis (336). A similar correlation is shown by STAT3 activation which is also used as a negative prognostic survival marker in CRC patients (337). To emphasize the prominent role of IL-6 in colon tumorigenesis, several reports show decreased tumor cell proliferation and reduced AOM/DSS tumor multiplicity in response to genetic ablation or pharmacological inhibition of IL-6 (338–343). IL- 6 was found to directly stimulate premalignant growth of enterocytes in ApcMin/+ mice, as well as in CAC models and several CRC cell lines (294, 344). The tumor promoting functions of IL-6 and TNF in intestinal carcinogenesis are mediated by protecting pre-malignant intestinal epithelial cells from apoptosis and promoting the proliferation of tumor-initiating colon cancer cells. Furthermore, these cytokines orchestrate myeloid cell recruitment to inflamed mucosa (331, 345, 346).

IL-6 is produced by Lamina propria macrophages and dendritic cells during early stages of CAC or produced by T cells at later stages of colitis mediated tumor progression (304, 331, 333, 347). In sporadic CRC, IL-6 (and IL-11) is mainly produced by cancer cells, tumor-associated fibroblasts and epithelial cells, but also by immune cells (316, 348).

IL-6 is induced by activation of transcription factors NFκB, C/EBP, CREB and AP-1. Its production is directly stimulated by PGEβ and TFG and indirectly via LPS (lipopolysaccharide) and IL-1 (349). The IL-6/JAK/STAT3 pathway may also induce autocrine IL-6 production. In turn, IL-6 potently activates STAT3, but also enables Ras/ERK and PI3K/Akt signaling (346).

Interestingly, the IL-6 mediated tumor promoting effect in premalignant cancer cells is activated by STAT3, while the transcription factor NFATc2 (nuclear factor of activated T cells P/c2) regulates IL-6 expression in T cells (332, 333). Besides, NFATc2 seems to be involved in CAC tumorigenesis, too (350). However in myeloid cells, NFκB activates IL-6 production (351). Thus, the aforementioned interaction of NFκB and STATγ is highlighted again by the paradigm of NFκB activation in immune cells leading to pro-inflammatory cytokine secretion (i.e. IL-6) which in turn promotes STAT3 activation in epithelial cells (304–306, 352, 353).

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STAT3 is constitutively activated in both tumor and immune cells and plays a significant role in colon carcinogenesis. STAT3 activation in cancer cells is not only induced by IL-6 but is also dependent on growth factors and cytokines in the tumor microenvironment including IL-11, IL-22, HGF (hepatocyte growth factor) and EGF (epidermal growth factor) family members, as wells as oncogenic tyrosin kinases (such as c-Met (mesenchymal-to-epithelial-transition) and Src) (330–332, 334, 345). Yet, STAT3 up-regulates and induces the expression of mitogenic factors involved in proliferation and cell cycle (c-Myc and cyclin D1, cyclin B1 and PCNA (proliferation cell nuclear antigen)) (331, 354) as well as of pro-angiogenic factors such as VEGF (vascular endothelial growth factor). It also promotes the induction of anti-apoptotic genes like Bcl-2 or Bcl-xL, survivin and XIAP (X-linked inhibitor of apoptosis protein). Furthermore, IL-6 induced STAT3 signaling simultaneously reduces caspase 9 activation and down-regulates the expression of the cdk inhibitor p21, thus promoting cell cycle entry (294, 345, 346, 355). IL-11, an IL-6 family member, is the other major cytokine which activates STAT3 via gp130 signaling. Also, IL-11 and IL-11Rα (IL-11 receptor alpha) are highly expressed in human colorectal cancer (356–359). Besides its direct effect on cancer cells, IL-6 also regulates the ratio of IL-12 and IL-23 in the tumor microenvironment and thus dictates local polarization of T helper cell subsets (360). Indeed, the IL- 6/STAT3 signaling pathway was shown to promote differentiation of IL-17A producing (pro- tumorigenic) Th17 cells as well as macrophages and DCs, while suppressing Treg activation (361).

A vast number of studies unequivocally links NFκB to tumor initiation and progression in CRC (362– 365). NFκB activation enhances the expression of genes that promote resistance to apoptosis (e.g. Bcl- xL, Bcl-2) or induce proliferation (e.g. cyclinD1) in addition to the induction of pro-inflammatory cytokines, adhesion molecules and ROS (366). Classical NFκB signaling in both inflammatory and malignant cells is activated by the tumor promoting cytokines TNF and IL-1 that are produced by inflammatory cells in the tumor micro-environment (TME). Noticeably, also the pro-tumorigenic cytokine IL-17 leads to NFκB activation (362, 367). Though to a lesser extent, NFκB can be additionally activated in response to autonomous genetic alterations and HIF-1α (hypoxia, inducible factor), especially in cancer cells (368). In turn, HIF-1α is induced in cancer cells by TNF and IL-1.

Conditional ablation of IKK- (IκB kinase beta) in enterocytes significantly attenuated AOM/DSS mediated tumor incidence via increased apoptosis in premalignant cells, suggesting that NFκB activation is particularly involved in early pathogenic events rather than in cancer progression (364). Specific inactivation of NFκB in tumor infiltrating myeloid cells (LP macrophages and DCs) blocked the production of tumor promoting cytokines IL-6, IL-11, IL-1 and TNF, highlighting the prominent role of NFκB (and inflammatory cells) in intestinal carcinogenesis (369). Interestingly, ablation of STAT3 in enterocytes also lowers the susceptibility of inflammation-mediated tumor development (331).

Of note, there is evidence that the maintenance of constant NFκB activation in tumors relies on STAT3 signaling (370). Therefore, NFκB and STATγ play potential overlapping roles in epithelial and malignant cells, as well as in surrounding immune cells due to their ability to regulate each other’s activity (332, 370). Since pro-inflammatory cytokines in immune cells are the main transcriptional targets of NFκB, these cells serve as the link between the NFκB and STATγ pathway in tumor cells (308, 371).

TNF initiates and perpetuates inflammation by inducing expression of various cytokines and chemokines from colonic cells, fibroblasts and leukocytes (342, 372). Furthermore, TNF efficiently recruits activated inflammatory cells to the colon mucosa, thus, contributing to the development of CRC. It was shown that TNF was abnormally increased in pre-neoplastic inflamed mucosal tissue and inhibition of TNF efficiently obstructed tumor formation in the AOM/DSS model (322).

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Furthermore, two other important pro-oncogenic cytokines IL-17A and IL-23, have been implicated in colorectal tumorigenesis. Both cytokines cause the attraction of lamina propria myeloid cells (macrophages, granulocytes) to the tumor site, which are the major source of IL-6 and TNF. The heterodimeric cytokine IL-23 is highly upregulated in CAC and CRC cancers, especially in tumor- associated macrophages (TAMs) upon STATγ and NFκB signaling. Albeit not acting directly on epithelial or adenoma cells, IL-23 serves as an important regulator in inflammation-mediated tumorigenesis (360, 373, 374). It could be shown that inhibition of IL-23 reduces tumor numbers and size in ApcMin/+ mice by reducing intra-tumoral levels of IL-6, IL-17A, IL-17F and IL-22. In addition, IL-23, TGF and IL-6 cooperatively stimulate naive CD4+ T cells to differentiate into Th17 cells, which produce the proinflammatory cytokine IL-17 (375). Of note, IL-23 is required for recruitment of Th17 cells to the tumor site (376, 377). On the other hand, IL-23 was also shown to be beneficial for induction of anti-tumor responses by stimulating the production of IFN upon STAT4 activation (375). Indeed, IL-23 secretion by colon cancer cells suppressed tumor growth, suggesting that a preferential IL-23 milieu would be better than accumulation of Th17 cytokines (378). IL-17A has been identified as one of the most important pro-tumorigenic cytokines in all different mouse models of colon carcinogenesis by regulating intestinal and intratumoral inflammation (310, 353, 379– 381). In inflammation induced mouse models of CRC it was shown that IL-17A deficient mice showed milder colitis, fewer and smaller tumors and expressed reduced levels of IL-6 and TNF (354). Moreover, ablation of IL-17RA, as well as genetic deficiency of IL-17A significantly reduced CRC formation (382–384).

Taken together, IL-6 and IL-17A may be directly implicated into immune cell – cancer cell interaction due to expression of their respective receptors on epithelial cells, whereas IL-23 may only orchestrate immune cells and the pro-tumorigenic cytokines in the tumor microenvironment without directly acting on transformed cells (353).

Immune cells in colon cancer tissue CRC and CAC tumors are infiltrated by diverse types of stromal and immune cells. Tumor-associated macrophages (TAMs) are the most important immune cell types within the pro-inflammatory tumor microenvironment and mainly constitute amplification of the inflammatory response by persistent secretion of pro-inflammatory cytokines such as TNF, IL-1, IL-6, IL-12 and IL-23 (308, 385–388). Additionally, mast cells, neutrophils, NK cells and DCs have been shown to contribute to the inflammatory response in colon cancer (304, 389–391). Despite innate immune cells, also cells of the adaptive immune system, especially T cells, are recruited into colon tumors, where they mainly exert tumor promoting roles (392). The clinical outcome and prognosis of CRC is strongly influenced by the type of immune response generated, especially with respect to T helper cell differentiation (Th1/Th2/Th17/Treg) (393, 394). For example, CRC tumors with a Th17-dependent transcriptional profile have a poor prognosis (6), while there is correlation of tumor- infiltrating memory T cells, Th1 cells, CTLs and local production of Th1-recruting chemokines with disease-free or improved survival of CRC patients (395). Th17 as well as Th1 cells were shown to further promote CAC development by increasing the expression of pro-inflammatory cytokines and chemokines, in particular TNF, IL-1, IL-6, IL-23 and IL-17 (396). However, it remains elusive whether immune-mediated mechanisms as opposed to environmental mutagens are the crucial driver of colon carcinogenesis or the consequence of the latter (397).

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Results

Immunoproteasome inhibition leads to anti-proliferative activity and induction of apoptosis in colon cancer cells in vitro To investigate the activities of ONX 0914 against colon cancer, we first employed in vitro analysis using the murine adenocarcinoma cell line MC38. Although, pulse treatment of the inhibitor was shown to yield irreversible covalent inhibition, we treated the cells for 24 hours with two varying concentrations of ONX 0914 followed by western blot analysis for proteasome subunits. In order to ensure proper inhibition of ONX 0914 and its selective binding to the immunoproteasome subunit LMP7, immunoblot against LMP7 was performed. Figure 6A shows a shift in electrophoretic mobility to higher molecular weight due to covalent binding of the inhibitor to Thr1 in the S1 pocket of LMP7. Noticeably, continuous treatment of either 200 nM or 300 nM ONX 0914 also resulted in inhibition of LMP2 indicating that both subunits are affected by inhibitor treatment (Figure 6A).

The UPS not only represents the major pathway for intracellular protein degradation, including misfolded, damaged or mutated proteins, but proteasomal activity is of crucial importance for regulation of expression level of multiple proteins involved in cellular proliferation, differentiation, survival, apoptosis and even antigen presentation. Thus, we investigated the influence of inhibitor treatment on protein degradation capacity. As early as 6 hours after continuous treatment with the inhibitor, an accumulation of poly-ubiquitinated proteins was observed. This was slightly more pronounced to pan- proteasome inhibition using the broad –spectrum inhibitor MG-132, while DMSO only led to Ubiquitin smears similar to untreated control cells (Figure 6C). A profound increase in poly-ubiquitin accumulation was evident after 24 hours of incubation with ONX 0914, while protein homeostasis apparently wasn’t affected in control cells. The complexity of various altered pathways and the accumulation of manifold molecular lesions that lead to tumor development or contribute to tumor progression is still not fully explored. However, the major hallmarks of cancer are perpetuated activation of proliferative signaling and resistance to cell death. So, we analyzed proteins of different molecular pathways that are critically involved in tumorigenesis. After 24 hours or 48 hours continuous treatment with either ONX 0914 or DMSO control, cell lysates were prepared and probed with antibodies recognizing the anti-apoptotic Bcl-2, the key component of the Wnt signaling pathway -catenin and one of its target genes cyclin D1, as well as p53. A significant increase in tumor suppressor p53 was observed after immunoproteasome inhibition (Figure 6B). Additionally, Bcl-2 expression was markedly downregulated after 24 hour inhibitor treatment in contrast to vehicle or untreated control cells. While incubation with DMSO led to induction of the cell cycle regulator cyclin D1 and -catenin, this was omitted in ONX 0914 treated cells (Figure 6B).

We then examined the impact of inhibitor treatment on cellular apoptosis by flow cytometric quantification using Annexin V/PI staining (Figure 6D). We could observe a highly significant increase in the number of apoptotic cells in response to inhibitor treatment, which is in concordance with the downregulation of the anti-apoptotic Bcl-2 as seen in Figure 6C. Notably, also the percentage of cells undergoing late apoptosis or necrosis were significantly increased after incubation with ONX 0914.

The possible anti-proliferative effect of immunoproteasome inhibition on MC38 colon cancer cells was assessed by analysis of CFSE dye dilution in subsequent generations of proliferating cells. Cells were continuously treated with the inhibitor or DMSO for 48 or 72 hours. After both intervals analyzed, ONX 0914 treated cells still exhibit higher median fluorescence intensity of the CFSE dye compared to DMSO

26 treated samples, indicating reduced or even suppressed proliferation of inhibitor treated cells (Figure 6E). Furthermore, we performed an MTT assay to assess the sensitivity of colon cancer cells to immunoproteasome inhibition. Cells were treated with the LMP7-inhibitor for 24 and 48 h. An evident 25% reduction in cell viability was observed already after 24 hours of continuous treatment with 300 nM ONX 0914 (Figure 6F). Thus, ONX 0914 mediated inhibition of the immunoproteasome leads to anti-tumor effects in murine colon cancer cells by reducing proteasomal capacity and potentially impairing protein homeostasis which lead to suppression of proliferation and induction of cell death.

Figure 6. The effect of immunoproteasome inhibition on proliferation and apoptosis in colon cancer cells in vitro. MC38 cells were stimulated with IFN for 48 h and further treated for 24 h with either 300 nM ONX 0914 (X), vehicle (0.3% DMSO (D)) or left untreated (NT) - unless otherwise indicated. A. Western blot analysis of immunoproteasomal subunits LMP7 and LMP2 after 24 h of incubation with either 200 nM (X2) or 300 nM ONX 0914 (X3) or respective amounts of DMSO (0.2% DMSO, D2; 0.3% DMSO, D3) or left untreated (NT, not treated). MC38 cells not stimulated with IFN- served as an additional control (CT, control). -tubulin represents the loading control. B. Western blot analysis of ubiquitinated proteins using anti- FK2 antibody following treatment of MC38 cells for 6 h, 24 h or 48 h with 300 nM ONX 0914 (X), 0.3% DMSO (D) or 10 µM MG-132 (MG). Untreated cells (NT) served as control. To confirm equal loading, the blot was reprobed with anti -Tubulin. C. MC38 cells were treated for either 24h or 48h with 300 nM ONX 0914 (X), 0.3% DMSO (D) and subjected to immunoblotting for the indicated proteins and -Tubulin as loading control. D. Flow cytometric analysis of Annexin/PI staining after 24 h incubation with either 300 nM ONX 0914 (ONX, black column) or vehicle (0.3% DMSO (DMSO, grey column)). Untreated (NT, white open columns) cells or cells incubated with 15 µM camptothecin (blue open bars) served as controls. Graphs indicate the percentage of either Annexin V (AV) – positive or Annexin V/PI - double positive cells. Representative dot plots of DMSO or ONX 0914 treated cells are shown on the right. E. Proliferation of MC38 cells was measured by CFSE fluorescence after 48 h or 72 h of continuous treatment with the inhibitor or DMSO. F. MC38 cells were incubated with increasing concentration of ONX 0914 and cell viability was determined by MTT assay. Graph shows 1x104 cells per well at 300 nM inhibitor treatment. Cells treated with 50 µM H202 served as positive control. Data are representative of three separate experiments yielding similar results. Error bars represent ± SEM. Statistical significance was determined by 2-way ANOVA or unpaired t-test with p-values *p < 0.05; ***p < 0.001; ****p < 0.0001.

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Treatment with the LMP7 inhibitor ONX 0914 suppresses the formation of inflammation-driven colon carcinogenesis in the AOM/DSS mouse model Previously, it has been shown that LMP7 deficiency and inhibition suppressed experimental colitis after induction with DSS (22, 23) as well as in T cell transfer colitis (21). In order to investigate the consequence of LMP7 inhibition in the setting of inflammation-associated colon tumorigenesis, we used the two-step AOM/DSS model.

ONX 0914 treatment started together with the first DSS cycle and mice were then treated with the inhibitor or vehicle control every other day for 8 weeks in total. In order to evaluate the effect of the inhibitor during all stages of AOM/DSS-mediated carcinogenesis, we analyzed mice at different time- points, namely on day 7, 14, 28, 35 and on the endpoint day 56. At first, we compared the clinical outcome in vehicle and ONX 0914 treated mice by comparing loss of body weight as a characteristic symptom of experimental colitis (Figure 7A). Even after the first DSS cycle, differences in body weight loss were detectable, as ONX 0914 treated mice were eminently protected from losing weight in the acute colitis phase with improved recovery to initial body weight. Similar to ONX 0914 treated wild type mice, LMP7-/- mice also develop only mild symptoms of the disease and were almost fully protected from weight loss during all cycles of DSS. Vehicle treated mice lost about 25% body weight due to DSS application (Figure 7A). The differences in loss of body weight between the vehicle and ONX 0914 treated groups of mice increased with the following DSS cycles during the course of the experiment, most prominently after the last cycle of DSS. This resulted in the need of sacrificing these mice due to induced chronic colitis accompanied with bloody stool and the occurrence of rectal prolapse. Treatment with the inhibitor resulted in extended survival of experimental mice compared to vehicle-treated mice (Figure 7B). Notably, 60% of the animals in vehicle treated groups died or had to be sacrificed before termination of the experiment even at early time points (day 22), whereas only one mouse of the ONX 0914 treated animals died during the course of 3 independent experiments. Moreover, LMP7 knockout (KO) mice also showed an increased lifespan in the AOM/DSS model. Consistent with the major weight loss, vehicle treated mice exhibited significant shortening of colons in the acute colitis phase as a hallmark of DSS-induced colonic damage. As well, colon shortening was still present at the endpoint of the experiment probably due to severe and persistent pathologic inflammation and increased fibrosis in colonic mucosal tissue. In contrast, inhibitor-treated mice, as well as LMP7 KO mice displayed colon lengths similar to naïve untreated mice (data not shown).

Notably, treatment with ONX 0914 attenuated or even suppressed tumor growth, whereas vehicle control mice exhibited a significant increase in tumor nodule number in the distal and mid colon with rare occasional lesions in the proximal colon (Figure 7C). In addition, average tumor sizes tended to be enlarged in vehicle treated mice compared to the two other experimental groups (Figure 7D, no graph shown). Also, genetic ablation of LMP7 blocked tumor multiplicity and dysplastic lesions similar to the effect of ONX 0914 treatment in C57BL/6 mice. In order to verify efficient inhibition of the LMP7 subunit by the inhibitor, whole colon tissue lysates of mice sacrificed on day 56 were analyzed for the typical shift in electrophoretic mobility, confirmed by western blotting. Although to a lesser extent, LMP2 was also inhibited as shown in Figure 7E. MECL-1 expression was not different between the different groups of mice (data not shown).

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Figure 7. Treatment with ONX 0914 suppresses AOM/DSS-induced colorectal tumor tormation. A. CAC was induced in 8-week old C57BL/6 or LMP7-/- mice by i.p. injection of AOM at 12 mg/kg and subsequent oral administration of 2% DSS (grey columns) in drinking water for 5 days in 3 cycles which was interrupted by 2-week periods without DSS challenge. Mice were treated with vehicle (grey triangles) or 10 mg/kg ONX 0914 (black dots) s.c. every second day from day 0 of the first DSS cycle. LMP7-/- mice (blue squares) were not inhibitor treated (n ≥ 5). Body weight (bw) was recorded daily during DSS cycles and every second day after recovery. Data are presented as percent loss of bw compared to the initial bw on day -5. Data are means ± SEM. B. Survival curve showing the percentage of mice that have survived until the end of the experiment on day 56. Mice were sacrificed due to disease symptoms such as extensive loss of bw, obstipation, rectal bleeding, rectal prolapse or moribund health condition. C. On day 7, 14, 28, 35 and 56 colons were removed and macroscopically analyzed for tumor lesions. Graph shows number of tumor lesions in the large intestine at the indicated time points by semi quantitative macroscopic assessment. Tumors are defined as lesion ≥ β mm. Representative gross pathology of the descending colon and rectum at the end of the AOM/DSS protocol on day 56 are shown on the right. E. Western blot analysis of LMP7 and LMP2 in whole colon tissue lysates (one mouse per lane) on day 56. -actin was used as loading control. LB, naïve large bowel, X, ONX, V, vehicle, L7, colon tissue of an AOM/DSS treated LMP7-/- mouse. Data are shown as means ± SEM of three independent experiments with *p < 0.05; **p < 0.01; ****p < 0.0001.

Inhibitor treated mice exhibited lower levels of inflammation as indicated by significantly decreased histology scores compared to mice treated with vehicle (Figure 8A, 8B). Histopathological analysis of colonic mucosa in vehicle treated mice show typical signs of colitis associated pathologies with the sequential process of acute, severe inflammation with destruction of the intestinal architecture with almost complete crypt loss and massive leukocyte infiltration into submucosal layers during acute colitis on day 7, inflammatory hyperplasia and dysplasia as first signs of carcinoma development due to chronic colitis on day 35 and the formation of adenoma at the end of AOM/DSS model on day 56 (Figure 8) Conversely, treatment with the inhibitor only led to moderate signs of inflammation, with restored crypt architecture and reduced inflammation in the lamina propria. Although, histology scores were not completely reduced to levels of naïve mice, ONX 0914 treatment limited tissue damage and suppressed the ensuing inflammatory response, which contributes to reduced tumor burden and disease pathology. In accordance with the macroscopically visible significant decrease in tumor multiplicity, histopathological quantification of intestinal adenomas supported reduction of gross tumor number in ONX 0914 treated mice, as illustrated in Figure 8A and 7C. Mice treated with the LMP7 inhibitor developed lower incidences of dysplastic regions compared to vehicle control. The majority of lesions were characterized as mild hyperplasia and low-grade adenomas with rarely occasional appearance of carcinoma in situ, displaying lesions in a less advanced stage with mostly focal, small sized residual

29 colitic lesions. Vehicle treated mice show a prevalence of high-grade dysplasia with frequent invasion into the submucosa. Evidence of adenocarcinoma as characterized by published histolopathological scoring systems (271) was only observed in vehicle-treated mice. Additionally, we also analyzed cell proliferation in colons and tumors and observed significant differences between ONX 0914 and vehicle treated mice (Figure 8D). ONX 0914 treatment reduced proliferation rates, as visualized by Ki-67 staining on colon sections on day 56 compared to colons of vehicle mice, where immunohistochemistry nicely revealed adenoma formation.

Induction of apoptosis was confirmed via western blotting (Figure 8E). Treatment with ONX 0914 led to activation of apoptotic signaling as shown by processing of caspase-3 to the cleaved active form in tumor adjacent areas compared to the absence of cleaved caspase-3 products in vehicle or naïve B6 colons. Cleaved caspase-3 in turn promoted activation of its target PARP (poly (ADP-ribose) polymerase). Cleavage of this classical apoptosis marker, resulted in increased levels of the 89 kDa C- terminal fragment of PARP in samples of inhibitor-treated mice. In accordance to that, the anti-apoptotic protein Bcl-2 was not overly affected by treatment with ONX 0914, while colon tissue of vehicle treated mice exhibited prominent induction of this factor. As well, p53 expression was upregulated in ONX 0914 treated animals, suggesting that increased apoptosis of malignant cells is probably mediated by upregulation of p53.

Furthermore, we analyzed the expression of pro-inflammatory cytokines in colon tissue and serum of diseased mice. As previously described, IL-6 is the crucial regulator of proliferation and survival of IEC (331, 347) and represents key importance in colon tumor development (294). We therefore assessed systemic level of IL-6 in serum by ELISA. As shown in Figure 8F (left panel), LMP7 inhibition led to significantly decreased IL-6 levels as compared to control mice. In addition, we analyzed the content of IL-6 and IL-17A in lysates of colon tissue (Figure F, right panel). IL-6 cytokine levels were reduced in mice treated with the inhibitor in the acute inflammatory phase (day 7), as well as at the endpoint of the experiment (day 56). Although, there was no significant difference observed for IL-17A in colon lysates on day 7, cytokine levels were reduced in ONX 0914 treated mice on day 56.

Collectively, this data show that selective inhibition of the immunoproteasome effectively suppresses tumor initiation and progression of CAC in vivo. The observed impact of ONX 0914 on the clinical outcome in AOM/DSS treated mice is most likely attributed to induction of pro-apoptotic effects in malignant cells. Additionally, the anti-inflammatory tumor microenvironment further prevents inflammation-mediated tumor development in colons of ONX 0914 treated AOM/DSS mice.

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Figure 8. Immunoproteasome inhibition reduces the pro-inflammatory tumor microenvironment A. Representative images of H&E stained paraffin-embedded colon sections. B. Histology score of colon sections was defined by colitis scoring at day 7, 14 and 28. In brief, intestinal inflammation was defined on the basis of 4 different parameters: Inflammation severity, extent of inflammation, crypt damage and involvement. C. H&E stainings of colon sections of vehicle or ONX 0914 treated mice on day 35 and day 56 (right panel of A.) were quantified according to the grading of the respective tumor types that are low-grade dysplasia (light grey), high-grade dysplasia (grey), intramucosal carcinoma (dark grey) or adenocarcinoma (black). The percentage of mice having no tumors are represented by white bars. D. Sections from distal colon of mice on day 56 of the AOM/DSS protocol were stained for anti-Ki-67 antibody via IHC. E. Western blot analysis of anti-cleaved caspase 3, anti- PARP, anti-Bcl-2 and anti-p53 antibodies in whole colon tissue lysates (one mouse per lane) on day 56. -tubulin was used as loading control. F. IL-6 levels in blood serum (left panel) and IL-6 and IL-17A cytokine levels of day 7 and day 56 in whole- colon lysates (right panel) of vehicle (grey columns) and ONX 0914 (black columns) treated AOM/DSS mice. Data are means ± SEM. Serum of naïve C57BL/6 mice was used as control (white columns). Data are representative of three independent experiments. *p < 0.05.

Treatment with ONX 0914 abrogates colitis-associated colon carcinogenesis in a therapeutic setting of the AOM/DSS mouse model In order to investigate a potential anti-tumorigenic effect of ONX 0914 on already established tumors in a therapeutic setting, we started ONX 0914 treatment in this AOM/DSS model at a time point when tumors already have established. Assessment of potential AOM/DSS-induced tumors on day 25 (Figure 9C, blue squares) revealed small, but macroscopically detectable tumors (data not shown). Hence, the onset of inhibitor treatment was the last day of the second DSS cycle. As shown in the body weight

31 assessment in Figure 9A, we observed faster recovery from body weight loss even after a single ONX 0914 treatment (Figure 9A, day 28). As well, reduced weight loss due to the last DSS exposure was observed in these mice compared to the control group which lost up to 20% of body weight. Moreover, ONX 0914 alleviated the shortening of the colon caused by the DSS treatment at the time-points analyzed (day 35 and day 56, graph not shown). Additional to alleviation of loss of body weight and a 100% survival rate (Figure 9B), treatment with the inhibitor also led to significantly decreased total tumor numbers compared to vehicle treated mice, already after one week of treatment on day 35 (Figure 9C). Noticeably, colon tumor numbers of ONX treated mice were similar to the multiplicity of tumors on day 35 suggesting suppression of further growth of already established tumors and almost complete inhibition of formation of new tumors. In contrast, vehicle control groups developed great tumor burden, as visualized by increased tumor numbers and sizes (Figure 9C and 9D) at the endpoint of the AOM/DSS on day 56.

Figure 9. Treatment with ONX 0914 suppresses already established AOM/DSS-induced tumor formation in a therapeutic setting. A. CAC was induced in 8-week old C57BL/6 by i.p. injection of AOM at 12 mg/kg and subsequent oral administration of 2% DSS in drinking water for 5 days in 3 cycles (grey background columns) which was interrupted by 2- week periods without DSS challenge. Mice were treated with vehicle (grey triangles) or 10 mg/kg ONX 0914 (black dots) s.c. every second day from day β5 (black triangle) until day 56 (n ≥ 5). Body weight (bw) was recorded daily during DSS cycles and every second day after recovery. Data are presented as percent loss of bw compared to the initial bw on day -5. Data are means ± SEM. B. Survival curve showing the percentage of mice remaining alive until the end of the experiment on day 56. Mice were sacrificed due to disease symptoms such as extensive loss of bw, obstipation, rectal bleeding, rectal prolapse or moribund health condition. C. On day 25, 35 and day 56, colons were removed and macroscopically analyzed for tumor lesions. Graph shows number of tumor lesions in the large intestine by semi quantitative macroscopic assessment. Tumors are defined as lesion ≥ β mm. Tumor numbers of untreated control C57BL/6 mice are shown on day β5 are shown as blue squares. D. Representative gross pathology of the descending colon and rectum at the end of the AOM/DSS protocol on day 56. Data are shown as means ± SEM of three independent experiments with *p < 0.05; **p < 0.01; ****p < 0.0001.

Taken together, ONX treatment suppresses CAC potentially by suppressing the pro-inflammatory environment that reduces or even inhibits hyper proliferation of transformed cells.

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LMP7 inhibitor treatment suppresses further colorectal tumor growth in the absence of inflammation Our study of ONX treatment using the AOM/DSS model of CRC suggests that the immunoproteasome is involved in initiation and progression of CAC, where inflammation plays a major contributing role to cancer development. Next, we addressed immunoproteasome inhibition as a therapeutic approach of already established colonic tumors in the absence of DSS-induced inflammation. To this aim, we adapted the AOM/DSS model such that the third DSS cycle was omitted and started treatment only after mice had regained their original weight after the second DSS cycle, to determine the influence of ONX 0914 after cessation of the acute inflammatory response. Untreated control mice subjected to the AOM/DSS protocol verified the formation of tumors on day 37 (Figure C) when we started ONX 0914 treatment every other day with sequential analysis of pathological alterations from day 42 until the endpoint of the AOM/DSS model on day 56. Already after a single application of the inhibitor, we observed an increase in body weight of mice treated with ONX 0914, whereas mice only treated with vehicle stayed after recovery from DSS-induced inflammation at a constantly low body weight level (Figure 10A). Intriguingly, treatment with ONX 0914 halted further progression of tumor growth, since tumor number arrested at the same level for the next three time-points analyzed (day 42, day 49 and day 56; Figure 10C) as compared to heavily progression of tumor numbers in control mice. ONX 0914- treated groups mainly if any exhibited colonic polyps of < 2 mm in diameter with no polyps exceeding 2 mm. Vehicle treated mice developed all sizes of polyps, including big tumor lesions exceeding 3 mm in size (Figure 10D, size distribution not shown). More interestingly, LMP7-deficient mice, which were as well protected from body weight loss during the two precedent DSS cycles (Figure 10A), showed similar tumor numbers as ONX 0914 treated animals (Figure 10C and 10D). Histological examination confirmed the presence of advanced stage colon tumorigenesis in vehicle mice, as visualized by aberrant crypt foci with hyper-chromatic nuclei, hyperplastic crypts and serrated adenomatous structures (Figure 10B). Compared to that, the mucosa of ONX 0914 treated animals only displayed mild forms of low- grade dysplasia. As shown in Figure 10D, vehicle treated mice showed high levels of proliferation in malignant cells, as well as in tumor adjacent areas, whereas Ki-67 staining of ONX 0914-treated colon sections was confined to the proliferative region of the crypts. In relation to that, we observed increased staining for cleaved caspase-3 after ONX 0914 treatment throughout the whole intestinal crypt as well as intense active caspase-3 staining at the top due to normal “shedding” and renewal of the intestinal mucosa. As illustrated in the H&E staining in Figure 10B, the tumor microenvironment in ONX 0914- treated colons showed less leukocyte infiltration into the submucosa compared to control mice. Therefore, we decided to further investigate the phenotype of the tumor-infiltrating cells and revealed significantly decreased or almost absent infiltration of F4/80-positive macrophages in colons of ONX 0914 treated mice (Figure 10D, lower panel). Furthermore, histomorphologic characteristics, as well as histochemical results of Ki-67 and cleaved caspase-3 staining in LMP7 deficient mice were comparable to ONX 0914 treated animals.

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Figure 10. Treatment with ONX 0914 suppresses tumor growth of already established adenomas in the absence of inflammation. A. CAC was induced in 8-week old C57BL/6 or LMP7-/- mice by i.p. injection of AOM at 12 mg/kg and subsequent oral administration of 2 % DSS in drinking water for 5 days in only 2 cycles (day 0-4, day 21-25, grey background columns). DSS cycles were interrupted by 2-week periods applying tap water only. Mice were treated with vehicle (grey triangles) or 10 mg/kg ONX 0914 (black dots) s.c. every second day from day 37 onwards (black triangle), when mice gained their original weight after the second DSS cycle. LMP7-/- mice (blue squares) were not inhibitor treated (n ≥ 4). The body weight (bw) was recorded daily during DSS cycles and every second day thereafter. Data are presented as percent loss of bw compared to initial bw on day -5. B. Representative images of H&E stained colon sections at the end of AOM/DSS protocol on day 56. C. On day 37, 42, 49 and 56, colons were removed and analyzed macroscopically for the presence of tumor lesions. The graph shows the numbers of tumor lesions in the large intestine of vehicle or ONX 0914 treated mice and LMP7-/- mice by semi quantitative macroscopic assessment. Additionally, AOM/DSS control mice (white diamonds) were analyzed on day γ7 for tumor incidence. Tumors are defined as lesion ≥ β mm. Representative gross pathology of the descending colon and rectum at the end of the AOM/DSS protocol on day 56 is shown below. D. Colon sections of day 56 were immunostained for cleaved caspase 3 (top row), Ki-67 (middle) and F4/80 (bottom). Data are means ± SEM with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Taken together, these data suggest that ONX 0914 treatment curtails tumor progression in the absence of exogenous acute inflammatory stimuli. Treatment with the LMP7 inhibitor counteracts further expansion of already formed adenomas but suppressed the generation of new colonic lesions.

Treatment with ONX 0914 reduces intestinal tumorigenesis in the ApcMin/+ mouse model Compared to the chemically induced and inflammation-associated AOM/DSS model, which is induced by activating mutations in epithelial pathways such as p53 and K-ras (295, 398), the ApcMin/+ mouse model is characterized by deregulation of the Wnt/-catenin signaling cascade in the first place,

34 therefore addressing another route of intestinal carcinogenesis development. The main disadvantages of the ApcMin/+ mouse model is the predisposition of adenoma progression in the small intestine, only occasional development to adenocarcinomas and low incidence of neoplastic lesions in the colon, compared to tumors in humans that are predominantly localized in the colon (399). Therefore, selective promotion of dysplastic crypts and neoplasms in the colon within 5 weeks was increased after initial receipt of 2% DSS in drinking water for 7 days (400). We started ONX 0914 treatment in ApcMin/+ mice at 5 weeks of age, as it is known that multiple intestinal polyposis spontaneously manifest early in life of these mice (245). However at this age, we could not detect macroscopically visible tumor nodules (own experience, data not shown). In addition, LMP7 KO mice were crossed onto ApcMin/+ background in order to investigate the influence of genetic LMP7 deficiency on intestinal tumorigenesis in the ApcMin/+ model. Either female or male mice were randomly distributed into vehicle or ONX 0914 treated groups while ApcMin/+LMP7-/- mice were left untreated. Treatment with the inhibitor started from day 0 of the experiment with continuous treatment every other day until the endpoint of the experiment on day 35. The profound weight loss of vehicle-treated mice after the DSS cycle was mostly prevented in ApcMin/+ mice treated with the LMP7 inhibitor (Figure 11A). In addition, mice of the vehicle group did not recover to original weight after regeneration post DSS treatment and stayed at a constantly low body weight level and lost weight again gradually from day 25 onwards until death or the endpoint of the experiment on day 35, likely due to exuberant tumor development in the colon, highlighted by the occurrence of rectal prolapse in more than 50% of vehicle treated animal. In contrast to the effect of LMP7 knock out in the AOM/DSS model, we observed a decrease in total body weight in ApcMin/+ LMP7-/- mice. However, not as severe as seen in ApcMin/+ mice of the vehicle group. ONX 0914 treatment, as well as genetic deletion of the LMP7 subunit in ApcMin/+ mice led to increased overall survival, whereas ¼ of animals which received only vehicle treatment died or had to be sacrificed before the experimental endpoint (Figure 11B). These mice probably succumb to the consequence of secondary effects due to tumor growth as a result of severe intestinal obstruction. Noticeably, macroscopic examination of colons at the endpoint of the experiment on day 35 showed that ONX 0914 treated mice developed none or significantly decreased colorectal tumor incidences compared to vehicle treated mice in a highly significant manner (Figure 11C). In comparison, vehicle treated ApcMin/+ mice show clear evidence of occult bleeding throughout colonic and rectal segments with extensive polyposis and multiple large tumors in the colon. Additionally, ONX 0914 treatment also had an effect on tumor size, suggesting that ONX 0914 suppressed tumor outgrowth, as well as tumor initiation (graph not shown). Moreover, the loss of LMP7 profoundly decreased the incidence and multiplicity of colonic adenomas in ApcMin/+LMP7-/- mice to similar levels as seen in LMP7 inhibitor treated animals. Thus, the loss of body weight apparently did not correlate with increased tumor development, but might be a sign of chronic colitis in these mice. However, this issue still has to be clarified by histologic assessment. Furthermore, colons of vehicle treated mice tended to differ in their length, i.e. we found slightly reduced colon lengths at the endpoint of the experiment compared to ONX 0914 treated or LMP7 KO control ApcMin/+ mice (graph not present). Histomorphologic analysis of the large intestine confirmed the macroscopically visible phenotype of reduced tumor numbers in ONX 0914 treated mice. Colon sections of these mice showed mostly preserved intestinal architecture, albeit enlarged crypts and low levels of immune infiltration can be detected in Figure 11D (bottom). On the contrary, colon sections of vehicle treated mice showed severe intestinal inflammation and high-grade hyperplasia characterized by crypt damage, necrosis of the epithelial layer, large follicles of lymphocyte infiltration, the loss of mucus- producing goblet cells and poorly differentiated lesions with hyperchromatic cells extending into the submucosa (Figure11D, top). As ApcMin/+ mice typically suffer from anemia, this commonly initiates splenomegaly formation due to splenic hematopoiesis in the context of disease progression (35). Thus, we measured spleen weight and size at sacrifice and found that vehicle treated mice had significantly

35 enlarged spleens compared to mice treated with the LMP7 inhibitor, as illustrated in Figure 11E. Spleen weights of ApcMin/+LMP7-/- mice were similar to those of ApcMin/+ mice treated with ONX 0914. Despite the phenotype of splenomegaly, no metastases to lymph nodes, liver or have been detected. Treatment of ApcMin/+ mice with the LMP7 inhibitor significantly increased levels of ubiquitinated proteins in tumor tissue, as shown Figure 11F. Nonetheless, biochemical analysis of tumor tissue was always an intricate issue, since inhibitor treated animals only developed low tumor numbers if at all. Immunohistological evaluation of colons of ONX 0914 treated mice, we observed that tumor areas in these mice show decreased proliferative indices as demonstrated by less intense staining of Ki-67- positive cells (Figure 11G). Staining of active proliferative areas were slightly more pronounced in ApcMin/+LMP7-/- mice which would correlate with the minor increased colon tumor numbers in these mice. In contrast to that, almost all parts of colons of vehicle treated ApcMin/+ mice showed strong Ki-67 staining in areas of neoplastic transformation, as well as in tumor-surrounding tissues.

Figure 11. Treatment with the LMP7 inhibitor ONX 0914 suppresses intestinal tumorigenesis in ApcMin/+ mice. A. Induction of colon tumorigenesis was accelerated by oral administration of 2% DSS for 7 consecutive days (grey background column). Five-week old ApcMin/+ or ApcMin/+LMP7-/- were treated every other day with either vehicle or 10 mg/kg ONX 0914 s.c. from day 0 of the DSS cycle until the endpoint of the ApcMin/+/DSS protocol on day 35. ApcMin/+LMP7-/- mice were left untreated (n ≥ 5). Body weight of vehicle (grey triangles) or inhibitor (black dots) treated ApcMin/+ mice and ApcMin/+LMP7-/- mice (blue squares) was recorded daily during the DSS cycle and every other day after recovering to original weight. Data are illustrated as percent weight loss (y-axis) plotted versus time (x-axis). B. Survival curve showing the percentage of vehicle or ONX 0914 treated ApcMin/+ mice and ApcMin/+LMP7-/- mice remaining alive until the end of the experiment on day 35. C. On day 20 or 35 after beginning of the experiment, colons were removed and opened longitudinally for semi quantitative macroscopic analysis of colonic tumor formation. Graph shows tumor number per mouse as defined by lesions ≥ β mm. Pictures are representative macroscopic views of colons on day 35. D. Individual spleen weight on treatment day 35 of vehicle or ONX

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0914 treated ApcMin/+ and ApcMin/+LMP7-/- mice. E. H&E stainings of colon sections of vehicle or ONX 0914 treated ApcMin/+ mice on day 35 were quantified according to the grading of the respective tumor types that are low-grade dysplasia (LGD, light grey), high-grade dysplasia (HGD, grey) or adenocarcinoma (black). The percentage of mice having no tumors are represented by white bars. F. Western blot analysis of poly-ubiquitinated proteins using anti-FK2 antibody of either whole colon tissue lysates of an untreated ApcMin/+ mouse (LB, large bowel) or colon tumors of ONX 0914 (X) or vehicle (V) treated ApcMin/+ mice on day 35 of the ApcMin/+/DSS protocol (one mouse per lane). Tumor tissue of an untreated 16 week old ApcMin/+ mouse (CT, control) was used as a control. Equal loading of γ0 µg protein was verified by reprobing against -tubulin. G. Colon sections of day 35 were immunostained for Ki-67. Data points represent means ± SEM and are compiled from two independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Strikingly, these data show that LMP7 inhibition as well as genetic deletion of this immunoproteasome subunit significantly suppresses intestinal polyp development in the ApcMin/+ mouse model of sporadic intestinal tumorigenesis.

Inhibition of the immunoproteasome is also efficacious in therapeutic treatment of already established tumors in the ApcMin/+ mouse model In order to further assess the anti-tumorigenic efficacy of ONX 0914 treatment in already established Apc-mediated tumors, we analyzed the effect of immunoproteasome inhibition in a therapeutic setting of the ApcMin/+ mouse model. In order to accelerate tumor formation specifically in colonic segment, we made again use of the ApcMin/+-DSS mouse model. Treatment with either vehicle or the LMP7 inhibitor started after mice had regained their original weight on day 14. The inhibitor was then applied every other day until the end of the experiment on day 35. Once more, we observed a rapid increase in body weight after initial injection of the inhibitor. After that, body weights of ONX 0914 treated ApcMin/+ mice steadily increased. Compared to that, vehicle treated ApcMin/+ mice started losing weight at day 7 and did not recover to initial body weight with significantly decreasing body weight levels in the last days of the experiment (Figure 12A). Furthermore, animals, which received only vehicle also showed significantly reduced survival (Figure 12B). Half of the mice succumbed to tumor complications, as seen by the severe phenotype accompanying disease progression with intestinal occlusion, excessive weight loss or rectal prolapse. Instead, mice treated with the inhibitor survived until the scheduled endpoint and showed ameliorated visible signs of the disease. As shown in Figure 12C, treatment of tumor-bearing ApcMin/+ mice with ONX 0914 led to a highly significant decrease in colonic tumor number compared to analysis of tumor number in vehicle treated ApcMin/+ mice which developed multiple large tumors in the distal and proximal parts of the large intestine. Additionally, we observed that spleen hypertrophy was reduced after treatment with the LMP7 inhibitor but not in samples from vehicle treated ApcMin/+ mice, as shown in Figure 12D. Collectively, we conclude that ONX 0914 treatment greatly suppressed clinical signs of the disease and tumor progression also in already adenoma-bearing ApcMin/+ mice.

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Figure 12. Therapeutic treatment with the LMP7 inhibitor ONX 0914 suppresses already established tumors in ApcMin/+ mice. A. Induction of colon tumorigenesis in ApcMin/+ mice was accelerated by oral administration of 2% DSS for 7 consecutive days (grey background column). B. Five-week old ApcMin/+ mice were treated every other day with either vehicle (grey triangles) or 10 mg/kg ONX 0914 (black dots) s.c. starting from day 14 (black triangle) (n ≥ 5). The body weight of individual mice was recorded daily during the DSS cycle or every other day after recovering to original weight. Data are illustrated as percent weight loss of the initial bw at day 0 of the experiment. B. Survival curve showing the percentage of mice remaining alive until the end of the experiment on day 35. C. On day 35 colons were removed and opened longitudinally for macroscopic assessment of tumor formation. Colon tumor numbers of vehicle (grey triangles) or ONX 0914 (black dots) treated ApcMin/+ scored on day γ5 as defined by lesions ≥ β mm are displayed. Images on the right are representative macroscopic views of colons on day 35. D. Individual spleen weights of vehicle or ONX 0914 treated ApcMin/+ on day 35 of treatment. Spleens of naïve 6-week old ApcMin/+ mice were used as a control (white squares). Representative images of individual spleen sizes of vehicle or ONX 0914 treated ApcMin/+ on day 35 are shown on the left. Data points represent means ± SEM. Data have been compiled from two independent experiments with *p < 0.05, **p < 0.01, ****p < 0.0001.

Genetic deficiency of the LMP7 subunit ameliorates spontaneous formation of intestinal polyposis in ApcMin/+ mice To investigate whether loss or inhibition of immunoproteasome subunits also interferes with intestinal tumorigenesis in a tumor microenvironment that did not develop due to preceding overt inflammatory stimuli, we examined intestinal polyposis in untreated ApcMin/+ mice. We evaluated tumor development after long-term inhibitor treatment three times a week, beginning at week of age 5 for a maximum of 11 weeks. As shown in Figure 13A, mean body weight was only modestly altered by ONX 0914 treatment in ApcMin/+ mice during the course of the experiment compared to vehicle treated ApcMin/+ mice. ONX 0914 treated mice continued to gain weight due to their normal development or maintained stable body weight levels later. Compared to that, ApcMin/+ mice of the vehicle group showed constantly reduced body weight already at early time-points of measurement, which became slightly more pronounced at later stages. Notably, since we did not observe a drastic loss of body weight compared to the ApcMin/+ DSS model (Figure 13A), we would assume that body weight loss is only a symptom of persistent inflammatory activity in response to DSS application. At 16 weeks of age, polyp formation in small and large intestine was found in all animals independent of the treatment group or genotype. Macroscopic analysis revealed that vehicle control mice developed multiple large polyps in the small intestine, whereas the multiplicity of small intestinal polyps was significantly reduced in ONX 0914- treated ApcMin/+ mice. Particularly, almost complete absence of polyps in the small intestines was

38 observed in ApcMin/+LMP7-/- mice (Figure 13B). Despite the well-known severe small intestinal phenotype of ApcMin/+ mice (401), colonic tumor lesions were less pronounced and showed variable infrequent tumor numbers in ApcMin/+ mice. However, the total tumor number in the small intestines and colons of ApcMin/+ wild type mice correlated with average tumor numbers observed in other studies (36). As shown in Figure 13C, ONX 0914 treatment as well as LMP7 deletion also led towards reduced or even suppressed colon tumor formation in ApcMin/+ mice. In contrast to small-sized polyps in ApcMin/+LMP7-/- mice, the average size distribution of colonic and small intestinal lesions was similar in ApcMin/+ mice treated with inhibitor or vehicle control (size distribution not shown). This may indicate that following loss of Apc, LMP7 deletion impedes tumor initiation and tumor growth in the absence of intestinal inflammation. Moreover, treatment with the inhibitor ameliorated indirect parameters of disease progression in ApcMin/+ mice, which were even more pronounced in ApcMin/+LMP7-/- mice. Profound signs of anemia were clearly reduced, since only vehicle treated ApcMin/+ mice showed pale feet and ears associated discoloration of internal organs (images not shown). We also noted that ONX 0914 treated ApcMin/+ mice did not develop splenomegaly compared to spleens isolated from vehicle ApcMin/+ mice, which also weighed significantly more. Remarkably, spleens of ApcMin/+LMP7-/- appeared without altered morphology compared to normal wild type spleens, suggesting the alleviation of systemic inflammation by ONX 0914 treatment as well as by LMP7 deficiency (Figure 13D).

In conclusion, these data indicate that the immunoproteasome contributes to initiation of intestinal carcinogenesis, since deletion of the LMP7 subunit ameliorates spontaneous development of Apc mutation-mediated polyposis in ApcMin/+ mice in the absence of experimental exposure to inflammatory agents. Hence, these results identify the immunoproteasome as a novel candidate drug target in the treatment of familial adenomatous polyposis.

Figure 13. Reduced intestinal tumor burden in response to pharmacological inhibition and genetic ablation of LMP7 in ApcMin/+ mice. A. 5-week old ApcMin/+ mice were treated with either ONX 0914 (black dots) or vehicle (grey triangles) three times a week for 11 weeks and were analyzed for colonic tumor formation and small intestinal polyps at 16 weeks of age. Body

39 weight measurements of male ApcMin/+ mice at indicated time-points are illustrated as percent change in body weight in relation to initial body weight (n ≥ 8). B. The whole intestine was removed and opened longitudinally for macroscopic assessment of tumor and polyp formation. Number of small intestinal polyps of ApcMin/+ or ApcMin/+LMP7-/- mice (blue squares, n = 5) are shown. C. Graph shows colonic tumor number, as defined by lesions ≥ β mm. D. Spleen weights of ApcMin/+ or ApcMin/+LMP7- /- mice were assessed. Representative pictures of individual spleens are shown on the right. Graphs represent data as means ± SEM. **p < 0 .01, ****p < 0 .0001.

The development of sporadic colon carcinogenesis is markedly suppressed by inhibition of the immunoproteasome with ONX 0914 Whereas the genetically modified ApcMin mouse model reflects hereditary aspects of CRC, we wanted to further investigate the effect of immunoproteasome inhibition on sporadic cancer formation based on inducible colonotropic AOM mutagenesis. AOM-induced carcinogenesis mimics human non-hereditary CRC development in many respects including predominant localization, morphology as well as at the molecular level (279). To reveal a potential direct anti-oncogenic activity of the LMP7 inhibitor, we performed the long-term AOM-mediated mouse model of colon carcinogenesis, which is characterized by repeated administration of azoxymethane leading to colon tumor formation after 24 weeks post the first AOM application. Treatment with the inhibitor ONX 0914 started at the day of the first AOM injection. In addition to the aforementioned experiments, LMP7 KO mice were further treated either with or without ONX 0914. Figure 14A shows changes in body weights after AOM treatment throughout the experimental period until day 200. The recommended regimen of six weekly intraperitoneal injections of 12 mg/kg AOM was well tolerated, however only by C57BL/6 mice treated with the inhibitor. Oddly, loss of body weight and deterioration of the overall health state was observed over the time period of the weekly AOM injections in vehicle treated mice, as well as in LMP7 KO mice of both treatment groups. These symptoms could possibly attributed to an accumulation of toxic metabolites in response to application of AOM. Intriguingly, LMP7 KO mice treated with vehicle control did not recover from body weight loss but rather developed a devastating wasting disease, which was accompanied by abusive water consumption and excessive micturition. These idiopathic comorbidities led to premature death of these animals (Figure 14B). Blood glucose levels or urine protein tests did not reveal evident differences to wild type control mice. Nevertheless, these symptoms probably developed independent of malignant neoplastic transformation or tumor burden in the intestine, since analysis of tumor numbers in these mice did not show increased tumor burden (Figure 14C). As well, no overt extra- colonic lesions could be detected at the time of sacrifice (not shown). Body weight steadily increased in ONX 0914 treated wild type mice, comparable to untreated wild type mice of the same age (Figure 14A, red line). Interestingly, from day 50 onwards, ONX 0914 treated LMP7 KO recovered from the AOM induced perturbations in body weight levels and body weight development was similar to wild type B6 mice treated with the inhibitor. Noticeably, around day 100, body weights of vehicle treated mice started to deviate from the ONX 0914-treated groups, which was even more striking during the last 20 days of the experiment. The loss of body weight is probably attributed due the compromised general health state including intermittent rectal bleeding which eventually led to death of the animals. Thus, vehicle control mice displayed significantly decreased survival compared to inhibitor treated mice (Figure 14B). The individual groups of mice were sacrificed 22 weeks after the last AOM injection and the large intestine was removed for further examinations. The luminal colon surfaces of vehicle treated B6 mice displayed exophytic or polypoid tumors that were localized to the distal and mid parts of the colon, but could not be detected within the proximal part. Tumor sizes ranged from 4 to 6 mm diameter with the big tumors being tanned red and exposing irregular surface. In contrast, ONX 0914 treatment either significantly reduced colon tumor number and size or complete blocked the formation of macroscopic lesions in wild type, as well as LMP7-/- mice. Coincidentally, we also observed splenomegaly formation in vehicle treated wild type mice, a symptom

40 that is actually characteristic to Min-associated pathologies in ApcMin/+ mice (Figure 14C). Histopathological assessment verified the observed increased tumor multiplicity in vehicle treated mice. The intestinal mucosa of these mice was completely devoid of characteristic crypt architecture and presented highly irrigated, polypoid and cauliflower-like adenomas with invasion through the submucosal membrane and even perforation into Lamina propria muscularis. Compared to that, the colon mucosa of ONX 0914 treated (wild type and LMP7–/-) mice appeared almost normal except for a few aberrant crypts or small, flat non-polyploid adenomas with low grade dysplasia, as visualized by H&E staining in Figure 14E. Furthermore, immunohistochemical analysis showed that levels of cleaved caspase-3-positive areas were increased in colon tissue sections of ONX 0914 treated B6 mice compared to vehicle control. Conversely, Ki-67+ epithelial cells were markedly decreased in animals treated with the inhibitor (Figure 14E).w

Figure 14. Long-term treatment with ONX 0914 suppresses AOM-induced sporadic colorectal tumor formation in C57BL/6 and LMP7-/- mice. A. CRC was induced in 5 to 6-week old C57BL/6 or LMP7-/- mice by i.p. injection of AOM at

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12 mg/kg weekly for 6 weeks (black triangles). Mice were treated with either vehicle (grey triangles) or 10 mg/kg ONX 0914 (black dots) s.c. every second day from day 0 of the first AOM injection. LMP7-/- mice were either left untreated (blue squares, left) or treated with the inhibitor (blue squares, right) or with vehicle control (blue open squares, right) (n ≥ 10). Naïve C57BL/6 (red dots) or LMP7-/- (red open dots) were left untreated (n=2). Body weight (bw) was recorded every second day. Data are presented as percent loss of bw compared to the initial bw on day 0. Bw curve on the left shows first experiment with the endpoint at day 120. Left graph shows body weight curve of a second, independent experiment lasting until day 200. B. Survival curve showing the percentage of mice remaining alive until the end of the experiment on day 200. C. At the endpoint, colons were removed and analyzed macroscopically for tumor lesions. Graph shows number of tumor in the large intestine by semi quantitative macroscopic assessment. Tumors are defined as lesion ≥ β mm. Representative gross pathology of the descending colon and rectum at the end of the only AOM protocol on day 200 is shown on the right. D. Spleen weight of individual mice at the endpoint. Images of representative spleens of either vehicle or ONX 0914 treated C57BL/6 mice are shown. E. Representative H&E stainings and IHC stainings (anti-Ki67 and anti-cleaved caspase-3) of colon sections of vehicle or ONX 0914 treated C57BL/6 mice on day 120 are shown. Data are shown as means ± SEM. Data are representative of 2 independent experiments. Significance was calculated by unpaired student’s t-test with **p < 0.01, ****p < 0.0001.

When applying the AOM model to ApcMin/+ mice, the obtained results were quite similar to the impact of immunoproteasome inhibition in wild type C57BL/6 mice. Again, we observed a drop in body weight as a response to repeated AOM injections, except for ONX 0914 treated ApcMin/+ mice. Interestingly, treatment with the inhibitor quickly led to restoration of the initial body weight in ApcMin/+LMP7-/- mice. Both groups of inhibitor-treated mice increased body weights according to normal development. Inhibitor of the immunoproteasome could additionally ameliorate the Min-associated body weight loss at later time points, which occurred in untreated ApcMin/+ or ApcMin/+LMP7-/- control mice as a result of Apc mutation - mediated tumor growth and intestinal obstruction (see approximately at day 50 in Figure 15A). This indicates that treatment with the inhibitor reduced overall disease symptoms accompanying AOM-induced augmentation of tumor development in ApcMin/+ mice. Instead, vehicle treated mice suffered from weight loss, particularly in the last stages of the AOM-associated tumor model. Furthermore, these mice were presented with a decreased general health state and poorer survival (Figure 15B). Although, ApcMin/+LMP7-/- (only treated with vehicle) did not develop the wasting disease observed in LMP7 KO mice (Figure 14), they nevertheless looked worse than ONX 0914 treated ApcMin/+LMP7-/- mice and showed decreased body scores, the occurrence of rectal prolapse and persistent rectal bleeding.

On day 90 after the initial AOM dose, mice were analyzed for colonic tumor lesions. This time point was chosen in accordance to well-established studies that show maximum tumor incidence in ApcMin/+ mice by 120 days after birth (267, 401). Furthermore, untreated ApcMin/+ control mice of this experiment succumbed due to severe intestinal tumor development at these days. Correlating to the observed differences in body weight development, vehicle treated ApcMin/+ mice and ApcMin/+LMP7-/- mice exhibited high tumor burden throughout the whole colon that was cobbled with coalescing lesions of different sizes with minimal leftover of the normal epithelium. In contrast, ONX 0914 treated ApcMin/+ mice significantly reduced tumor formation in the colon, which was even more pronounced in ApcMin/+LMP7-/- mice treated with the LMP7 inhibitor – also compared to the already suppressed tumor formation in untreated ApcMin/+LMP7-/- mice (see Figure 13 and 15C). Noticeably, the reduced health state of ApcMin/+LMP7-/- mice treated with vehicle control did not reflect tumor development, since these mice also showed significantly low numbers of colon tumors compared to vehicle treated ApcMin/+ mice, suggesting that clinical signs developed independent from tumor progression, but are rather mediated by the AOM treatment. This tumor growth was otherwise rescued by treatment with the LMP7 inhibitor. Additionally, as a direct visible symptom of the liability of Min-associated colon carcinogenesis, which was observed decoloration of extremities in ApcMin/+ mice only treated with vehicle control (for example pale feet, Figure 15C. Furthermore, we observed splenomegaly formation in tumor-bearing vehicle treated ApcMin/+ mice, compared to a spleen size and weight reduction in response to ONX 0914 treatment (Figure 15D). Interestingly, spleen size and weight were reduced in ApcMin/+LMP7-/- mice

42 irrespective of the individual treatment group. The reduced tumor multiplicity in ONX 0914 treated ApcMin/+ mice was further verified by H&E staining, as well as by histochemical analysis of strongly reduced Ki-67-positive cells and intense staining of cleaved-caspase 3 opposed to that in comparison to vehicle treated mice (Figure 15E).

These data strikingly revealed an anti-tumorigenic effect of ONX 0914 in a mouse model of sporadic colon cancer, even in the absence of preceding, tumor-promoting intestinal inflammation. This indicates that ONX 0914 treatment is effective in all types of colon cancer.

Figure 15. ONX 0914 treatment inhibits AOM-induced sporadic colorectal tumor formation in ApcMin/+ mice and ApcMin/+LMP7-/- mice. A. CRC was induced in 5 to 6-week old ApcMin/+ or ApcMin/+LMP7-/- mice by i.p. injection of AOM at 12 mg/kg weekly for 6 weeks (black triangles). Mice were treated with either vehicle (grey triangles) or 10 mg/kg ONX 0914 (black dots) s.c. every second day from day 0 of the first AOM injection. ApcMin/+ LMP7-/- mice were either left untreated (blue squares, left), treated with the inhibitor (blue squares, right) or vehicle treated (blue open squares, right) (n ≥ 10). Naïve ApcMin/+ (red dots) or ApcMin/+ LMP7-/- (red open dots) were left untreated (n=2). Body weight (bw) was recorded every second day.

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Data are presented as percent loss of bw compared to the initial bw on day 0. For better visualization two independent experiments are shown. B. Survival curve showing the percentage of mice remaining alive until the end of the experiment on day 90. C. Colons were removed and analyzed macroscopically for tumor lesions. Graph shows number of tumor lesions in the large intestine by semi quantitative macroscopic assessment. Tumors are defined as lesion ≥ β mm. Data are means ± SEM. Representative gross pathology of the descending colon and rectum, as well as feet at the end of the only AOM protocol are shown on the right. D. Spleen weight and size of individual ApcMin/+ mice at the endpoint of the AOM-induced model on day 90. E. Representative H&E stainings and IHC stainings (anti-Ki67 and anti-cleaved caspase-3) of colon sections of vehicle or ONX 0914 treated ApcMin/+ mice on day 90 are shown. Data are shown as means ± SEM. Data are representative of 2 independent experiments. Significance was calculated by unpaired student’s t-test with **p < 0.01, ***p < 0.001.

Patient-derived CRC specimen showed elevated expression of immunoproteasome subunits Since we are eager to provide reasonable relevance of translating ONX 0914 treatment for application of human CRC, we investigated the composition of proteasomal subunits in primary intestinal tumors of patients suffering from colon colorectal carcinoma. Therefore, colon tumor tissue and adjacent healthy tissue from 10 patients were analyzed by immunoblotting against the respective subunits. Figure 16 shows a representative blot from colon tumor (T) and adjacent normal tissue (H) of three different patients. By visual inspection, all tumor tissue samples showed increased expression of immunoproteasome subunits LMP7, LMP2 and MECL-1, whereas less pronounced signals of the respective subunits were obtained from matched adjacent normal colon tissues lysates.

In order to analyze whether the possibly increased incorporation of the immunoproteasomal subunits is the limiting factor for expression of the constitutive counterpart, whole tissue lysates were also analyzed for the corresponding subunits 5c, 1c, βc. Compared to healthy tissue, protein levels for the constitutive subunits of the 20S proteasome were also upregulated in adenocarcinoma tissue, indicating that the increase of i subunits did not result in decreased expression of the constitutive subunits. Since immunoproteasomal subunits compete with their constitutive counterparts for incorporation into the 20S proteasomes, we quantified the c/i ratio in tumor and adjacent control tissue after normalization of signal intensities to the loading control as shown in the lower panel of Figure 16. We observed a significant increase of LMP7 and LMPβ expression relative to the constitutive subunits in tumor tissue, while MECL-1 expression in adenocarcinoma was yet upregulated but not significantly. However, elevated levels of LMP7 and LMP2 could also be detected in adjacent colon tissue. Interestingly, western blot analysis showed decreased MECL-1 expression levels in contiguous normal tissue.

Collectively, we observed elevated expression levels of the catalytically active immunoproteasome subunits LMP7, LMP2 and MECL-1 in adenocarcinomas compared to the conterminous tissue. Thus, these data provide evidence of prerequisites for potential application of ONX 0914 in human colorectal carcinoma.

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Figure 16. Elevated levels of immunoproteasome subunits in primary samples of human CRC. Top. Analysis of protein expression of immunoproteasome subunits and constitutive proteasomal subunits in 30 µg tissue lysates of adenocarcinoma and matching adjacent healthy colonic tissue of CRC patients (n = 10). Proteins were resolved on 15% SDS gels and detected using anti-human antibodies for LMP7, LMP2 and MECL-1, as well as for correlating constitutive subunits 5c, 1c, βc. - actin was used as loading control. Results are shown from three CRC patients with obvious differences in expression levels (PT5, PT6, PT7; PT, patient; H, healthy; T, tumor). Blots on the right show NIR immunoblot of the same indicated samples with proteasomal subunits in green and -actin in red acquired at emission wavelengths of 800 nm and 700 nm, respectively. Bottom. Relative ratio of proteasomal subunits 5c/5i, 1c/1i, βc/βc of individual CRC patients in adenocarcinoma tissue (black columns) and respective adjacent healthy tissue (grey columns) (n = 10). The results are presented as normalized signal to loading control and were quantified by fluorescent signal intensities in the NIR immunoblot. Data are presented as means ± SEM. Significance was calculated by unpaired student’s t-test with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (ns, not significant).

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Discussion and future implications

The proteasome is the key player of intracellular protein homeostasis and essential for almost every cellular process. Hence, dysregulation of the UPS degradation system plays an essential role in development and progression of human cancer (247, 402, 403).

The UPS is implicated in regulation of virtually every molecular pathway in the pathogenesis of CRC such as proliferation, apoptosis, angiogenesis and metastasis (4, 22). Oncogenic transformation and rapid tumor growth impose heavy demand on proper protein quality control systems in colon cancer cells. All of the cancer-associated intracellular mechanisms, including genetic mutations, accelerated protein synthesis and turnover rates or metabolic alterations can lead to misfolded or damaged proteins which need to be eliminated to maintain tumor cell viability (404). Additionally, selective degradation of tumor suppressors is favored in cancer cells (405). Thus, proteasome inhibition may be of reasonable therapeutic impact in colon cancer.

During the past decade proteasome inhibition has been shown to be highly implicated as a valuable drug target in cancer therapy. Malignant cells are significantly more sensitive to proteasome inhibition compared to normal cells as they heavily increase their proteasomal dependency due to increased protein turnover and proliferation (406–410). In general, proteasome inhibitors enhance anti-proliferative, pro- apoptotic, anti-tumor and anti-angiogenic effects by influencing a plethora of cellular responses such as inhibition of NFκB activation and blocking subsequent transcriptional regulation of pro-inflammatory cytokines, dysbalance of pro- and anti-apoptotic factors resulting in activation of the caspase-mediated cell death, as well as accumulation of tumor suppressor proteins and stabilization of cell cycle promoters. All these effects increase ER-stress levels and suppress the anti-apoptotic response which eventually results in terminal unfolded protein response (UPR), cell cycle arrest and apoptotic cell death upon proteasome inhibitor (PI) treatment (169).

To date, three FDA approved drugs, the dipeptide boronic acid analogue Bortezomib (BZ, PS-341, Velcade®), the irreversible inhibitor carfilzomib (CFZ, PR-171, Kyprolis®) and the reversible PI Ixazomib show clinically therapeutic impact in the treatment of B cell and plasma-cell related hematologic diseases (180, 411, 412). These proteasome inhibitors induce several apoptotic pathways and inhibition of transcription factors, including NFκB or c-Myc in cancer cells while sparing non- malignant neighboring cells. Accompanied downregulation of pro-inflammatory cytokines such as IL- 6 contributes to inhibition of tumor growth by proteasome inhibitors. Several groups also reported an upregulation of p53 and mediation of cell cycle arrest by increased expression of cdk inhibitors p27 and p21 during proteasome inhibitor-induced anti-myeloma therapeutic treatment (413).

Despite the tremendous impact on induction of anti-tumor responses, PI treatment is only limited to certain types of blood cancer. BZ initially showed tumor growth inhibition and apoptotic cell death induction in several colorectal carcinoma cell lines (LOVO, KM12L4, WiDR) via phosphorylation and subsequent cleavage of Bcl-2 (414). However, these encouraging pre-clinical data did not translate into clinical trials against solid tumors (411). Bortezomib, as well as the more advantageous CFZ treatment of advanced stage CRC hampered clinical efficacy and did not provide improved overall survival of CRC patients (415, 416). Even combination therapies using BZ with the topoisomerase I inhibitor irinotecan (mainly used for the treatment of colorectal carcinoma), gemcitabine, docetaxel, or carboplatin and paclitaxel did not improve anti-tumor responses in CRC patients (226, 247). The non- existing activity in solid tumors may be due to altered tumor-antigen presentation which resulted in impaired DC function and reduced tumor-specific T cell responses (417). As well, red blood cells might

46 act as a collecting vessel for the slowly irreversible inhibitor BZ, thus limiting further tissue distribution. Additionally, off-target effects and drug resistance emerged after long-term treatment and limited applicability of either BZ or CFZ.

The immunoproteasome has recently gained substantial interest as a potential therapeutic drug target to improve current anti-cancer therapies as it is selectively expressed in cells of the immune system and inflamed tissues. At least in mice, selective immunoproteasome inhibition has a much broader therapeutic window with improved pharmacokinetics and pharmacodynamics and less adverse side effects due to selective compared to general proteasome inhibition. Cutting edge studies report the involvement of immunoproteasomes in development and progression of a magnitude of diseases such as neurodegenerative diseases, autoimmune diseases and inflammatory disorders as well as in cancer (37, 208, 418, 419). Immunoproteasomes regulate pivotal pathways that are involved in tumor development and progression including apoptosis and the induction of inflammatory responses. Selective inhibition of the immunoproteasome was shown to block growth of leukemic cell lines and patient-derived primary cells by triggering apoptosis. Additionally, LMP2 selective inhibition of the immunoproteasome by the compound UK-101 has been shown to exert anti-tumorigenic effects in the aggressive prostatic cancer cell line PC-3 by inducing apoptosis and cell-cycle arrest – independent of NFκB activation (199). These pre-clinical results indicate that tumor therapy via selective immunoproteasome inhibition may be effective not only in hematological malignancies but also in solid tumors with the advantage of not targeting the constitutive proteasome in otherwise healthy tumor- surrounding cells.

ONX 0914 was shown to induce anti-leukemic effects during preclinical evaluation in vitro (420–422) and reached now application in hematological malignancies. Being the first pharmaceutical company taking a selective inhibitor of the immunoproteasome into the clinic, Kezar Life-Science entered clinical Phase 1b studies with an orally available derivate of ONX 0914, KZR-616, only recently. This compound is supposed to be tested as well against autoimmune diseases and inflammatory disorders (423, 424).

The importance of the immunoproteasome in regulation of colorectal tumorigenesis is further highlighted by the fact that activity and abundance of immunoproteasomes is generally up-regulated in many cancer types, particularly after being exposed to the inflamed tumor microenvironment (103, 404, 420, 425–428). Additionally, inflamed mouse and human tissue shows increased expression levels of immunoproteasome subunits probably contributed by the release of TNF or IFN from infiltrating lymphocytes and monocytes already constitutively expressing immunoproteasomes (72, 107, 138, 147).

Besides evident upregulation of proteasome activity or abundance across many cancer types, the most prominent genes that are amplified in malignant tumors encode proteasomal subunits (429, 430). High IP expression was shown to be a marker of susceptibility to selective proteasome inhibitors, as shown by massive accumulation of poly-ubiquitinated proteins and reduction in viability upon ONX 0914 treatment in cells overexpressing immunoproteasome compared to the absent effects in low IP expressing cells (420). This would perfectly suit to the supposed therapeutic benefit of the anti-tumor activity of ONX 0914. Increased proteasomal activity is especially associated with malignant progression (431). High activity of proteasomes together with increased proteasome subunit expression (PSMD4, PSMA5) was particularly shown for CRC probably due to enhanced activation of Nrf2 (nuclear factor E2-related factor 2) (432). Additionally, PSME3, a member of the PA28 family, was shown to be significantly upregulated in colorectal tumors compared to adjacent normal tumor tissue (427). Among the α-subunits of the 20S proteasome, particularly PSMA7 (which encodes the 20S subunit αγ) is highly upregulated in colon cancer, which also validated PSMA7 as a verified prognostic

47 indicator of poorer outcome after CRC surgery (433). Among the proteasome activators, the REG regulatory particle has been shown to be particularly relevant to colon cancer due to facilitated degradation of cancer-associated substrates (434). Given that colorectal lesions are mostly affected by perturbations in genes such as Apc, -catenin, TCF4, K-ras, Smad4 (DPC4 deleted in pancreatic cancer 4), p53 as well as HIF-1α and members of the Bcl-2 family, of which all of them are regulated by proteasomal degradation, proteasome inhibition is further suggested to exert anti-oncogenic effects. Direct evidence of participation of the IP in regulating tumor development is entailed in HPV- independent tumor uterine leiomyosarcoma (435). Spontaneous development of this tumor is seen in LMP2 KO mice starting at 6 months of age with a 40% incidence at 14 months. Correlative to the mouse model, LMP2 expression is absent in human leiomyosarcoma tumors (436). In vitro data determined the association of LMP2 and IRF-1 as critical factors in this type of cancer, since IRF-1 regulates proteins that are involved in cell-cycle progression and proliferation (435). While the immunoproteasome is known to be upregulated in hematological malignancies, expression levels of proteasomal immunosubunits in solid cancers have not been examined well so far.

Analysis of subunit content in human colorectal tissue specimen revealed increased expression of all three immunoproteasome subunits in malignant tissue compared to matching healthy controls. LMP7 appeared to be the predominant form of the expressed proteolytically active subunit in colon tumors despite variations between individual patient samples (see Supplementary Figure 2 and 3). In addition, the total pool of proteasomes in cancer tissue was increased as well, as illustrated by upregulation of the proteasome α-subunit iota (see Supplementary Figure 3). However, the constitutive catalytic subunits 5c, as well as 1c were induced as well in tumor tissue though to a lesser extent compared to their IP counterparts. Constitutive expression of IP in non-tumor tissue was already described by Guillaume et al. and others (102, 103, 107). The percentage of IP in normal colon mucosa was about 49%, as measured by subunit-specific ELISA. Within this approach the authors state that only 2% of the proteasome pool consisted of standard proteasome (102). Interestingly, the leftover percentage account for the presence of intermediate proteasomes of the 1-β-LMP7 type, whereas LMP2-β-LMP7 containing mixed proteasomes are absent (102, 103). The existence of mixed proteasomes in colon tissue is in accordance with our data. For healthy colon tissue, standard proteasome subunits were almost not present, but – in huge contrast to already described proteasome subtypes, the majority of 20S proteasomes in healthy tissue were probably mixed proteasomes of the LMP2 – β – LMP7 subtype. Nevertheless, the most abundant proteasome subtype in colon tumor tissue are immunoproteasomes. Previously, the group of Steinhoff also identified an evident increased expression of IP in inflamed colonic tissue of Crohn’s Disease patients with preferential incorporation of especially LMP2 (72, 138). Since CD is one of the risk factor for the development of CAC, these data together with our findings suggest the meaningful involvement and activity of the immunoproteasome in the pathogenesis of chronic inflammation and consequential formation of colon cancer. The authors further suggest that IP-mediated accelerated NFκB activation is the driving force of sustained and progressive intestinal inflammation in IBD (137). Apart from the tremendous role of IP in inflammatory disorders in relationship with IκB degradation and NFκB activation (137, 437, 438) the involvement of LMP2 in activation of the canonical NFκB pathway has been controversial. Recently, Bitzer et al. verified non-existent influence of immunoproteasome subunits in canonical NFκB pathway (133). Interestingly, one study could state frequent expression of LMP2 in colorectal (and pancreatic) cancerous tissue compared to nonmalignant colonic tissue from matching donors (145). Related to that, another report could demonstrate elevated expression of βc in colon adenocarcinoma, while being expressed only at low levels in healthy tissue. Additionally, they identified nuclear and cytoplasmic localization of βc-positive staining (439). Moreover, Kondakova et al. stated increased chymotrypsin-

48 like activities present in malignant CRC tissue due to increased expression of LMP7 and LMP2 subunits. As well, they found high levels of the regulatory sub complex PAβ8 in CRC that potentially contributes to increased proteasomal activity in these tissues (440, 441). All of the mentioned studies only focused on one or two subunits. So, to our knowledge, this is the first analysis of all six catalytically active proteasome subunits in colorectal adenocarcinoma.

Admittedly, we did not analyze all tissue specimen yet, but we will further improve the analysis readout of leftover samples by analysis of all proteasome subunits either by examination of mRNA expression levels or via two-dimensional gel electrophoresis. So, we can directly compare expression of either constitutive of immunoproteasome subunits on one gel. Additionally, it would be interesting to perform immunohistochemical analysis of LMP2 and LMP7 of colon section, in order to identify localization or even cell type-specific expression of these subunits in tumor tissue, since cancer tissue is a heterogeneous complex of neoplastic cells, benign epithelial cells, mesenchymal and immune cells. In conclusion, malignant tissue shows upregulation of the immunoproteasome subunits LMP7, LMP2 and MECL-1. Immunoproteasome formation is slightly more pronounced compared to surrounding control specimen. Thus, inhibition of the immunoproteasome or especially ONX 0914 treatment would constitute a considerable therapeutic approach in the treatment of colorectal carcinoma in humans.

Prior to investigating the role of selective immunoproteasome inhibition in colon cancer-related malignancies, we first examined the potential anti-tumor response of ONX 0914 treatment to EL-4 thymoma as well as to the aggressive B16F10 melanoma in a protective tumor model with inhibitor treatment starting at the day of tumor cell inoculation. Albeit absent therapeutic effects in the B16F10 subcutaneous solid tumor model and EL-4 – mediated formation of liver metastasis, we observed significant reduction of tumor growth and metastasis formation in the lung upon inhibitor treatment presumably due to a reduction of tumor promoting Th17 cells (Supplementary Figure 1) (376, 442, 443). These preliminary result ensued to further identify the anti-tumor effect of ONX 0914 in more relevant mouse models of cancer.

Studies of analysis of proteasome content revealed, that the basal expression of immunoproteasome subunits is not necessarily restricted to immune cells but is also present in cancer cells (66, 69). Cancer cell lines derived from solid tumors only express low amounts of LMP7 (444). Elevated protein levels of MECL-1 were detected in human Caco-2 colon cancer cells, whereas no basal expression of LMP2 and LMP7 mRNA was detectable in the SW620 colon cancer cell line (445). Without prior induction of immunoproteasomal subunits in the used murine colon adenocarcinoma cell line MC38, LMP2 and to a lesser extent also MECL-1 was already present. IFN stimulation leads to increased expression of aforementioned subunits and induction of LMP7. Interestingly, also 5c was induced, suggesting the potential presence of intermediate proteasome subtypes present in these cells (data not shown).

We could show that ONX 0914 treatment was associated with enhanced cytotoxicity by impairing protein homeostasis which led to suppression of proliferation and induction of cell death (Figure 6). Tumor suppression was visualized by lesser decrease of the intracellular fluorescent dye CFSE compared to DMSO control (Figure 6E) and was accompanied by downregulation of the cell cycle promoter cyclin D1 (Figure 6B). As well, the measurement of cell viability by MTT assay could show increased cytotoxicity in MC38 cells upon inhibitor treatment (Figure 6F). The induction of cell death was verified by a two-fold higher percentage of apoptotic and necrotic cells (Figure 6D) and decreased expression of the anti-apoptotic factor Bcl-2 in ONX 0914 treated cells (Figure 6B). These cytotoxic effects are probably due to suppression of the proteasomal capacity that culminated in the accumulation of poly-ubiquitinated proteins and potentially toxic substrates (Figure 6A). Noticeably,

49 accumulation of ubiquitin-protein conjugates were also seen in tumor tissue of ApcMin/+ mice (Figure 11F). It was already reported that IFN simultaneously mediates upregulation of IP subunit and levels of damaged proteins upon oxidative stress which increases the sensitivity of ONX 0914 treatment (9, 155, 421, 422). However, the constrained and physiologically unrelated high amounts of IFN we used in vitro nevertheless indicate that the pro-inflammatory (tumor) environment in the intestine favors immunoproteasome assembly in colon cancer cells which then constitute an ideal target for ONX 0914 mediated cytotoxicity.

The antitumor effect of ONX 0914 is likely to be connected to the observed interference with ER stress signals and induction of proteotoxic stress, illustrated by the accumulation of damaged proteins in MC38 cells, as well as in tumor tissue of ApcMin/+ mice. ER stress leads to disruption of peptide processing by either changing calcium levels or by interfering with glycosylation which eventually leads to accumulation of unfolded or misfolded, non-functional proteins. Subsequent formation of protein aggregates eventually results in apoptosis, which has been shown to be implicated in the anti-tumor effect of other proteasome inhibitors. The formation of poly-ubiquitin protein accumulation may result from an increase in the release of free radicals from oxidized proteins due to impaired efficiency of the immunoproteasome to remove these proteins. Oxidative stress preferentially induces the formation of immunoproteasome since IP induction increases turnover efficiency of oxidatively damaged proteins or aggregates to restore the imbalance of pro-oxidants and anti-oxidants and to up-regulate antigen presentation during cell stress – independent of pro-inflammatory (IFN) signaling (9, 446, 447). And in turn, immunoproteasomes show increased activity to favor selective degradation of oxidized protein which is improved by binding of PA28 to the immunoproteasome (448, 449). Nevertheless, IP deficiency was attributed to increased levels of ROS and accumulation of polyubiquitin conjugates. Oxidative stress and enhanced generation of ROS play an important role in all stages of colon carcinogenesis. It was found that the human colorectal tumors have increased levels of important markers of oxidative stress and protein oxidation, such as increased levels of ROS per se, nitric oxide (NO) or 8-oxodG (8-oxodeoxyguanosine) in DNA. Besides colon tumor-specific upregulation of these markers, similar effects were found on immune cells in intestinal mucosa and in serum of CRC patients (450). Thus, omitting the specialized ability of immunoproteasomes to relief oxidative stress levels, immunoproteasome inhibition with ONX 0914 would further potentiate the anti-tumorigenic role of the inhibitor, especially in colon tumor cells.

Further, profound accumulation of poly-ubiquitinated substrates triggers activation of the UPR (unfolded protein response) pathway. This intracellular stress signaling is supposed to reduce overall protein synthesis and to enhance removal of misfolded proteins (402). However, persistent activation of the UPR in presence of overt accumulation of ubiquitin conjugates triggers apoptosis and subsequently leads to pro-apoptotic signaling. As oncogenic transformation usually develops and progresses in hostile tumor microenvironment, the UPR is activated to accelerate protein folding capacity. In this way, the UPR confers as a survival strategy of cancer cells from stress induced cell death. The UPR comprises three parallel signaling pathways that are activated by binding and sequestering of misfolded proteins to the immunoglobulin heavy-chain binding protein BiP. Subsequently, activation of UPR-dependent transcription further leads to attenuation of protein synthesis and NFκB activation. Furthermore, protein trafficking through the ER and protein degradation via ERAD (ER-associated degradation) or autophagy is increased. If protein accumulation and the protein-folding defect cannot be solved with these adaptive mechanism the cell enters into apoptosis (451, 452). Especially the highly secretory colon cancer cells are prone to constant UPR activation, as for example loss of XBP1(X-box binding protein 1) was shown to promote both colitis-associated and APC mutation-mediated intestinal tumorigenesis (453). In

50 addition, the UPR is required to support malignant progression and cancer cell survival by ameliorating ER stress (451). Furthermore, it was shown that ER-stressed cancer cells secrete soluble factors that promote the M2 macrophage phenotype of TAMs which in turn fosters tumor growth (451, 454). It would be further interesting to analyze the contribution of ONX 0914 treatment on UPR signaling, for example via analysis of expression levels or activation of typical UPR chaperones such as of eIFβα or the PKR-like endoplasmatic reticulum kinase (PERK). One of the hallmarks of ER-stress induced cell death is the upregulation of XBP-1, which leads to accumulation of the pro-apoptotic protein CHOP(C/EBP homologous protein) or its transcription factor ATF4 (activating transcription factor 4) that is required for apoptosis both in vitro and in vivo (175, 455). Indeed, inhibition of the proteolytic activity of the proteasome has been shown to induce ER stress in MM and other tumor cells mediating apoptosis and cell death (455–457). With respect to colon cancer, it was shown that BIP and CHOP were correlated with high malignancy and advanced stage of human CRC (451). Indeed, it could be shown that an impaired and defective UPR signaling was the major cause of ER stress which subsequently increased the susceptibility of developing intestinal inflammation and inflammation- mediated tumor formation (458, 459). This suggests that an increase in protein synthesis after ONX 0914 treatment may further exacerbate ER stress and UPR-mediated apoptosis by either increasing or decreasing UPR signaling selectively in stressed tumor cells.

Besides impairment of the UPR response, yet others claim a predominant role of p53 and its target gene the BH3-only protein PUMA (p53 upregulated modulator of apoptosis) for propagation of cell death upon protein stress in colon cancer cells (460). These findings would relate to our data of accumulating p53 expression and simultaneous induction of apoptosis in MC38, as well as in whole colon lysates of AOM/DSS mice upon IP inhibition using ONX 0914 (Figures 6B and 8E). On the other hand, increased levels of the tumor suppressor p53 have been shown to trigger autophagy activation leading to cell death upon proteasome inhibition (461). Considering that the UPS and autophagy represent the two main proteolytic pathways, another interesting aspect would be to additionally analyze a potential contribution of autophagy in the anti-tumor response of ONX 0914 in colon tumors. Since inhibition of autophagy was shown to enhance proteasome inhibitor induced cell death in studies using CFZ or ONX 0912 (457), a possible role of autophagy for executing anti-cancer related effects upon immunoproteasome inhibition seems plausible. Autophagy has been identified as a key tumor-suppressor through inhibition of tumor promoting inflammation and simultaneous activation of anti-tumor responses (462). In addition, autophagy confers important roles in the colitis associated colon carcinogenesis, as autophagy-related 16-like 1 (ATG16L1) gene has been linked to Crohn’s Disease susceptibility and CRC risk (463). Autophagy perturbation in macrophages resulted in increased levels of the CRC-tumor promoting cytokines IL-1 and IL-18 (464) and high microbial load due to impaired xenophagy stimulated IL-23 and IL-17 production that directly drives CRC development (268). As well, defective autophagy in epithelial cells was shown to enhance oxidative stress. And in fact, accumulation of polyubiquitinated proteins has been shown to be a direct consequence of autophagy deficiency (465).

In relation to that, activation of caspase-3, a key enzyme during apoptotic cell death, was analyzed to elucidate the basis of ONX 0914-mediated cell death. In fact, ONX 0914 treatment resulted in significant active caspase-3 upregulation in colon tissue of mice. In AOM/DSS mice treated with the inhibitor the expression of cleaved caspase-3 was consistent with increased cleavage of PARP that is hydrolyzed by caspase-3. Furthermore, we observed counteracting decreased expression of the anti-apoptotic Bcl-2 in whole colon tissue of ONX 0914 mice (Figure 8E). A pronounced downregulation of Bcl-2 was also demonstrated in vitro (Figure 6B). It would be worthwhile to further analyze the opposite apoptotic pathways with respect to a possible increase in pro-apoptotic proteins such as Bik (Bcl-2 interacting killer), Bim (Bcl-2 like protein 11) or Bax (Bcl-2 associated X protein). Histochemistry of cleaved

51 caspase-3 positive staining on colon sections verified the increased apoptotic signaling in colon tissue of ONX 0914 treated mice. We showed higher apoptotic activity in the tumor-adjacent areas after ONX 0914 treatment (Figures 10D, 14E, 15E), which implies that ONX 0914 increases apoptotic rates also in the tumor microenvironment to destroy pre-neoplastic lesions and further promote the tumor- suppressive environment. To determine a direct effect of ONX 0914 treatment on the tumor cell compartment, it would be necessary to further analyze signs of apoptosis and/or proliferation in tumor areas of established early and advanced lesions upon single administration of the inhibitor and subsequent analysis of colon (cancer) tissue several hours later.

In the present thesis, we could clearly show that immunoproteasome inhibition offers great promise for the treatment of CRC using different mouse models of colon cancer. ONX 0914 treatment lead to significantly decreased tumor burden in colitis-associated cancer (Figure 7 and 8). In a therapeutic setting, ONX 0914 blocked tumor growth progression and suppressed further development of intestinal colonic polyposis (Figures 9 and 10). As well, treatment with the inhibitor was beneficial in ApcMin/+ mouse models of either protective (Figure 11) or therapeutic settings (Figure 12). Furthermore, we could state that ONX 0914 impedes colorectal tumorigenesis in long-term treatment schedules of the sporadic CRC model in the absence of overt inflammatory conditions (Figures 14 and 15). And we identified a protective role of LMP7 deficiency in spontaneous tumorigenesis upon loss of Apc (Figure 13), as well as for the establishment of adenomas and/or adenocarcinomas in inflammation-mediated tumor models (Figure 11). These findings suggest an important role of the chymotrypsin-like activity of LMP7 in regulation of colon cancer initiation, development and progression. Since also LMP7 deficient mice are protected from colon tumor formation, this demonstrates that ONX 0914 exerts its effect by directly targeting LMP7 and not due to an ‘off target’ effect. Additionally, it shows that LMP7 has a pivotal function during the pathogenesis of chronic colitis and colon cancer, which cannot be accomplished by compensatory incorporation of the constitutive subunit 5c into the 20S proteasome. These data contain supporting evidence that LMP7 inhibition also plays a protective role in the tumor microenvironment and that ONX 0914 may assist the endogenous response to resolve (residual) tumor-promoting inflammation and pro-cancerogenous responses, thereby limiting colorectal cancer formation.

Although definite underlying mechanisms of treatment efficacy of immunoproteasome inhibition still need to be elucidated, we surmise the anti-inflammatory action of ONX 0914 as the main potential to combat CRC, since all tumors are characterized by an inflammatory tumor microenvironment even if tumor development is not subsequent to chronic inflammation.

20% of all cancers arouse within chronic inflammatory conditions and also solid tumors contain inflammatory infiltrates with high levels of cytokine expression. Inflammation appears to be one of the major mechanisms associated with the development of colorectal tumorigenesis and cancer progression (390), underlined by the fact that chronic inflammation is supposed to be the driving force for development of IBD-associated CRC (392). Compared to CAC, where inflammation proceeds CRC development, most sporadic CRC malignancies trigger an intrinsic inflammatory response which builds up upon the pro-tumorigenic microenvironment (308, 466). Both, adenoma-carcinoma mediated and inflammation-associated tumorigenesis are characterized by inflammatory dysregulation that eventually leads to intestinal adenoma formation. Experimental models of inflammation-associated colon carcinogenesis as well as murine models with germline Apc mutations frequently show massive infiltration of inflammatory cells as well as elevated levels of cell-derived pro-inflammatory cytokines and growth factors directly promoting colorectal tumor cell proliferation, survival and angiogenesis (255, 467).

52

We show that ONX 0914 treatment reduces IL-6 levels in the acute colitis phase as well as in later stages of tumor progression (Figure 8F). Decreased IL-6 secretion probably correlates with CRC regression or blockage of tumor formation, as the change of the cytokine profile in the colon even at early time points might create the formation of an anti-tumorigenic microenvironment. Also, polyp formation is accompanied by increased inflammation in ApcMin/+ mice despite the initiation of adenoma development by mutation of the tumor suppressor gene (342). Downregulation of inflammation was further verified by histology, since ONX 0914 treated colon sections of ApcMin/+ mice exhibit reduced inflammatory infiltrates and less advanced pathological grading of the tumor microenvironment (Figure 11D), thereby suppressing colon tumorigenesis. Additionally, we detected increased numbers of lymphoid follicles in distal colons of vehicle treated ApcMin/+ mice (Figure 11D). The increase of lymphoid tissue in the intestinal mucosa was shown to correlate with the development of gross tumor lesions (468). We state, that the primary therapeutic effect of the LMP7 inhibitor is presented with downregulation of por- inflammatory cytokines, which control the immune and inflammatory milieu to enhance tumor progression. Thus, targeting IL-6 by ONX 0914, as the major regulator of tumor-associated inflammation, reduces intestinal inflammation and therefore lowers the subsequent risk for colon cancer formation.

The role of the immunoproteasome is not only dependent on inflammation, but also actively participates in inflammatory responses by regulating cytokine profiles as well as cytokine production, for example via transcription factors GATA3 and T-bet (T-box expressed in T cells) in response to inflammatory challenge (127, 128). Furthermore, genetic abrogation of immunoproteasome subunits in thioglycollate- elicited macrophages from LMP7/MECL-1 double deficient mice secreted less IL-6 and showed markedly reduced NO levels (469).

Tumor-promoting cytokines, such as IL-6, TNF as well as Th17-related cytokines like IL-17A, IL-21 or IL-23, are highly upregulated in the intestinal tumor microenvironment of human CRC and experimental mouse models of colon cancer and play important roles in pro-inflammatory responses which contribute to formation of a tumor-supportive microenvironment and enhancement of inflammation-related tumor progression (306, 317, 380).

Of particular note, IL-6 is highly upregulated in all types of murine colon cancer models (294, 344). IL- 6 was shown to directly promote cancer cell proliferation, as well as indirectly being implicated in cell survival, cell cycle progression, and in the induction of anti-apoptotic and proliferation-associated genes by mediating STAT3 activation in cancer cells and immune cells (332, 334, 355). Particularly in sporadic CRC, the role of IL-6 comprises transition from chronic colitis to cancer via the linkage between innate and adaptive immunity in the intestine. The cytokine-driven AOM/DSS model as well as the ApcMin/+ mouse model are associated with increased levels of IL-6 that is mainly produced by lamina propria macrophages to promote proliferation and survival of premalignant intestinal epithelial cells, thus enhancing initiation and progression of CRC (470). Inhibition of IL-6 signaling significantly reduced AOM/DSS tumor development suggesting that IL-6 trans-signaling also plays a pivotal role in the development of CRC (343). In turn, loss of the IL-6 signaling suppressor and negative regulator of STAT3, SOCS3 was inversely correlated with further CAC progression (335). Likewise, human CRC exhibits increased IL-6 levels in both serum and tumor biopsies and has been also found to positively correlate with tumor load in colon cancer patients (471). The well characterized pro-tumorigenic functions of IL-6 is mainly exerted by activation of the oncogenic transcription factors STAT3 and NFκB in epithelial cells (331, 347). Recently, IL-6 was reported to aid in differentiation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). These cells are present in all types of colon cancer and might be implicated in suppression of

53 intratumoral immune response while inducing tumor growth instead. Especially in solid cancers, MDSCs have been identified as key-driver of not only tumor immune evasion but also of tumor progression and metastasis (472). MDSCs represent an immature, phenotypically heterogeneous population of immune cells originating from myeloid progenitors and are recruited to tumor tissue by CXCL5 (CXC chemokine ligand 5) and CXCL12. These cells are characterized by suppression of the cytotoxic function of CD8+ T cell and NK cells primarily by expression of nitric oxide synthase (Nos2) and arginase (Arg1). Additionally, they induce Treg development and shift tumor-associated macrophage (TAM) polarization towards the M2 phenotype. Together with type 2 NKT (natural killer T) cells, MDSCs majorly contribute to the immunosuppressive repertoire in the TME of colon cancer (473–477). It would be of great interest to investigate the role of ONX 0914 in suppressing MDSC recruitment into tumor tissue, which might further contribute to the anti-tumor efficacy of selective inhibition of the immunoproteasome in colon cancer.

Furthermore, we also observed decreased secretion of IL-17A in ONX 0914 treated colon tissue of AOM/DSS mice compared to vehicle control (Figure 8F). Increased expression levels of IL-17A in blood and cancer tissues have been shown to be clearly associated with the development of CAC (380). IL-17A was shown to promote angiogenesis by upregulation of pro-angiogenic factors secreted from tumor cells, subsequently promoting carcinogenesis (478). CRCs with a Th17-associated immune profile always have poor prognosis in human patients (395). Inflammation-associated mouse models of colon cancer show that IL-17A-/- mice exhibit milder sign of colitis and tumor growth and have reduced levels of the pro-inflammatory cytokines IL-6 and TNF (354). Thus, immunoproteasome inhibitors would be effective in the therapy of CRC due to their suppressive impact on Th17 responses and the production of IL-23 or IL-6 by myeloid cells. Since IL-6 (in combination with TGF) is an important cytokine in promoting Th17 development (479), we expect that reduced IL-6 levels in ONX 0914 treated mice also reduces pro-inflammatory Th17 cell population and their related cytokines such as IL-17A. Further evidence for the anti-inflammatory potential of ONX 0914 treatment in the ApcMin/+ mouse model is provided by the phenotype of restored normal spleen sizes after ONX 0914 treatment. Splenomegaly develops due to polyposis-associated inflammation that has become systemic, in agreement with elevated levels of pro-inflammatory cytokines in the serum. Alongside with splenomegaly formation, Chae et al. (380) observed thymic atrophy in ApcMin/+ mice. Ablation of IL- 17A corrected both of these immune abnormalities suggesting that T cell mediated responses contribute to suppression of spontaneous intestinal tumorigenesis. Thus, ONX 0914 treatment as well as LMP7 deficiency probably influence IL-17A cytokine levels in ApcMin/+ mice, thus suppressing tumor growth. Although we did not yet analyze the cytokine and chemokine expression profile of the tumor microenvironment in the ApcMin/+ mouse models, Baltgalvis et al. (470) showed the importance of Th17- related cytokines in the regulation of tumorigenesis in ApcMin/+ mice. Inhibition of IL-17A and the Th17 stabilizing cytokine IL-23 inhibited intestinal hyperplasia and tumor formation in ApcMin/+ mice arguing in favor of a cytokine-dependent mechanism of this carcinogenesis model (268). Moreover, TNF and IL-6 directly control and contribute to tumor promotion in early lesions of ApcMin/+ mice by inhibition of apoptosis and up-regulation of angiogenic mediators. Thus, even in the absence of chronic inflammation, CRCs exhibit immune inflammatory infiltrates known as ‘tumor-elicited inflammation’ (306). To highlight previous result using the LMP7 inhibitor in preclinical models of inflammatory disorders, ONX 0914 was shown to substantially affect inflammatory cytokine production and T helper cell differentiation (78). Selective inhibition or genetic ablation of LMP7 resulted in diminished Th1 and Th17 differentiation while enhancing the development of regulatory T cells (126, 127, 144). Furthermore, LMP7 blockage as well as genetic deficiency of immunoproteasome subunits led to reduced secretion of IL-βγ (by monocytes), IFN and IL-2 (by T cells) as well as IL-4 and IL-10 (by splenocytes) among others (127, 128, 437, 469). In addition, we observed overall decreased of pro-

54 inflammatory cytokine secretion of IL-1 and TNF, as well as GM-CSF in sera of ONX 0914 treated mice subjected to the AOM/DSS model and the ApcMin/+/DSS model (Supplementary Figures 5-7). We hypothesize, that ONX 0914 treatment significantly down-regulates pro-inflammatory cytokine secretion thus inhibiting tumor formation in mouse models of colon cancer.

As shown in MC38 cells (Figure 6A), as well as in colon lysates of the AOM/DSS model (Figure 7E), we observed dual inhibition of LMP7 and LMP2 upon ONX 0914 treatment. The cell permeable epoxyketone was first reported to be an LMP7-selective inhibitor. Selectivity was determined by a subunit-specific ELISA to analyze occupied active sites of the proteasome, as well as using LMP7 deficient mice. Muchamuel et al. demonstrated a 20-40 fold higher selectivity compared to the second most susceptible proteolytically active subunits 5c and LMP2 (127). Thus, LMP2 can be co-inhibited by ONX 0914 albeit with IC50 values approximately 15-fold larger than for LMP7 (127). This selectivity was further shown by crystallographic analysis of the subunit structure and the inhibitor bound counterpart. As expected, the three subunits which lack CT-L character, namely 1, 2, and 2i, did not exhibit structural requirements for strong inhibitor binding (79, 113). Insight into structural features show that the morpholine adduct of ONX 0914 favorably fits into the more spacious S1 substrate binding pocket of LMP7. Otherwise, it would require rotation of the side chain of Met45 in the S1 pocket of the 5c subunit. In case of LMP2, the hydrophobic residues (P1) needed to stably bind ONX 0914 are given, however, inhibitor binding would induce unfavorable steric hindrance with the Phe31 residue of the LMP2 binding pocket (79).

Interestingly, several other groups observed considerable effect on the LMP7 activity using LMP2- specific inhibitors (57, 198, 480). Since ONX 0914 is an irreversible inhibitor of the immunoproteasome, repeated or long-term treatment may possible result in cumulative co-inhibition of other (immuno)proteasome subunits, probably due to high structural homology between the catalytic subunits. Using a variant of ONX 0914 with 600-fold selectivity for LMP7, it was demonstrated, that long-term exposure to this irreversibly peptide epoxyketone potentially increased the risk for inhibition of unrelated targets, as for 5c in this case (181, 481, 482). Most proteasome inhibitors were initially designed to block chymotrypsin-like activities (LMP7, LMPβ, 5c). Surprisingly, it was shown that selective inhibition of LMP7 seems to be insufficient to induce therapeutic benefit in MM cell lines, but requires co-inhibition of either 1c/LMPβ or βc/MECL-1 (480). Additionally experimental results using PR-924 demonstrated that sole inhibition of LMP7 alone was not sufficient to induce cytotoxicity or proteasome substrate accumulation in neoplastic cells, which would require similar blockage of LMP7 and 5c (207, 444, 483). Importantly, blockage of the CT-L activity needs to be given, but only inhibition of both subunits might be the actual prerequisite to mediate the observed anti-inflammatory and anti-tumorigenic effects in CRC. These findings suggest that the observed cytotoxic and anti- tumorigenic effects in colon cancer might potentially result from dual inhibition of LMP7 and LMP2 by ONX 0914. This hypothesis could be verified by repetition of experiments alongside using the highly selective (and exclusive) LMP7 inhibitors LU-015i and LU-005i (484).

Inexpicably, repeated injections of a standard dose of 12 mg AOM per kg body weight were not well tolerated by LMP7 knockout mice. Instead, LMP7-/- mice possessed disease symptoms more reminiscent of diabetes, which was already reported – however, not in AOM-induced carcinogenesis models (448). Although these mice did not die within the first days after receiving the AOM dose, lethality may not be due to acute toxicity of AOM. Interestingly, nucleotide polymorphism of the PSMB8 gene has been associated with autoimmune type I diabetes in humans (485). Differences in the intestinal microflora, genetic heterogeneity of mouse inbred strains as well as high lot-to-lot variabilities contribute to increased AOM toxicity. Additionally, a variety of potential molecular mechanisms may contribute to the differential AOM susceptibility and subsequent tumor formation including different rates of AOM

55 metabolism but may be also an imbalance of apoptosis and proliferation in colonic crypts. However, ONX 0914 treated LMP7–/- mice with the exact same dose of AOM gained weight comparable to wild type mice treated with the inhibitor. These observations suggest an additional effect to ONX 0914 in reducing toxicity of the carcinogenic agent AOM. It was suggested that lack of adaptive immunity in SCID mice renders those mice highly susceptible to carcinogenic toxicity (486). Thus, maybe the interaction of AOM carcinogenesis together with the lack of LMP7 alters T cell populations present in the intestine. Matkowskyi et al. found that AOM is also a hepato-mitochondrial toxin and induces fulminant hepatic failure (FHF) – which causes excessive and lethal hepatic failure with centrilobular liver necrosis and associated encephalopathy, albeit only at 5-fold higher doses of the standard AOM dose (487). Disease progression was associated with an increase in serum bilirubin and a significant decrease in serum glucose, which we didn’t observe (data not shown). Further experiments would require a dose reduction to 8 mg/kg, which is currently being tested. Additionally, histophatologic analysis of contemplable organs hopefully will reveal the underlying cause of this severe wasting diseases that is obviously ameliorated by an off-target of ONX 0914, since disease symptoms are clearly dependent on the lack of LMP7. Anyway, the use of LMP7 KO mice engenders several drawbacks including the potential for compensatory mechanisms, as loss of LMP7 is completely rescued by incorporation of the constitutive subunit 5c (488). This fact might also complicate proper interpretation, also partly due to the absence of LMP7 preceding disease onset. Secondly, the genetic ablation of a proteasomal subunit might alter the structure of the 20S and therefore affect proteolytic activity, substrate specificity or binding of regulatory molecules. Interestingly, in some mouse models, it was shown that the loss of LMP7 does not resemble ONX 0914-mediated inhibition of LMP7, as shown in the EAE mouse model (202). Furthermore, LMP7 KO do not share characteristic disease symptoms of the human PSMB8-associated inflammatory disorders, where the main causes of these disorders are elevated levels of IL-6 and IFN inducible protein (IP)-10 (153). The reduced survival in ApcMin/+LMP7- /- mice of the AOM model for sporadic CRC is probably a result additional excessive polyposis formation in the small intestine due to the underlying ApcMin/+ phenotype. However, we only analyzed colon tumor formation in this model. Undoubtedly, the pharmacological activity of ONX 0914 to inhibition of proteolytic activities of individual catalytic subunits needs to be further clarified using colon tissue lysates of the AOM model. This could be accomplished by monitoring inhibition of the different catalytic activities with fluorogenic peptide substrates (according to the protocol of Basler et al. (418)).

The contribution of the intestinal microflora is not only important for AOM-mediated tumor formation. Microbes have been shown to be directly involved in tumor development by production of excessive amounts of bacterial genotoxins and metabolites influencing DNA integrity, cell cycle, or inflammatory responses (311, 312). Unfortunately, we did not investigate the contribution of immunoproteasome inhibition on regulation of intestinal permeability and concomitant changes in number or diversity of commensal microbiota. In fact, it was shown that increase in specific bacterial strains changes intestinal homeostasis into noxious microenvironment perpetuated by further dysbiosis and eventually defects in the intestinal immune system leading to microbial driven CAC (353). Intestinal bacterial - glucuronidases play crucial roles in metabolic activation of carcinogens and mutagens, including AOM. Therefore, intestinal microbiota may enhance the exposure to carcinogens (279, 284). As shown in the AOM/DSS model and ApcMin/+ colon cancer models, the absence of intestinal bacteria in germ-free mice significantly decreases oncogenic mutations and tumor formation, suggesting a drastic relevance for CAC and CRC related colon cancer (489). This was emphasized by the fact that colonic levels of certain microbes directly correlate with more severe colon cancer in human CRC (e.g. Fusobacterium nucleatum, Clostridia bacteria) (490, 491). Although B. fragilis is no causative effect of CRC promotion,

56 it is tempting to speculate that its ability to disrupt epithelial barriers promotes a localized inflammatory response by stimulating cytokine production in immune cells accompanied by enhancement of -catenin signaling and oncogenic transformation (310).

Disruption of mucosal integrity by altered of tight junction proteins or cytokines leads to impaired epithelial barrier function and thus to enhanced microbial stimulation of pro-inflammatory responses (492). A defective intestinal barrier, erosion of the mucus layer and loss of tight junctions causes translocation of the commensal microflora into the colon lumen, which is the classical mode of action using DSS to induce microbial-dependent intestinal inflammation and tumor development (493, 494). In fact, selective inhibition of the immunoproteasome by ONX 0914 or using MECL-1 knock-out mice restored intestinal permeability in the TNBS (trinitrobenzene sulfonic acid) colitis model (495). Thus, the anti-tumorigenic effect of ONX 0914 in the colon cancer models may potentially attributed to sustaining epithelial integrity, which could be verified in additional experiments using the fluorescent probe FITC-dextran in these mouse models.

Since we observed almost complete absent F4/80-positive macrophage staining in inhibitor treated colon sections of the AOM/DSS model (Figure 10D), ONX 0914 may exert its suppressive and anti-tumor effect via suppressing the recruitment of pro-inflammatory cells such as neutrophils or macrophages into the TME. These cells were shown to be pivotal contributors to development and progression of adenomatous polyps (496). The frequency and subtype of colon tumor-infiltrating leukocytes, as well as the expression of inflammatory mediators have been shown to correlate to conditions in human CRC (497).

By pure coincidence, the group of Visekruna simultaneously published corresponding results, as they also showed that either ONX 0914 mediated immunoproteasome inhibition as well as genetic inhibition of the proteasome subunit LMP7 suppressed CAC-mediated tumor formation by dampening the pro- tumorigenic tumor microenvironment (498). The authors further observed reduced NFκB activity LMP7-/- mice which resulted in decreased secretion of the pro-inflammatory mediators IL-17A, TNF and IL-6 by Th17 and innate lymphoid cells, respectively. Importantly, reduced expression of VCAM- 1 (vascular 1), CXCL1, CXCL2 and CXCL3 inhibits the recruitment of tumor- associated leukocytes, such as CD11b+Ly6+ neutrophils, as well as macrophages in the absence of LMP7. Activated neutrophils and macrophages produce large amounts of ROS and RONS which cause DNA damage and tissue destruction during chronic inflammation. Macrophages are the major source of MIP-1 (macrophage inflammatory protein-1 gamma) which was shown to directly promote CRC cell growth via NFκB activation (499). Additionally, the neutrophil chemoattractant CXCL-1 has been shown to be increased in adenomas and adenocarcinomas, thus in more advanced dysplastic lesions, which is regulated by TNF and IL-1 levels (500). Previous studies have demonstrated that upregulation of several chemokines such as CCL7, CCL20, CCL25, CXCL1 and CCL26 and chemokine receptors like CCR8, CCR6 and CXCR2 in tumor tissue was directly linked to tumor growth by facilitating pro- tumorigenic leukocyte trafficking into the tumor microenvironment (501). The data of Vacharjani et al. suggest that inhibition of the immunoproteasome has major impact on the expression profile of chemokines in tumor tissue (498) and should further be analyzed as well in our experiments in order to outline the contribution of ONX 0914 treatment on the chemokine profile in the tumor microenvironment.

Tumor-associated macrophages (TAMs) infiltrating the colon cancer tissue play indispensable roles in inflammation and colon carcinoma development by directly promoting tumor growth, angiogenesis and metastasis (385, 502). These cells represent up to 50% of tumor mass and predominantly produce protumorigenic inflammatory mediators (503, 504). Developing from circulating monocytes, TAMs are

57 attracted into the colon tumor microenvironment by MCP-1 (CCL-2, monocyte chemo-attractant protein 1) which has been shown to be an important mediator of tumor growth and immune regulation. MCP-1 expression is upregulation in advanced large lesions of ApcMin/+ mice and correlates with TAM frequency (342). Similar findings were also obtained in the AOM/DSS model using MCP-1 deficient mice (505). Additionally, the number of macrophages that are accumulating especially in hypoxic areas of tumors directly corresponds to the magnitude of the pro-inflammatory cytokine response in the tumor microenvironment (506) and was associated with advanced stages of CRC progression (507). The final phenotype and activity of these macrophages, which can be distinguished by type M1 and M2 macrophages, is further influenced by cytokines and chemokines produced by cancer cells (e.g. M-CSF, macrophage/monocyte-colony stimulating factor) (385, 386, 466, 508). CRC tumor promotion is likely unleashed due to an unaltered balance between M1 and M2 macrophages. M2 macrophages, that are characterized by secretion or expression of IL-1γ, CDβ06, TGF, CCL17 and angiogenic factors which promote tumor progression by inhibition of the anti-tumor response. On the other hand, M1 type macrophages rather create a pro-inflammatory microenvironment by releasing corresponding cytokines such as IL-1, IL-6, IL-12, IL-18, IL-23 and TNF (388, 475, 508, 509). Noticeably, a CRC tumor environment which consist of high levels of type 1 TAMs was associated with improved overall survival (510), while in turn massive infiltration of TAMs of the type 2 phenotype is associated with a poorer prognosis for CRC patients (511). Of note, it was reported that the proteasome regulates macrophage function. Since Chen et al. found direct effect of ONX 0914 on (alveolar) macrophage polarization (512), it could be that ONX 0914 alters either general recruitment or macrophage phenotypes in the colon tumor microenvironment. Although unrelated to colon cancer, ONX 0914 inhibitor treatment was shown to reduce monocyte/macrophage infiltration inflamed heart tissue upon coxsackievirus B3 (CB3)-induced myocarditis (513). However, LMP7 subunit deficiency resulted in the opposite effect (118). It could be assumed that ONX 0914 treatment reduces macrophage infiltration into the tumor microenvironment which might contribute to decreased intestinal inflammation and subsequent reduction of tumor burden. Thus, a greater insight of the potential effect of IP inhibition by ONX 0914 on the generation of macrophage subsets and immune regulation within the tumor microenvironment would be necessary.

Besides, there are a lot of further cellular mechanism to investigate in order to fully exploit the potential of ONX 0914 treatment as an anti-cancer drug. For example, we did not focus on Tregs that are also of high importance in colon cancer formation. In contrast to other solid cancers, increased frequencies of CD4+Foxp3+Tregs correlate with increased antitumor immunity and improved outcome in CRC patients (514). The protective role of Tregs in colon LP is possibly explained by counteracting pro-tumorigenic IL-17 that is induced by high bacterial load and reduced intestinal barrier function, as shown in human and murine CRC tumors (268, 380). However, a phenotypic change in the tumor-associated Tregs from immunosuppressive IL-10 producing cells to pro-inflammatory IL-17 secreting Tregs induces polyposis in the presence of only moderate increase in Th17 cells (515, 516). Therefore, the role of Tregs may primarily depend on the tumor microenvironment. Akeus et al. revealed an altered chemokine balance in ApcMin/+ derived adenomas with higher CCL17 and reduced CXCL11 and CCL25 expression which might contribute to accumulation of Tregs by lymphocyte-recruiting chemokines (517). Thus, we could further investigate the role of ONX 0914 on the differentiation of the Treg phenotype in colon tumors.

Although further investigations of ONX 0914 mediated effects on initiation and progression of inflammation and colon cancer are warranted to understand the specific mode of the present anti- tumorigenic action of the inhibitor, these results clearly demonstrate that inhibition of the LMP7 subunit suppresses the pathogenesis of CRC of either hereditary, inflammation-mediated or of spontaneous origin in preventive as well as therapeutic preclinical models.

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Furthermore, our data suggest, that the immunoproteasome plays remarkably significant roles in the pathogenesis of CRC development and progression. The anti-tumorigenic effect of ONX 0914 in colon cancer is characterized by the induction of a powerful anti-inflammatory response in the colon mucosa and the modulation of the pro-tumorigenic tumor microenvironment (TME) probably by acting on immunoproteasome-expressing colon cancer cells as well as on infiltrating immune cells. Furthermore, direct cytotoxic effects on pre-neoplastic and malignant cells curtail the emergence of tumor initiation and progression. Based on the given preclinical data, treatment with the immunoproteasome-selective inhibitor ONX 0914 provides a valuable approach for treatment of life-threating colorectal cancer.

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Material and Methods

Cell lines and cell culture methods The mouse colon adenocarcinoma cell line MC38 was generously obtained from Prof. Onur Boyman, UZH, Zurich and was maintained in DMEM (Dulbecco’s minimal essential medium) supplemented with 10% heat-inactivated FCS (fetal calf serum), 100 units/mL penicillin and 100 µg/mL streptomycin

(Gibco, Thermo Fisher Scientific) at 37°C and 5% CO2. Sub-confluent cultures were passaged by detachment of adherent cells with trypsin-EDTA (0.05%;, Gibco, Invitrogen). To induce immunoproteasome subunits, MC38 cells were treated with 100 U/mL mouse IFN (Peprotech) for a minimum of 48 hours. Animals and ethics statement Female C57BL/6 (H-2Kb) were obtained from Charles River (Sulzfeld, Germany). LMP7-/- mice were kindly provided by Dr. John J. Monaco (Department of Molecular Genetics, Cincinnati Medical Center, OH, (64)). C57Bl/6J-ApcMin/+[CD44] mice (264) on a pure C57BL/6 background were obtained from Prof. Jan Paul Medema (University of Amsterdam, The Netherlands). Male ApcMin/+ mice were bred with female C57BL/6 mice to generate heterozygous transgenic mice. LMP7-/- mice were bred with ApcMin/+ mice to obtain ApcMin/+ LMP7-/- mice. Animals were housed in the air-conditioned, accredited animal facility of the University of Konstanz under specific pathogen free conditions on a 12 hour light/dark cycle with lights on at 7 a.m. Animals were provided ad libitum access to autoclaved standard chow and water. Mice were allowed to adapt to the housing conditions for at least one week before experiments were started. Animal experiments have been conducted in accordance with ethical standards and have been approved by the animal experimentation ethical committee of the Regierungspräsidium Freiburg.

Genotyping of mice Genotypes of mice were verified via PCR using genomic DNA extracted from tail biopsies of 4-5 week- old mice to determine LMP7 knockout and Apc heterozygous state. Tail biopsies were lysed in 600 µl of 50 mM NaOH at 95°C under constant agitation at 900 rpm for 1 hour. After that, the digestion was neutralized using 50 μl of 1 M Tris pH 8.0 before centrifugation at 1γ000 rpm. β μl of the supernatant were used as DNA template. For the LMP7 PCR, DNA has to be further purified by DNA precipitation using 1/10 volumes of 3 M NaOAc (pH 5.2) and 1/6 volumes of isopropanol per 100 µl of digestion supernatant. The mixture was thoroughly mixed, incubated at -80°C for approximately 1 hour, and centrifuged at 13000 x g for 30 minutes at 4°C. The supernatant was carefully discarded and the obtained DNA pellet was rinsed with 1 ml of 70% EtOH. After an additional centrifugation step at 13000 x g for 30 min at 4°C, the supernatant was removed, the DNA pellet was air-dried for a maximum of 10 minutes and finally, DNA was dissolved in dH20 and used for genotyping PCR. Standard genotyping protocol was performed using GoTaq® G2 Flexi DNA polymerase (Promega). Expected PCR products of the LMP7 knockout allele are at 700 bp and 600 bp for the LMP7 wild type allele. The expected PCR product size for the ApcMin/+ mutant allele is 300 bp. Primer pairs were synthesized at Microsynth and are displayed below.

Primer Sequence (5’-3’) LMP7 wild type forward GGA CCA GGA CTT TAC TAC GTA GAT G LMP7 wild type reverse CTT GTA CAG CAG GTC ACT GAC ATC G LMP7 KO forward CCG ACG GCG AGG ATC TCG TCG TGA LMP7 KO reverse CTT GTA CAG CAG GTC ACT GAC ATC G

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ApcMin/+ wild type TTC TGA GAA AGA CAG AAG TTT ApcMin/+ mutant TTC TGA GAA AGA CAG AAG TTA ApcMin/+ common TTC CAC TTT GGC ATA AGG C

PCR cycling conditions for LMP7-/- Temperature Time 95°C 7 min 94°C 30 sec 63°C 30 sec X 32 72°C 45 sec 72°C 7 min 4°C ∞

PCR cycling conditions for ApcMin/+ Temperature Time 94°C 3 min 94°C 30 sec 55°C 30 sec X 40 72°C 60 sec 72°C 2 min 4°C ∞

Human CRC samples Fresh clinical tumor tissue samples of colon adenocarcinoma and matching adjacent tissue of 0.5 cm3 to 1 cm3 were collected from 20 patients undergoing colonscopy at the Chirurgische Kinik, Kantonsspital Münsterlingen (Prof. Florian Martens). Tissue samples were immediately snap-frozen in liquid nitrogen after surgery and transferred to -80°C for long term storage. The study was taken in collaboration with the Institut für Pathologie, Kantonsspital Münsterlingen (Prof. Achim Fleischmann). Volunteers were given informed consent to participate in accordance with an ethic vote. In total, tissue specimen of 10 patients harboring different stages of CRC were analyzed in this thesis.

Immunoproteasome inhibition in vitro and in vivo ONX 0914 was generously provided by Dr. Christopher J. Kirk (Onyx Pharmaceuticals). For in vitro inhibition, a 10 mM stock solution of inhibitor in DMSO was prepared and stored at -80°C. A total concentration of 300 nM in DMSO was added into the cell culture medium. The equal amount of DMSO was used as vehicle control. For in vivo studies, the inhibitor was formulated in an aqueous solution of 10% (w/v) sulfobutylether--cyclodextrin (Captisol, CM Fine Chemicals) and 10 mM sodium citrate (pH 6). 10 mg/kg of the inhibitor was administered subcutaneously in a volume of 100 µl. Subcutaneous injection of 100 µl vehicle (Captisol) were used as control. Mice were treated with either vehicle or 10 mg/kg ONX 0914 s.c. every second day.

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Animal models of colon carcinogenesis The AOM/DSS tumor model The establishment of the AOM/DSS mouse model was conducted as previously described (505). CAC was induced in 6 to 8 weeks old female C57BL/6 mice or LMP7-/- mice by a single intraperitoneal injection of 12 mg/kg azoxymethane in 0.9% NaCl (AOM, Sigma-Aldrich). Five days later, a subsequent oral administration of 2% dextran sulfate sodium salt (DSS, MW 36000-500000 Da, MP Biomedicals) in non-acidified drinking water ad libitum was given for five consecutive days in three intermittent cycles. These DSS cycles were interrupted by two-week recovery periods without DSS challenge. A body weight of 19 g was considered as threshold for treatment. Mice were sacrificed by cervical dislocation either 7, 14, 21, 28, 42 days after the initial AOM injection or 8 weeks after the first day of the first DSS cycle (endpoint). Body weight development was recorded every day during DSS cycles and every other day thereafter. Mice were monitored for clinical signs of illness including stool consistency, rectal bleeding, rectal prolapse, reduced activity, general appearance and body weight loss every day during the DSS cycles and every other day afterwards. Upon sacrifice at indicated time points, colons were excised completely from the ileocecal junction to the anal verge to verify uniform recording of colon lengths. Fat appendages were carefully removed without damaging the colon. After that, colons were opened longitudinally and flushed several times with ice-cold PBS. The presence of gross colonic lesions was examined macroscopically and evaluation of tumor development was performed by measuring quantity, size and location of tumors within the large bowel. According to their position, tumors were assigned to either proximal, intermediate or the distal third of the colon. Colonic tumors with a size of ≥ β mm in diameter – as measured by a caliper - were counted to obtain overall numbers of lesions per group. Separately excised tumors as well as adjacent normal tissue were collected in 1% DEPC/PBS and stored at -80°C for further biochemical analysis. Colon tissue, along with flat colon tumors were rolled into concentric circles (“Swiss rolls”) and stored in formalin for further histopathological analysis (see below). ApcMin/+ mice and the ApcMin/+-DSS mouse model ApcMin/+ mice of different sex with a starting age of 5 weeks were administered orally with 2% DSS in the drinking water for 7 consecutive days in order to accelerate tumor initiation, as previously reported (400). Following sacrifice, the small intestine and the colon were resected, opened longitudinally and macroscopically examined as described above. The small intestine was divided by length into three equal sections (proximal, middle, and distal). Polyps in each intestinal segments were counted. Subsequently, a quantity of colonic and small intestinal lesions were resected and stored at -80 °C. To assess and compare tumor incidences in all parts of the intestine, the middle intestinal sections as well as the colon were embedded together in order to recapitulate intestinal construction. Furthermore, since proximal and distal sections are morphologically different, these two parts were embedded simultaneously. Additionally, spleen weight and size were recorded AOM-induced mouse model of sporadic colon carcinogenesis Colon cancer was induced in 5 to 6 weeks old C57BL/6 mice, LMP7-/- mice, ApcMin/+ mice or ApcMin/+ LMP7-/- mice by six weekly intraperitoneal injection of 12 mg/kg azoxymethane in 0.9% NaCl (AOM, Sigma-Aldrich). To standardize potential confounding factors, we only used female mice. Furthermore, AOM and inhibitor treatment were not applied at the same time, but required 10 hours in between. Body weight development was recorded every other day. Mice were sacrificed by cervical dislocation 250 days after the initial AOM injection. At the endpoint, colons were excised and processed for histological and biochemical analysis as described above.

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Histology Preparation of tissue sections Mouse colons and small intestinal segments were Swiss-rolled and fixed in freshly prepared 10% formalin/PBS for 24-36 hours. After that, tissue was embedded in paraffin after dehydration in ascending EtOH series. Paraffin sections of 4 µm thickness were cut with a motorized rotary microtome equipped with a special section transfer system of sections (HM 355 S, MICROM, Walldorf). Sections were mounted on adhesive-coated glass slides (SuperFrost® Plus for H&E or Histobond® for IHC, Marienfeld) and air-dried for approximately 12-18 hours. Sections were kept at 25°C until further analysis. Histopathologic analysis of neoplastic lesions and degree of dysplasia were assessed blindly according to previously published standard criteria and classification of human adenomas of the colon assessed by Suzuki et al. (518). Tumor multiplicity, intestinal pathology, as well as histology scoring was determined microscopically using either the light microscope DM-RB (Leica) and ImageQuant LAS 4000 software (GE Healthcare) or the AxioObserver microscope equipped with Axiocam MRm camera with CCD sensor (Zeiss) following overlay of blue, red and green color channel to display true color images. In brief, intestinal inflammation and histopathological assessment was assessed as previously defined by Horino et al. (519) on the basis of 4 different parameters: Inflammation severity (0-3) + extent of inflammation (0-3) + Crypt damage (0-4)] x % involvement (0-4).

Haematoxylin and eosin (H&E) staining For H&E staining, paraffin-embedded tissue sections were first deparaffinized in three successive baths of xylene for five minutes each followed by rehydration in decreasing concentration of ethanol (100%, 90%, 80%, 70%; five minutes each) and tap water for five minutes. The slides were incubated in hematoxylin solution (according to Mayer, Sigma) for five minutes, washed under running tap water for another five minutes and incubated in EosinG solution (0.5%, C.Roth) for four minutes. Dehydration was performed in ascending ethanol baths of 70%-80%-90%-100% for 20 seconds, each and xylene for three times three minutes. Slides were subsequently covered with mounting medium (Organo/Limonene Mount™, Sigma) and coverslips.

Immunohistochemistry (IHC) For immunohistochemistry, histological slides were deparaffinized and rehydrated by sequential incubation in xylene, 100% EtOH and 90% EtOH. Unmasking and recovery of possibly blocked antigenic binding sites was achieved by boiling of deparaffinized and rehydrated sections in 0.01 M sodium citrate buffer (pH 6.0) for 5 minutes followed by sub-boiling for another 10 minutes in a common microwave oven (400W). Slides were allowed to slowly cool down to RT for at least 30 minutes. For the F4/80 staining, antigen retrieval was performed using Proteinase K in TE-buffer for 5 minutes at

RT. After that, slides were washed three times for five minutes in dH20. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes under constant shaking. Slides were then washed with wash buffer (TBS-0.1%Tween). Nonspecific binding sites were blocked with antibody dilution buffer (5% goat serum in TBS-T). Incubation with primary antibody diluted in antibody dilution buffer was performed overnight at 4°C in a humidified atmosphere to avoid evaporation of the antibody solution. Primary antibodies used include: anti-cleaved caspase-3 (1:100; #9661S, Cell Signaling), anti- F4/80 (1:100; #14-4801, Invitrogen) and anti-Ki-67 (1:400; #12202, Cell Signaling). For detection and visualization of the immunostaining, slides were incubated for two hours in the dark using secondary antibody (biotinylated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories)) at concentration of 1:100 in antibody dilution buffer. Slides were washed in TBS-T and signal detection was performed using horseradish peroxidase coupled avidin-biotin conjugate (Vectastain Elite ABC Kit, Vector Laboratories). Finally, the immunoreaction was developed using peroxidase substrate solution

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(ImmPACT DAB, Vector Laboratories) according to the manufacturer's directions. Sections were counterstained with hematoxylin and were subsequently dehydrated and embedded in mounting medium (Organo/Limonene Mount™, Sigma).

Analysis of cytokine expression in serum or intestinal tissue Blood was collected by cardiac puncture at the time of animal sacrifice into serum separator tubes (Microvette®, Sarstedt). Serum was collected after centrifugation at 13000 rpm for five minutes at RT. Supernatant was immediately transferred into chilled 1.5ml tubes, snap-frozen using liquid nitrogen and stored at -80°C. Lysed intestinal tissue or serum samples were analyzed for mouse cytokines by ELISA according to the manufacturer’s instructions (eBioscience).

Analysis of cell viability and cell proliferation The MTT assay The MTT assay is based on conversion of the water-soluble tetrazolium salt MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into insoluble purple formazan crystals by living cells via cleavage of the tetrazolium ring by dehydrogenase enzymes. Cells were seeded in 96 well-plates at different cell densities in a final volume of 200 µl medium (starting concentration of 1x105 cells per well). Cells were cultured with different concentrations of ONX 0914 or DMSO. Blank controls were included by using medium alone and untreated cells. Positive control of unviable cells was gained after treatment of cells with 50 μM H2O2. After incubation, MTT stock solution (5mg/ml in PBS, Sigma- Aldrich) was added to equal one-tenth of the original culture volume (here 20 µl) and incubated for additional four hours at 37 °C until purple formazan crystals have formed. At the end of the incubation period, the medium was removed by centrifugation and the crystals were carefully solubilized by adding 100 µl DMSO. After 10 minutes, the absorbance of the converted dye was measured at a wavelength of 570 nm with background subtractions at 630 nm in a plate reader (Infinite 200 PRO, Tecan). The concentration of the converted dye directly correlates with the number of viable cells possessing mitochondrial activity. The viability of MC38 cells is given as percentage of baseline values of untreated control cells. CFSE labeling for assessment of cell proliferation Carboxyfluorescein succinimidyl ester (CFSE) retains within the cell due to covalent coupling of intracellular molecules via its succinimidyl group. Thus, fluorescent CFSE is diluted with every proliferation step to dividing cells. MC38 cells were harvested and resupended in PBS/0.1% FBS at a final concentration of 5×107 cells/ml. A CFSE (Biolegend, U.K.) working solution in PBS was added to the cells at a final concentration of 5 μM per ml cells. Cells were incubated for 10 minutes at γ7°C with intermediate mixing to ensure uniform labeling. In a next step, the staining was quenched by adding five times the original staining volume of ice-cold medium containing 10% FCS. Finally, the cells were washed three times with cell culture medium and plated out before being treated with the inhibitor. Downstream analysis of median CFSE fluorescence was assessed after 48 hours and 72 hours using flow cytometry.

Flow cytometric analysis Annexin V/PI staining for flow cytometric analysis of apoptotic cell death Apoptotic cells were detected by labeling cells with Annexin V and propidium iodide which either bind to translocated external phosphatidylserine at the membrane or to DNA in the nucleus due to destructed

64 cell membrane integrity. To induce apoptotic cells as a control, cells were incubated with 15 μM camptothecin (Sigma-Aldrich) for approximately six hours. Cells were harvested 24 hours after inhibitor treatment, washed once in ice-cold PBS and resuspended in 1x Annexin V binding buffer (in dH20, BD Bioscience) at a concentration of approximately 1x106 cells/ml. PI and Annexin V-APC (BD Bioscience) were added to the cells at 1 µg/ml each in 1x Annexin V binding buffer and incubated for 15 minutes at RT in the dark before quantifying apoptotic cells by two-color flow cytometric analysis using the BD LSR Fortessa™ (BD, Biosciences) that is provided at the FlowKon Facility, University of Konstanz. To exclude cell debris and doublets, only single cells were analyzed by doublet discrimination. Living cells will not show positive staining for either Annexin V or PI. Cells which are in the early apoptotic process will be stained by Annexin V. Cells in late apoptosis and/or in an early necrotic state will be stained by both Annexin V and PI.

Molecular Biological Methods - western blotting Protein extraction for western blotting Cell pellets were lysed in ice-cold protein lysis buffer (RIPA buffer: 20 mM Tris-HCl (pH 7.5) 150 mM

NaCl, 1 mM Na2EDTA 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate; freshly supplemented with protease inhibitor mixture (cOmplete™, Roche Pharmaceuticals)). Snap frozen colon or small intestinal tissue fractions were homogenized using a pellet pistil in approximately 500 µl ice-cold RIPA buffer supplemented with 2 mM Na3VO4, 10 mM NaF and 1 mM PMSF. Homogenates were incubated for 30 min on ice including intermediate mixing of the samples during incubation. Sonicated samples were subsequently centrifuged for at least 15 minutes at 13000 rpm and 4°C in order to separate cell lysate from cell debris. The supernatant was transferred into fresh, chilled 1.5 ml tubes and small aliquots were used for protein quantification. Protein quantification using the BCA assay Total protein was quantified using Pierce™ bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s recommendations. First, a bovine serum albumin (BSA) standard was prepared in RIPA buffer with final BSA concentrations ranging from 2000 μg/ml to 25 μg/ml. The working reagent was prepared as recommended in the protocol. 2.5 μl of each supernatant and standard were pipetted into a flat bottom 96-well plate. In a next step, 200 μl of the working reagent was added to the respective wells and the mixture was incubated at 37°C for 30 minutes. After that, the plate was cooled to room temperature and the absorbance was measured spectrofluorometrically at a wavelength of 595 nm (Infinite 200 PRO, Tecan). Protein concentration was determined using the established standard curve. Samples were subsequently adjusted to a final concentration of 30 µg with 1 x Laemmli buffer (300 mM Tris-HCl, pH 6.8, 10% (w/v) SDS, 50% (v/v) glycerol, 0.05% (w/v) bromphenole blue, 5% (v/v) -mercaptoethanol) and protein was denatured at 95°C for 5 minutes. Samples were stored at -20°C until further processing using SDS-PAGE. Before use, samples were thawed to room temperature and shortly centrifuged.

SDS- polyacrylamide gel electrophoresis (SDS-PAGE) Electrophoretic resolving of proteins under reducing conditions and subsequent western blot analysis were performed using the Mini-Protean® 3 Cell System (Bio-Rad). Equal amounts of protein lysates were loaded onto the gel, together with a protein standard to determine the molecular size of the proteins (PageRuler™ Prestained Protein Ladder, Thermo Fisher Scientific). The SDS gel consisted of an upper collection gel of 4% polyacrylamide and a lower separating gel of 8%, 12.5% or 15% polyacrylamide - depending on the expected protein size. Detailed components and composition of SDS gels are given

65 elsewhere (520). Pre-prepared gels were stored at 4°C for a maximum of one week. Electrophoresis was carried out in SDS running buffer (25 mM Tris-HCL, 192 mM glycine, 0.1% (w/v) SDS) at an initial voltage of 50 V until the running front entered into the upper resolving gel. Then, the voltage was increased to 100 V until the protein dye front reached the estimated position for displaying the protein of interest.

Wet blotting procedure and protein detection Protein transfer after SDS-PAGE was carried out by wet blotting in the Mini Trans-Blot Cell™ System (Bio Rad) onto nitrocellulose membranes (0.45µm) or nitrocellulose-FL membranes (0.45µm), especially suitable for NIR (near-infrared) detection (GE Healthcare). Membranes and whatman filter were equilibrated in pre-cooled blotting buffer (25 mM Tris-HCl, 192 mM glycine, 20% (v/v) Methanol) prior to assembly into a lattice cassette, where SDS gels and membranes were then placed between two pieces of blotting buffer soaked whatman paper, covered by two sponges. The assembled transfer unit was put into an electrophoresis container filled with ice cold blotting buffer. Western blot was performed at 110 V for 1.5 hours on ice. Successful protein transfer was identified by visible transfer of the pre- stained molecular weight marker on the membrane. After transfer, the membrane was washed for two minutes in 1x TBS and air-dried on whatman filter for 1 hour in the dark. After that, membranes are incubated in blocking buffer (1x Roti®-Block (C. Roth) in TBS/0.05%Tween®-20) or in Odysee® blocking buffer (LI-COR Biosciences) when using the NIR system. Blocking of unspecific protein background was carried out for one hour at room temperature under constant gentle rotation. Primary antibody incubation in Roti®-Block/TBS-T was performed overnight at 4°C under constant rotation. Primary antibodies include:

Antibody against Dilution Distributor m (mouse) LMP7 1:5000 made in the lab mLMP2 1:3000 made in the lab mMECL-1 1:500 made in the lab hu (human) LMP7 1:5000 made in the lab huLMP2 1:5000 made in the lab huMECL-1 1:1000 Thermo Fisher Scientific PSMB5 1:1000 Cell Signaling PSMB6 1:1000 Cell Signaling PSMB7 1:1000 Cell Signaling Iota (IB5) 1:3000 provided by Prof. K. Scherrer PSMA7 1:1000 Biomol Bcl-2 1:1000 Cell Signaling cleaved caspase-3 1:500 Cell Signaling PARP 1:1000 Cell Signaling p53 1:1000 Cell Signaling -catenin 1:1000 Cell Signaling FK2 1:3000 Sigma-Aldrich HSP90 1:1000 Cell Signaling -actin 1:5000 Cell Signaling -Tubulin 1:5000 Sigma-Aldrich

Following incubation, membranes were washed three times with TBS-T for 10 minutes each on a shaker to remove remaining primary antibody that was not tightly bound. Afterwards, membranes were incubated for one hour at RT with either HRP-conjugated secondary anti-rabbit or anti-mouse Ig antibody (1:3000, Dako) or fluorescently labeled IRDye® secondary antibodies (IRDye® 680, IRDye®

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800, 1:15000, LI-COR Biosciences) when using NIR immunoblotting. Blots were then again washed three times as described above. Development of specific bands was carried out by chemiluminescence after incubation with Pierce™ ECL Super Signal West Chemiluminescent Substrate (Thermo Fisher Scientific) using the Gel Doc™ system and Quantity One 1-D analysis software (BioRad). NIR fluorescence was detected using the Odysee Infrared Imaging system (LI-COR Biosciences) at 700 nm or 800 nm. Quantification of band intensities was performed with the aid of the Image Studio Lite software (LI-COR, Biosciences) equipped at the Odysee Imager. To ascertain equivalent loading of the lanes and to normalize respective protein levels to internal loading controls, either -actin (1:5000, Cell Signaling) or -Tubulin (1:5000, Sigma-Aldrich) was used.

Statistical analysis For animal models, statistical significance of comparisons between ONX 0914 and vehicle treated mice was determined by applying two-tailed Student’s t-test with P values p < 0.05 scored as statistically significant. For all other analyses, unless noted in figure legends, data from at least three experiments were presented as means ± SEM and analyzed by applying Student’s t-test, one-way or two-way ANOVA, where appropriate with p < 0.05 as statistically significant. For ANOVA, we used Bonferroni post hoc analysis to compare groups. The Kaplan-Meier method was used to estimate survival distribution between the groups and log-rank tests were applied to compare survival rates. Statistics were performed using GraphPad Prism6 software (GraphPad Software).

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Supplementary Information

Supplementary material and methods Tumor challenge and bioluminescence imaging (BLI) Female C57BL/6 (H-2b) mice of 8-10 weeks were used (n = 5 per group). B16F10-Luc+ cells (521) were maintained in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% heat inactivated FCS, 1% antibiotics (penicillin and streptomycin), L-glutamine and 0.1% -mercaptoethanol. To establish lung metastasis, cells were harvested by trypsination, washed three times with PBS and resuspended in ice-cold PBS at a concentration of 1x105 cells per 100 µl. The cell suspension was injected intravenously into the tail vein using a 16 G’’ needle. To avoid light absorption and scattering, fur was removed at desired body parts prior to the start of the experiment. Bioluminescence signals were acquired at indicated time points by a CCD camera IVIS® imaging system (IVIS200, Perkin Elmer). For that, mice were anaesthetized by inhalation of Isofluran (1 ml/ml, CP Pharma) in oxygen through a nose cone (5% (v/v)) at a flow of 1 l/min. For anesthesia maintenance, Isofluran was reduced to 2.5% at a flow rate of 0.5 L/min. ThiloTears® (Novartis) was applied onto the eyes to avoid desiccation. Prior to BLI, D-luciferin (Synchem) was dissolved in PBS and was injected intraperitoneal at dose of 150 mg/kg in 400 µ, 10 minutes before measurement of luminescence. Images were acquired using a 20 cm field of view, small binning and 180 seconds exposure time. Region of interest (ROI) were manually drawn around the signal on pseudo-color luminescent images using the Living Image 4.0 software. The emitted signal intensity (photon flux) was integrated over these ROIs and it is expressed as relative light units (RLU, photons per second per cm2) with the lower signal threshold set to 5% of the maximum signal value. Intracellular staining of IL-17A in tumor-infiltrating lymphocytes (TILs) After dissection, lung tissue was stored in HBSS (Hank’s balanced salt medium) at room temperature (RT) before being minced and smashed using scissors or plungers. HBSS containing 0.25 mg/ml collagenase IV, 1 U/ml hyaluronidase and 25 µg/ml DNase IV (Sigma-Aldrich) was added to the homogenized cell suspension and incubated at RT for 2 hours on a shaker with intermitting pipetting to further dissociate cells. After that, cell suspension was centrifuged at 50 x g for 10 min at RT. The supernatant was collected and was centrifuged again at 200 x g for five minutes. The obtained cell pellet was washed with PBS and centrifuged again (200 x g, five minutes). To deplete red blood cells, the pellet was resuspended in 1 ml of ACK (ammonium-chloride-potassium) lysis buffer (150 mM ammonium chloride; 10 mM potassium bicarbonate; 0.1 mM EDTA; pH 7.4) for 2 minutes at RT. After addition of several volumes of PBS, cells were centrifuged at 200 x g for five minutes. After that, cells were centrifuged with discontinuous Percoll (GE Healthcare) gradient of 40% and 80% in PBS at 325 x g at RT for 30 minutes (ascending rate: 5; descending rate: 5). Leukocytes were collected from the interface for further analysis directly passed through a 40 μm nylon cell strainer. Finally, the cell suspension was centrifuged at 425 x g at RT for 10 min. Roughly 5×106 were resuspended in FACS buffer (2% FBS, 1 mM EDTA, 0.1% NaN3 (sodium azide) in PBS) and stained against the cell surface marker CD4 for 20 minutes at 4°C. Cells were washed twice with FACS buffer, centrifuged again and resuspended in 1 ml of 1x Fixation/Perm buffer (eBiosciences). Cells were then incubated either overnight or for 3 hours at 4°C in the dark. Cells were subsequently washed twice with permeabilization buffer (2% FBS, 1 mM EDTA, 0.1% NaN3 (sodium azide), 0.1% saponin in PBS) and incubated in APC- conjugated anti-IL17A mAb (1:200, clone #eBio17B7, eBiosciences) in permeabilization buffer for 3 hours in the dark at 4°C. Cells were washed again two times with permeabilization buffer with a final washing step in FACS buffer. Finally, cells are resuspended in appropriate volumes of FACS buffer for flow cytometric analysis.

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Analysis of cytokine expression in serum samples using multiplex immunoassay Samples were analyzed using Bio-Plex Pro™ Mouse Group I βγ-Plex magnetic bead panel for IL-1, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN and TNFα on pre-coated 96 well plate (Bio-Rad Laboratories). The Bio-Plex assay was run as detailed in manufacturer’s instructions with 50 µl of γ-fold diluted serum samples incubated under constant shaking (600rpm) for 30 minutes at RT. Washing steps were performed using a hand-held magnetic block. Data were acquired on a validated and calibrated Bio-Plex 200 system (Bio-Rad Laboratories) and analyzed with Bio-Plex Manager 6.1 software (Bio-Rad Laboratories) according to the manufacturer’s instructions with double discriminator (DD) gates of 8000-23000. Sample wells for the individual analyte replicates with bead counts ≤ β0 were neglected. Standard curves were fitted using five-parameter logistic regression with default weighting (power law variance), as optimized by the software. Lower and upper limits of quantification (LLOQ and ULOQ) were determined as the highest and lowest fitted standards with recovery of 80-120%.

Supplementary Figures

Supplementary Figure 1. Treatment with the LMP7 inhibitor ONX 0914 reduces formation of lung metastasis. A. Female C57BL/6 mice were injected i.v. with 1x105 Luciferase expressing B16F10 melanoma cells and treated with 10 mg/kg ONX 0914 (black dots) or vehicle control (grey triangles) every other day. Animals were imaged for bioluminescent signals immediately after i.p. injection of D-Luciferin at different time points after tumor cell inoculation (day 0, 5, 10, 12, 16, 18, 20 and day 21). The luminescence signal intensities were averaged from values of ROI analysis and are represented as photons/seconds/cm2/steradian. Bioluminescence imaging (BLI) of representative animals on day 21 are shown on the right panel. B. Graph shows BLI values at the endpoint of the lung tumor model on day 21. C. Immediately after sacrifice, lungs are resected and melanin-black tumor foci are counted manually on the surface of every lung lobe. The right panel shows representative images of lungs on day 21. Exemplary lung of vehicle treated mice exhibiting high tumor load with undeterminable foci number was autonomously set to 500 as maximum value of tumor number. D. Graph shows individual weights of lungs after resection on day 21. E. Lung tissue was homogenized in presence of an enzyme cocktail to obtain tumor- infiltrating lymphocytes, which were stained for intracellular IL-17A and analyzed by flow cytometry. Error bars represent mean ± SEM (n= 8) with *p < 0.05, ***p < 0.001, ****p < 0.0001.

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Supplementary Figure 2. Normalized expression levels of proteasomal subunits 5c and 5i (LMP7), 1c and 1i (LMPβ), βc and βi (MECL-1) of individual CRC patients measured in adenocarcinoma tissue (black columns) or adjacent healthy tissue (grey columns). Constitutive subunits are presented as open columns, while immunoproteasome subunits are illustrated as filled columns. The results are presented as normalized signal to -actin loading control as quantified by fluorescent signal intensities in the NIR immunoblot.

Supplementary Figure 3. Analysis of protein expression of constitutive and immunoproteasome subunits in patient-derived CRC samples. Adenocarcinoma (T, Tumor) and adjacent healthy (H) colonic tissue of CRC patients are shown (n = 10, respective patient ID is shown at the top). Western blot analysis was performed using anti-human antibodies for LMP7, LMP2 and MECL-1, as well as for correlating constitutive subunits 5c, 1c, βc and the 20S subunits iota and PSMA7. -actin was used as loading control. Representative results from two out of 3 experiments.

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Supplementary Figure 5 related to Figure 7 and 8. The effect of immunoproteasome inhibition on systemic inflammatory mediators in AOM/DSS treated mice. Cytokines were quantified from serum of ONX 0914 (black dots), vehicle treated (grey triangles) or LMP7 KO (blue squares mice) using a multiplex sandwich immunoassay kit (Bio-Rad). Naïve C57BL/6 mice served as control (black open squares). Top. Inflammatory mediators (IL-1, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN and TNFα) were quantified from sera of AOM/DSS mice on day 7 after the first DSS cycle Bottom. Inflammatory mediators (IL-1, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN and TNFα) were quantified from sera of AOM/DSS mice at the endpoint of the AOM/DSS protocol on day 56. Quantitative data are shown as means ± SEM with p-values determined by students t-test between ONX 0914 and vehicle treated samples with *p < 0.05.

Supplementary Figure 6 related to Figure 10. The effect of immunoproteasome inhibition on systemic inflammatory mediators in the absence of Inflammation after omitting the 3rd DSS cycle of the standard AOM/DSS protocol. Cytokines were quantified from serum of ONX 0914 (black dots), vehicle treated (grey triangles) or LMP7 KO (blue squares mice) using a multiplex sandwich immunoassay kit (Bio-Rad). Naïve C57BL/6 mice served as control (black open squares). Inflammatory mediators (IL-1, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN and TNFα) were quantified from sera at the endpoint of the AOM/DSS protocol on day 56. Quantitative data are shown as means ± SEM with p-values determined by students t-test between ONX 0914 and vehicle treated samples with *p < 0.05, ***p < 0.001.

Supplementary Figure 7 related to Figures 11 and 12. The Effect of immunoproteasome inhibition on systemic inflammatory mediators in ApcMin/+ mice. Cytokines were quantified from serum of ONX 0914 (black dots), vehicle treated (grey triangles) ApcMin/+ mice or ApcMin/+LMP7-/- mice (blue squares) using a multiplex sandwich immunoassay kit (Bio-Rad). Naïve age-matched ApcMin/+ mice served as control (black open squares). Inflammatory mediators (IL-1, IL-2, IL-4, IL-5, IL- 10, GM-CSF, IFN and TNFα) were quantified from sera at the endpoint of the ApcMin/+/DSS protocol on day 35 (top) or of the therapeutic setting (bottom). Quantitative data are shown as means ± SEM with p-values determined by students t-test between ONX 0914 and vehicle treated samples with **p < 0.01, ***p < 0.001.

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Chapter II

The Role of Autophagy in Cross-presentation of Antigens encapsulated into biodegradable PLGA Microspheres

Abstract

With emerging success in fighting cancer, chronic infections and autoimmune diseases, immunotherapy became a promising therapeutic approach compared to conventional therapies such as surgery, chemotherapy, radiation therapy or immunosuppressive medication. Anti-tumor immunotherapy requires the induction of tumor associated antigen (TAA)-specific CD8+ cytotoxic T lymphocyte (CTL) responses which recognize and specifically destroy tumor cells. Due to the unique ability of dendritic cells (DCs) to stimulate naïve T cells, DC-based immunotherapy is auspicious in modern vaccine research. Nevertheless, DC-mediated immunotherapies have faced several obstacles which eventually led to the development of novel strategies that utilize immune modulators such as adjuvants or cytokines in combination with appropriate delivery systems. To overcome limited delivery of exogenous antigens for cross-presentation by professional antigen presenting cells (APCs) for the induction of CTL immunity in vivo, our laboratory has developed an efficient antigen delivery system for T cell vaccines. Biodegradable poly (D, L-lactide-co-glycolide) microspheres (PLGA MS) exhibit ideal properties for the uptake by DCs and macrophages. The encapsulated protein or peptide antigens are presented on either major histocompatibility complex MHC class II for CD4+ T cell help or cross-presented on MHC class I to CD8+ T cells. We could show that co-encapsulation of a protein antigen (ovalbumin) together with toll-like receptor (TLR) ligands yields potent and long lasting CTL and T helper cell (Th) responses in vivo leading to effective anti-tumor activity in several tumor mouse models. Particularly, we could demonstrate that immunization of PLGA MS co-encapsulated with TRAMP-prostate derived tumor lysate and toll-like receptor (TLR) ligands CpG and polyI:C as adjuvants yielded effective anti-tumor activity in tumor bearing mice and led to reduced tumor burden. However, it is still a matter of debate how encapsulated antigens escape from the into the cytoplasm to enable cross-presentation of antigens on MHC class I. Thus, we aimed to elucidate the influence of autophagy on internalization, subsequent processing and presentation of encapsulated protein antigens due to reasonable evidence that provide participation of the autophagy pathway in antigen cross-presentation. Our data show that inhibition of autophagy leads to decreased presentation of encapsulated MHC class I epitopes either by chemical inhibition of autophagy or via using BMDCs genetically deficient for autophagy factors Atg5 and Atg7. Concerning autophagy’s role in cancer, the link between MHC class I restricted antigen presentation and autophagy will become of interest in improving PLGA MS mediated anti-tumor therapy in vivo.

I

Zusammenfassung

Im Gegensatz zu herkömmlichen Behandlungsmethoden wie beispielsweise der chirurgischen Entfernung des Primär-Tumors mit begleitender Chemo- oder Strahlentherapie, gilt die Immuntherapie mittlerweile als vielversprechende Therapiemöglichkeit für diverse Tumorerkrankungen. Die Immuntherapie richtet sich dabei besonders auf zytotoxische T Zellen (CTLs), welche Tumorzellen spezifisch erkennen und zerstören können. Eine der bedeutendsten Entwicklungen im Bereich der Immuntherapie sind Vakzine auf der Grundlage von tumor-assoziierten Antigenen (TAA), welche auf dem Haupthistokompatibilitäts-Komplexen der Klasse I den CTLs präsentiert werden. Daher ist bei der Generierung einer Antigen-spezifischen Immunantwort gegen Tumorzellen letztendlich die Funktionalität und Effektivität der induzierten T Zellen der entscheidende Faktor. Tierexperimentelle Studien konnten eindrucksvoll zeigen, dass eine Immunisierung mit Tumor-Antigen tragenden dendritischen Zellen spezifische und reaktive T Zell Antworten stimulieren konnte. Dies korrelierte jedoch nur wenig mit dem klinischem Ansprechen jener Vakzine im Menschen. Trotz der Identifizierung einer Vielzahl solcher TAAs und deren CTL Epitope, sind die Immunantworten bei Krebspatienten enttäuschend gering und nicht in der Lage das Tumorwachstum zu kontrollieren. Verschiedene neue T Zell-Vakzinierungsstrategien zielen darauf ab diese Immuntoleranz zu durchbrechen. Darunter gehört die gleichzeitige Verabreichung von immunstimulierenden Hilfsstoffen (Adjuvanzien), welche die Immunogenität der Tumor-Antigene erhöhen sollen, sowie die Strategie einer spezifischen Aktivierung von dendritischen Zellen (DC) in vivo. DCs sind hochpotente, professionelle Antigen-präsentierende Zellen (APCs), die die Möglichkeit haben, die aufgenommenen Antigene zusammen mit ko- stimulierenden Molekülen den CTLs zu präsentieren. Dadurch wird eine effektive Aktivierung der CTLs gewährleistet. Biologisch abbaubare Mikrosphären (MS) bestehend aus poly (D,L)-Laktat und Glykolat (PLGA) sind eine vielversprechende Möglichkeit die gleichzeitige Aufnahme von verkapselten Antigenen zusammen mit Reifungs-Stimuli wie z.B. Toll-like Rezeptor Agonisten durch DCs zu erreichen und nachfolgend eine CTL Antwort zu generieren. Da die verkapselten Antigene über mehrere Tage sowohl auf Klasse II bzw. mittels beschriebener Wege der Kreuz-Präsentation auch auf dem MHC Klasse I Molekül präsentiert werden können, kommt es zu einer Antigen-spezifischen Immunantwort, die in ihrer Stärke optimal amplifiziert und intensiviert wird. PLGA Mikrosphären zeichnen sich durch vollständige Abbaubarkeit im Organismus aus und sind bereits für den klinischen Einsatz im Mensch zugelassen. In vitro und in vivo konnte bereits erfolgreich gezeigt werden, dass eine PLGA MS basierte Immunisierung mit Tumor-Antigenen den klinischen Ausgang verschiedener Tumor Modelle in Mäusen positiv beeinflusste. Zum Beispiel führte die Applikation von verkapseltem Tumorlysat eines Prostata Karzinoms in transgenen Prostata-Tumor tragenden Mäusen zu einer fast kompletten Tumor-Remission. Mehrere Studien konnten belegen, dass partikuläre Antigene neben dem klassischen Weg der Antigen- Prozessierung und Präsentation auch durch Wege der Autophagozytose (Autophagie) auf MHC Moleküle der Klasse I präsentiert werden. So soll die funktionelle Relevanz und mögliche Synergie des Autophagie-Weges bei der Beteiligung der Präsentation verkapselter Antigene identifiziert werden. Es konnte gezeigt werden, dass durch die Blockierung der Autophagie mittels pharmazeutischer Inhibitoren oder durch einen genetischen Ausfall der relevanten Gene, die Kreuz-Präsentation PLGA MS vermittelter Antigene signifikant reduziert wurde. Im Hinblick auf die prominente Rolle, welche Autophagie im Zusammenhang mit Krebs spielt, soll die Akzentuierung der Antigen Prozessierung nach PLGA MS vermittelter Vakzinierung verstärkt werden, um somit eine effektive und dauerhaft funktionelle anti-tumorale Immunantwort zu ermöglichen.

II

Content

Abstract ...... I Zusammenfassung ...... II Introduction ...... 94 Dendritic cells and their role in immunity ...... 94 Subtypes of dendritic cells ...... 94 Dendritic cell maturation and migration ...... 95 The role of dendritic cells in T cell priming ...... 96 Modern approaches in immunotherapy ...... 97 Dendritic cell – mediated immunotherapy ...... 98 PLGA microspheres as vaccine delivery system ...... 100 Antigen processing and presentation ...... 102 Molecular mechanisms of autophagy ...... 105 The role of autophagy in immunity and antigen presentation ...... 107 Results ...... 110 Inhibition of autophagy reduces cross-presentation of PLGA MS encapsulated antigens ...... 110 Autophagy may enhance cross-presentation by promoting phagocytosis of particulate antigens ...... 113 Discussion and future implications ...... 115 Material and Methods ...... 119 Cell lines and cell culture methods ...... 119 Animals and ethics standard ...... 119 Mouse genotyping ...... 119 Agarose gel electrophoresis ...... 120 Antibodies and flow cytometry ...... 120 Generation of CD8a+ bone marrow-derived dendritic cells (BMDCs) using Flt3 Ligand (Flt3L) ...... 121 Preparation of PLGA microspheres ...... 122 Detection of antigen presentation by LacZ T cell hybridoma assay ...... 122 Confocal laser scanning microscopy (LSM) ...... 122 Preparation of cells for immunofluorescence microscopy ...... 122 Molecular biological methods – western blotting ...... 123 Protein extraction and quantification ...... 123 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ...... 124 Wet blotting procedure and protein detection ...... 124 References ...... 125 List of Abbreviations ...... 137 Record of Contribution ...... 140 List of Publications...... 141 Acknowledgments ...... 142

Introduction

Dendritic cells and their role in immunity DCs are the most important type of professional antigen presenting cells (APCs) that act as key mediators for initiation and regulation of T cell mediated primary immune responses to foreign antigens and tumors (1). Among their unique ability to prime naïve T cells, DCs possess the exceptional capacity for uptake, processing and transport of antigens. However, besides stimulating the induction of adaptive immune responses, priming of T lymphocytes by DCs can also lead to tolerance and anergy (2). Hence, they also play an important role in maintaining central and peripheral tolerance to self-peptides (3). Depending on the signals they receive from their cellular environment, DCs have an outstanding role in regulating the nature and extent of T cell responses by orchestrating innate and adaptive immune responses. Subtypes of dendritic cells Considering their diverse functions within the immune system, DCs are a heterogeneous cell population consisting of a multi-lineage system with several subtypes. DCs derive from common myeloid and lymphoid precursors, which are constantly produced in the bone marrow and further differentiate in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and fms-related tyrosinase kinase-3 ligand (Flt3L) (4). Although myeloid and lymphoid DCs differ in their phenotype, functional properties and localization, these two subpopulations are distinguished by only one marker, namely CD8α, which is expressed on CD11c+ DCs as an αα-homodimer unlike the α-heterodimer that is typical for T cells. Since CD8a+ DCs derive from lymphoid precursors, expression of CD8 is absent on myeloid DCs (5). Depending on their developmental pathway, DCs are further subdivided into conventional (cDCs, CD11c+) and plasmacytoid DCs (pDCs) (6). The latter are characterized by intermediate expression of CD11c (CD11clow), while expressing high levels of CD45RA (B220+) and Ly6C. They play an important role in anti-viral immune responses by secreting large amounts of type I interferons (IFN) (7). cDCs are strategically located at sites that are exposed to the environment such as skin or mucosal tissue. Especially, secondary lymphoid organs (SLO) such as spleen and lymph nodes (LNs) embody enriched numbers of cDCs. These cells can further be classified into migratory and resident DCs on the basis of the cell phenotype (8, 9) Since this thesis only refers to conventional DCs, the term DCs only covers this type of dendritic cells from here on. Immature migratory DCs (iDCs) are largely distributed in most peripheral, non-lymphhoid tissues, where they act as “sentinels” of their environment by constantly sampling foreign and endogenous antigens. They comprise two main subpopulations, CD103+CD11b− and CD103−CD11b+ DCs (10) and two other subsets that are exclusively located in the skin or associated subcutaneous LNs, namely epidermal Langerhans cells (LC) and interstitial or dermal DCs (intDCs) (11). Migratory DCs are highly specialized in the uptake of endogenous and pathogenic antigens. Upon antigen encounter, iDCs internalize antigens and shuttle them to local draining lymph nodes (dLNs) through the afferent lymphatics. Migration from peripheral tissue to sites of antigen presentation is directly associated with phenotypic and functional changes in response to different stimuli during activation and further development.

Resident DCs are phenotypically and functionally different from iDCs and reside within lymphoid organs such as spleen, LN and thymus - without ever entering peripheral tissue (10, 12). This DC subset encompasses a less mature phenotype but shows superior activity in T cell priming (13) and can be divided into two subsets on the basis of CD8+ expression, namely CD8+CD11b− and CD8−CD11b+ DCs (10).

94

CD8α+ DCs are described to be the most efficient DC subtype for capturing and processing antigens from the extracellular environment that are rerouted from the endosomal pathway into the cytosol for degradation by the proteasome back into the ER for MHC class I binding, a mechanism called cross- presentation (14). The peptide/MHC class I complexes are subsequently delivered to the plasma membrane for activation of CD8+ CTLs. Thus, cross-presenting DCs play tremendous roles in priming of naïve CD8+ T cells in response to tumor or viral antigens as well as to antigens derived from apoptotic cells, immune complexes or other pathogens (15–17). Beyond that, cross-presentation implicates the tolerance to self (18, 19). As well, CD8α- DCs and macrophages were published to display the ability to cross-present particulate antigens (15, 20, 21), whereas migratory DCs are rather inefficient in cross- presentation. Recently, a third mechanism for priming of CD8+ T cell responses has been proposed. Migratory DCs have the ability to “cross-dress” other DCs, which refers to the transfer of their MHC/peptide complex onto LN resident CD8+CD11c+ DCs (22). Dendritic cell maturation and migration In order to execute their miscellaneous functions, DCs need to undergo maturation from immature, antigen-capturing DCs to mature antigen presenting APCs. Only then, priming and activation of naïve T cells is possible, which culminates in T cell clonal expansion and activation of CD8+ or CD4+ T cell immune responses (23). Maturation can be induced by T cell-derived factors, pro-inflammatory cytokines secreted by other immune cells (tumor necrosis factor (TNF), Interleukin 1 (IL-1)) or chemokines, as well as immuncomplexes (IC) and especially via pathogen-derived factors (3, 24–26). Immature DCs have an exceptional capacity to internalize endogenous or microbial antigens via phagocytosis, macropinocytosis or receptor-mediated endocytosis by expression of DC-specific surface receptors such as C-type lectin receptors (e.g. the mannose receptor DEC-205), Fc or scavenger receptors (27, 28). An additional “danger signal” is needed to ensure maturation. These so-called DAMPs (damage-associated molecular patterns) or PAMPs (pathogen-associated molecular patterns) are released by damaged tissue or invading pathogens and include viral nucleotides or bacterial proteins such as LPS (lipopolysaccharides). All these ligands bind to NOD-like receptors (NLRs, such as RIG- 1), cytosolic receptors or membrane-associated lectins and particularly to pathogen-recognition receptors (PRR) called toll-like receptors (TLR) (29), which are highly expressed on iDCs (30). TLRs are innate type I transmembrane proteins, characterized by horseshoe-like arranged leucine-rich repeats in the extracellular domain, and are involved in recognition of molecular patterns. To date, 13 TLRs have been identified in mammals, of which 12 TLRs are expressed by mouse DCs (31–33) and are located either on the cell surface (TLR 1, 2, 4 and 6) or on endosomal membranes (TLR 3, 7, 8 and 9) (34). Upon ligand binding, TLRs generally trigger downstream signaling via their cytoplasmic TIR (Toll-IL-1 receptor) domain. The signaling is further mediated through the adapter protein myeloid differentiation primary-response protein 88 (MyD88), IL-1 receptor associated kinase (IRAK) and TNF receptor-associated factor 6 (TRAF6) and triggers subsequent activation of transcription factors such as nuclear factor κB (NFκB) and activator protein 1 (AP-1) leading to upregulation of type I interferons, proinflammatory cytokines, such as IL-1, IL-1β and TNFα, as well as additional genes, e.g. costimulatory molecules which results in DC maturation (25, 35–38). Noticeably, TLR3 signaling follows a MyD88 independent signaling pathway via the TIR-domain-containing adaptor protein inducing IFN- (TRIF) which finally results in activation of interferon regulatory factor (IRF3). Simultaneous to downregulation of phagocytic receptors as a first step in DC maturation, DCs alter their chemokine receptor (seven-transmembrane-domain-G-protein-coupled CC chemokine receptors (CCRs)) profile in order to respond to chemokines that regulate directed migration to LNs. While immature DCs express CCR1, CCR2, CCR5 and CCR6, which respond to macrophage inflammatory protein 1α (MIP-1α), MIP-1 and MIP-γα and define the location of DCs at defined sites in peripheral tissues (39, 40), DCs temporarily upregulate CC chemokine ligands (CCLs) CCL3, CCL4 and CCL5, 95 which recruit DCs to the site of inflammation. Additionally, recruitment to sites of antigen encounter is mediated via CCR1 and CCR5 on DCs which respond to MIP-1α expressed on activated T cells. At later stages of maturation, DCs acquire the “homing-receptor” CCR7 which enables directed migration of DCs from inflamed tissue to lymph nodes. Homing is facilitated by expression of the CCR7 ligands CCL19 (MIP-γ, ECL, Epstein-Barr virus-induced molecule-1 ligand chemokine) and CCL21 (SLC, secondary lymphoid organ chemokine) on the luminal side of high endothelial venules (HEV) and T cell rich zones (23, 39, 41). Furthermore, DC maturation is associated with upregulation of surface MHC class I and class II molecules, elevated expression of co-stimulatory molecules of the B7 superfamily and enhanced processing and presentation capacity (3, 42). The role of dendritic cells in T cell priming In general, endogenous antigens are restricted to MHC class I presentation via the ER pathway or may be presented on MHC class II after degradation by autophagy, while exogenous antigenic material is loaded on MHC class II molecules via the endocytic pathway in order to initiate T cell help. Efficient stimulation and activation of T cells which results in clonal expansion and differentiation into effector cells is tightly controlled by three signals in an intimate interaction between DCs and antigen-specific T cells (depicted in Figure 1). Upon maturation, DCs express high levels of surface MHC class I and MHC class II molecules which present processed peptides to naïve CD8+ or CD4+ T cells, respectively (signal 1). T cells directly recognize and specifically bind with high affinity to peptide presenting MHC complexes via their T cell receptor/CD3 complex, which is facilitated by several adhesion molecules such as integrins 1, β or ICAM-1 (intercellular adhesion molecule 1, CD54) (26, 43, 44). During the process of maturation, DCs further up-regulate their levels of co-stimulatory molecules such as CD80 (B7.1) and CD86 (B7.β), which constitutes “signal β” by facilitating interaction with TCRs in order to sustain T cell activation and amplify T cell responses (45). CD80/CD86 have either co-stimulatory or co-inhibitory specificity by binding to either the constantly expressed stimulatory receptor CD28 on T cells or the inhibitory receptor CTLA-4 (cytotoxic lymphocyte antigen-4, CD125), which is immediately upregulated upon T cell activation (46). As a result to CD28 engagement, the expression of IL-2 and IFN synergizes with TCR signaling to promote T cell activation and T cell survival. Additionally, activated DCs further up-regulate the constitutively expressed DC licensing factor CD40, which binds to the TNF-family transmembrane cytokine CD40 ligand (CD40L, CD154) expressed by T helper cells (47). CD40L-CD40 signaling (so called “licensing”) leads to further activation of DCs, displayed by enhanced production of cytokines such as IL-12 and the increased expression of CD80/CD86, MHC molecules, as well as expression of other costimulatory accessory molecules such as CD70, 4-1BB ligand (4-1BBL) and OX40 ligand (OX40L) (48). These factors all complement each other in mediating cell-to-cell contact to primary activated CD8+ T cells. Thus, licensing of DCs mediated via CD4+ T cells during the priming phase leads to additional stimulation of CD8+ T cell activation, proliferation and differentiation into effector CTLs. Moreover, the co-stimulatory context provides DC survival (49–51).

The intimate interplay of DCs and CD4+ or CD8+ T cells finally leads to differentiation of antigen- specific T cells into various effector T cells and thus shapes the type of T cell response. Activated CD4+ and CD8+ T cells further differentiate into either T helper cells or CTLs, respectively. This final step of T cell activation is accompanied by the increased capacity of DCs for pro-inflammatory cytokine secretion (signal 3) (52, 53). Based on the cytokine profile, that is elicited by DCs, CD4+ cells can be subdivided into distinct populations. Type 1 cytokines such as IL-12 promote the differentiation of CD4+ T cells into T helper 1 (Th1) cells. Th1 mediated immune responses are characterized by secretion of IFN, TNF, as well as IL-2 and play a crucial role in immunity to viruses, intracellular bacteria or parasites. On the other hand, type 2 cytokines which count for IL-4 and IL-10 mediate Th2 cell development which is associated with humoral immune responses by secreting IL-4 and IL-5. DCs 96 additionally influence the differentiation of Th17 and Tfh (follicular helper T cells), as well as the generation of regulatory T cells (Tregs). Tregs are mainly involved in controlling the immune system by inhibiting both CD8+ and CD4+ T cell responses and maintaining peripheral tolerance to self- peptides. DCs also stimulate innate immunity via activation of natural killer (NK) and natural killer T (NKT) cells (54, 55). In contrast to T lymphocytes, NKT cell activation is mediated by presentation of lipid antigens on the non-classical, invariant MHC molecules CD1d (56).

Figure 1. Molecular mechanism of T cell priming by cross-presenting dendritic cells. DCs present antigens to CD4+ T helper (Th) cells via MHC class II and cross-present it to CD8+ cytotoxic T cells (CTLs) through MHC class I molecules by recognizing the peptide/MHC complex via their respective TCR (signal 1). Licensed DCs up-regulate the expression of co- stimulatory molecules such as CD80 or CD86 (signal 2), which bind to CD28 on T lymphocytes, while simultaneously down- regulating inhibitory molecules like programmed cell death ligand (PD-L1) or Toll like receptor (TLR) ligands which further increases the cross-presenting activity. Activated CD4+ Th cells further stimulate CTLs through interleukin 2 (IL-2) secretion. Cross-priming through CD40 ligand (CD40L)-CD40 interactions with CD4+ T cells results in further licensing of DCs for cross-priming of CTLs. The secretion of IL-1β and type I IFNs (only in response to activation of LTR (lymphotoxin beta receptor) signaling) additionally stimulates and activate CTLs or T helper cells (signal 3, not shown). Cross-primed CTLs are programmed for survival and cease TNF-related apoptosis-inducing ligand (TRAIL) production. 'Helpless' CTLs activated by unlicensed DCs die following secondary encounter with antigen in their effector phase (not shown) (57).

Modern approaches in immunotherapy The immune system possesses the unique ability to defend the host against infection or tumor development. Thus, modulation of immune responses to activate the host’s own immune system to fight against numerous diseases is the basis of immunotherapy. To date, promising approaches in immunotherapy of cancer, chronic infectious diseases or autoimmune disorders have been developed. Vaccination is one of the oldest and most successful methods in immunotherapy as shown by global eradication of diseases such as smallpox or poliomyelitis (58). Whereas the prevention of infections is mediated by humoral immune response which relies on neutralizing antibodies produced by B cells, the classical vaccination strategy has limitations in fighting against chronic intracellular infections or cancer. In contrast to passive immunotherapy which aims at delivering neutralizing antibodies, active immunotherapy is supposed to induce cell mediated immunity by simultaneous activation of APCs, CD4+ and CD8+ T cells, as well as B cells and innate immune cells, for instance NK cells. Especially, immunotherapeutic antitumor vaccines aim at activation of antigen-specific potent CTL responses together with a supporting immune environment and CD4+ T cell help. The ultimate goal is the recognition and destruction of tumor cells by specific effector CD8+ T cells against the tumor antigen. In fact, effective antitumor therapy is still a great challenge today. Most tumor immunotherapies fail at breaking underlying tolerance mechanisms, since tumor cells derive from self-tissue and are not recognized as foreign. Tumors themselves can escape immunosurveillance (59, 60) by downregulation 97 of HLA class I and II or co-stimulatory molecules, as well as through defective antigen processing and presentation machinery. Direct inhibition of antitumor activity is achieved by production of immunosuppressive cytokines (e.g. IL-10, transforming growth factor (TGF-) or vascular endothelial growth factor (VEGF)) within the tumor microenvironment. Various approaches in immunotherapy have been accomplished during the last years. Recombinant viral vectors encoding tumor antigens are potentially ideal vectors for the delivery of tumor associated antigens, due to their ability to infect and activate APCs (61, 62). The main disadvantage relies on the large amount of viral sequences inside the vectors leading to antigenic competition of viral antigens with the incorporated CD4+ and CD8+ epitopes (63–66). The viral vector may also contain intrinsic genes capable of suppressing host immune response by antibody response to viral envelope proteins (67). T cell mediated antitumor immunity is by far the most promising approach. The FDA approved adoptive T-cell therapy (ACT) was shown to be successful in treatment of many types of cancer such as melanoma or lymphoma (68–70). This whole cell polyvalent vaccine utilizes ex vivo derived autologous tumor cells which contain a large repertoire of TAAs in order to induce immune responses against a broad spectrum of multiple epitopes including those that are unique to the patient’s tumor. The ex vivo expansion has the possibility to add cytokines (e.g. GM-CSF or IL-2) as adjuvants. This concept was further improved by using tumor-infiltrating lymphocytes (TILs) which exhibit high functional avidity (71). Major drawbacks were seen in suboptimal antigen presenting capacity of tumor cells, uncertainty in antigen processing or simple lack of autologous tumor samples (72, 73). Additionally, these regimens were extremely cost- and labor-intensive and lack broad applicability. Allogeneic tumor cell vaccines could overcome the drawback of being limited to the patient’s haplotype, as they are independent of HLA (human leukocyte antigen) restriction. For instance, Melacine® which consists of an allogeneic melanoma tumor cell line was administered in combination with the water-in-oil emulsion DETOX®, that comprises bacterial components as immunostimulatory adjuvants. This vaccine showed relapse free survival in melanoma patients (74, 75). However, clinical phase I and II trials revealed inconsistency in therapeutic success of using Melacine® (75). Mentionable, there have been promising immunotherapeutic approaches using allogeneic tumor lysates, which were first conducted in 1988 by Mitchel and colleagues (73, 76). This led to the development of GVAX®, a vaccine for prostate cancer composed of two allogeneic prostate cancer cells lines transfected with GM-CSF. Another approach to make ACT available for different types of cancer is the use of T cells that are transduced with retro- or lenti-viruses encoding HLA-A*0201 restricted, high affinity TCRs specific for e.g. melanoma antigens MART-1 or gp100 (77) or cancer testis antigen, such as NY-ESO-1 (78). Nevertheless, there is a need for the development of new vaccines with the ability to trigger powerful cellular immune responses that are also broadly applicable and less expensive or labor-intensive. Dendritic cell – mediated immunotherapy Due to the unique ability of DCs to prime and activate naïve T cells, DC-based immunotherapy has become a promising cellular vaccine in modern immunotherapy research. Immunotherapy that requires the induction of efficient CD8+ T cell responses is initiated by antigen delivery to APCs, predominantly DCs. Thus, the major strategy of immunotherapeutic approaches is the delivery of exogenous antigens to DCs in order to cross-prime CTLs. First approaches have been achieved by utilizing ex vivo derived immature DCs loaded with synthetic MHC class I-restricted tumor-derived proteins or peptides to trigger antigen-specific T cell responses (79). In one of the earliest trials for treatment of melanoma patients, Mukherji et al. (80) induced antigen- specific T cell responses after immunization with MHC class I-restricted MAGE-1 peptide-pulsed autologous DCs. The promising strategy was characterized by its safety record but clinical responses remained inefficient leading mainly to weak and transient T cell responses. External pulsing of DCs with peptides has several limitations such as peptide degradation, rapid turnover of peptide/MHC 98 complexes or dissociation of peptide from MHC during DC preparation/injection (71). This was likely to be attributed to the fact that only a limited number of peptides with few if any T helper peptides were used (81–84). Furthermore, the re-administered DCs displayed poor migratory capacity, limiting the amount of antigen presented to T lymphocytes in local dLN (85). With respect to limitations of peptide-pulsed DC vaccines, putative advance came across with the use of tumor antigens or tumor lysate-pulsed DCs. Although enhanced T cell responses and partial remission of tumor load was observed, the downside of this approach was the lack of adequate specificity. Clinical use is still limited as ex vivo DC generation is labor-intensive and expensive and lacks universal applicability, because in vitro manipulation of DCs is patient-specific and only some DC subsets can be utilized (79, 86, 87). Nevertheless, the development of DC-based therapeutic vaccination has led to recently approved treatment of hormone-resistant prostate cancer with Sipuleucel® (88) utilizing ex vivo loading of mature autologous DCs with prostate cancer-associated antigens (PAP-GM-CSF fusion protein) which are subsequently re-administered into patients (89). In order to circumvent the limitations associated with in vitro manipulation of DCs, direct in vivo targeting of DCs along with appropriate adjuvants for simultaneous activation of DCs has gained major focus. T cell activation in the absence of costimulatory molecules or pro-inflammatory cytokines will result in an induction of T cell tolerance (90). Thus, targeting solely antigens to DCs will result in tolerance induction. A well-established vaccination strategy uses a combination of antigen and immunostimulatory adjuvants. The most common vehicle and adjuvant which has been introduced for vaccination trials over 60 years ago is the water-in-oil emulsion incomplete Freund’s adjuvants IFA (91), commonly used as Montanide ISA-51 in clinical trials, which represents a similar formulation to IFA (92). Besides complete Freund’s adjuvant (CFA), which already contains several immunostimulatory factors, IFA is commonly administered in DC-based immunotherapy (93). Although IFA is primarily known to induce Th2-biased responses and stimulates long-term IgG production, it can also stimulate CTL or Th1 immunity directed against the antigen that is emulsified in IFA (94). Nevertheless, IFA is not approved for routine immunotherapy, due to emerging adverse effects such as local skin reactions, abscesses, inflammation or granulomas at the injection site (95). Besides the Th2-polarizing adjuvant alum, MF-59 (another IFA formulation, which is an emulsified squalene together with Tween and Span85) are the only two carrier system approved for clinical use in humans (96). The adjuvant effect of IFA relies on the formation of an antigen depot from which the antigen is slowly released (97). Although providing long-term antigen presentation, most of its formulations show poor therapeutic efficacy. As already outlined, DCs need several stimuli to experience activation and maturation and eventually prime naïve T cells. One of the most commonly used immunostimulatory adjuvants to strongly activate DCs and promote desired Th1 responses are TLR ligands. As most of the TLR ligands are fully characterized by their molecular pattern, they can be easily synthesized and included in vaccine formulation. Many different TLR ligands such as oligonucleotides, single-stranded RNA (ssRNA), flagellin or lipids, like Pam3CSK (98) have been tested in various vaccine studies (99). Enhancing vaccine efficiency is accomplished via multiple mechanisms. At first, TLR stimulation results in an increase of peptide/MHC complexes on the surface of DCs. Together with upregulation of co-stimulatory molecules and secretion of pro-inflammatory cytokines these are the three signals required for T cell activation (100). The choice of TLR ligand in a vaccine critically determines the outcome of the underlying immune response. For instance, a Th2-skewed polarization is rather unwanted in the induction of an anti-tumor response. We use two prominent examples of synthetic TLR ligands, which are successfully utilized in research: Oligodeoxynucleotides (ODN) containing cytosine- phosphorothioate-guanine (CpG) motifs and polyriboinosinic:polyribocitidylic acid (polyI:C) (101). CpG-ODNs mimic the stimulatory effect of double-stranded bacterial DNA and are strong ligands for TLR9 within phagosomes of dendritic cells (102). Upon ligand binding, TLR9 signaling promotes DC activation and maturation via direct induction of IL-12 secretion and by triggering the upregulation of

99 co-stimulatory molecules (103). The potency of CpG-ODNs to induce maturation and activation of DCs and subsequently enhance CTL responses is even more pronounced if administered in combination with other TLR ligands or together with an antigen (104, 105). For example, Heit et al. (106) showed the immunostimulatory capacity of inducing antigen-specific responses by CpG-conjugated OVA. Despite the use of highly immunogenic model antigens, CpG together with the melanoma associated Melan- A/MART-1 self- peptide emulsified in IFA enhanced the Melan-A/MART-1 specific CTL response, as well (94). In particular, CpG ODNs were also described to promote cross-presentation (107) and are known to polarize CD4+ T cell differentiation towards Th1 (108, 109). PolyI:C is a double-stranded RNA-like molecule and a ligand for its cognate receptor TLR3. As with CpG ODNs, TLR3 triggering by polyI:C also promotes cross-priming by DCs and increases immune responses when co-administered with antigens (110). Additionally, it has been reported that co-administration of TLR3 and TLR9 ligands and vaccine enhances cross presentation of the internalized exogenous antigen. Thus, CpG ODNs and polyI:C are promising adjuvants for DC mediated CTL responses, since they promote DC activation and maturation via upregulation of co-stimulatory molecules and thus, enhance their capacity in eliciting a substantial T-cell response. Besides the advantages of DC mediated vaccines, in vivo targeting of DCs needs several maturation and activation stimuli to sufficiently prime T cell responses. In the absence of maturation or activation stimuli, antigen loaded DCs remain in an immature, “resting” state and become tolerogenic (50). In contrast, triggering DC maturation and/or activation by “danger” signals enhances the underlying adaptive immune response. It has been demonstrated that simultaneous administration of adjuvants, such as TLR agonists, in close proximity or physically associated with antigen is required to efficiently activate DCs in vivo (111, 112). This in turn emphasizes the necessity of a carrier device that is not only efficiently internalized by DCs, but also able to carry a mixture of adjuvants and antigens. PLGA microspheres as vaccine delivery system Considering antitumor therapy in particular, the delivery of exogenous antigen for cross-presentation to CD8+ T cells has become a main strategy of modern T cell vaccine design. To overcome the limited cross-presentation of soluble antigens by DCs, research is concentrated on finding suitable antigen delivery devices. A promising candidate for such antigen delivery systems are biodegradable poly (D, L)-lactide-co-glycolide acid microspheres (PLGA MS), which encompass eminent immunoadjuvant properties. The copolymer PLGA has many advantages over other polymers and is pharmaceutically accepted with excellent safety records (113). Thus, PLGA MS are already approved for use in humans by the US Food and Drug Administration (FDA) (114). Almost any stable adjuvant or antigen including peptides, proteins, RNA or DNA can be microencapsulated into PLGA MS by several different procedures (115–117). PLGA MS are produced by the pharmaceutically well applicable spray-drying process, which produces spherical particles of 1-10 µm in diameter (116, 118, 119), thereby exhibiting ideal properties for phagocytic uptake by APCs, mainly DCs and macrophages (120, 121). Encapsulated antigens and adjuvants are rapidly and efficiently taken up by human monocyte-derived DCs (moDCs), murine immature bone-marrow derived DCs (BMDCs), as well as macrophages in vitro and in vivo (122, 123) and the entrapped content is transported from peripheral tissue to the site of antigen- presentation in SLOs (124). Uptake by human moDCs in vitro does not negatively influence biological properties, such as survival, migratory capacity to SLOs, cytokine secretion or antigen presentation in these cells or subsequent T cell stimulation (122, 125). Inside a cell, the aliphatic polyester PLGA slowly hydrolyzes in the endosomal compartment over a period of about 30 - 60 days (118). The polymer is thereby degraded into its monomeric components lactic and glycolic acid, two natural metabolites of the citric acid cycle. The controlled release of encapsulated substances over time generates a depot effect at the site of injection and serves for prolonged antigen presentation and effective T cell stimulation which circumvents the need for booster vaccination (116, 126, 127). There are several additional advantages for the use of PLGA MS in a vaccine design. Encapsulated material is protected from degradation by proteases, RNAses or DNAses (128). Upon uptake of PLGA MS, epitopes of microencapsulated 100 exogenous antigens are efficiently presented on MHC class I to CD8+ T cells via cross-presentation as well as on MHC class II for CD4+ T cell help, thereby stimulating both cellular and humoral immune responses (129, 130). Hydrolysis of PLGA MS in endolysosomes leads to processing of encapsulated proteins by lysosomal proteases (41, 131). As a result, those peptides presented on MHC class II molecules are recognized by CD4+ T cells. There are three proposed pathways for cross-presentation, namely the TAP- and proteasome dependent “cytosolic” pathway, the processing of antigenic peptides using endolysosmal proteases in the “vacuolar” pathway and lastly, some extracellular proteins bypass the endocytic system and directly enter the proteasome (Figure 2). Peptides generated via this latter mechanism could presumably associate with MHC class I via the cytosolic route (132, 133).

Figure 2. Intracellular pathways for cross-presentation. Left. The phagosome-to-cytosol pathway with ER loading. After phagocytosis, exogenous antigens are exported from the phagosome into the cytosol through the Sec61 translocon. Following, the classical MHC class I processing pathway is initiated by proteasomal degradation, TAP-mediated entry into the ER and MHC class I loading to the plasma membrane (PM) via the Golgi. The ER-Phagosome fusion pathway with phagosomal loading. Phagosomes fuse with components of the ER including TAP and pMHC class I. Antigens are then translocated back to the cytosol for proteasome-dependent processing, followed by re-entry into the ER-phagosome, loading of MHC class I and transport of pMHC class I complexes to the plasma membrane. Right. The vacuolar pathway. Exogenous antigens can be directly processed into peptides in the early phagosome via cathepsins and are then loaded onto MHC class I molecules that are already present in the phagosome and subsequently transported to the PM. TAP, transporter associated with antigen processing. Adopted from (134). It has been reported that PLGA nanoparticles (NP) could escape from endo- into the cytosol facilitated by reversion of the anionic surface charge in the acidic lysosomal compartment which leads to interaction with the endo-lysosomal membrane (128). Since presentation of protein and peptide antigens from PLGA MS is dependent on the proteasome and on TAP activity and is sensitive to brefeldin A, the “cytosolic escape” pathway is most likely involved in cross-presentation for PLGA encapsulated antigens (135, 136) compared to ER-phagosome fusion and endosomal class I loading (132, 137, 138). Thus, the excellent efficiency for cross-presentation by PLGA MS mediated delivery of co-encapsulated antigen and adjuvant by DCs (116, 126) is favored by the particulate formulation of PLGA MS (139) and is likely to be attributed to the release of antigenic cargo into the cytoplasmic compartment after escape of PLGA MS from the phagosome (123, 140, 141). Simultaneously, cytosolic escape protects the antigenic content from lysosomal degradation (128). In addition to that, the maturation of phagosomes associated with TLR triggering may accelerate release of antigen into the 101 cytosolic compartment and may therefore also protect antigen from lysosomal degradation and premature release (142). Another important advantage comes along with recent studies, which declare that PLGA MS are also capable to rescue impaired DCs from tumor induced immunosuppression (143). Moreover, PLGA has the advantage of great flexibility with respect to possible manipulation of physiochemical properties of the polymer. Thus, degradation kinetics and hydrophobicity can be determined by changing monomer ratio, molecular mass or end-group chemistry (144). Our group and others showed that co-encapsulation of antigen (ovalbumin) together with the TLR9 ligand CpG ODN as adjuvant into PLGA MS yields in more potent CTL responses than a mixture of MS loaded separately with either antigen or adjuvant (111). The co-delivery of antigen and adjuvant to DCs therefore is required to enhance vaccine efficiency, which manifests in prolonged presentation of antigen derived epitopes and superior anti-tumor responses in mice (111, 112, 145). PLGA MS mediated antitumor immunotherapy of various syngeneic tumor models in mice is favorably compared to IFA (146), even by a single administration of co-encapsulated OVA/CpG. Improvement of the PLGA MS system by adding a second, separately encapsulated, TLR ligand was shown to positively influence T cell – mediated immune responses by targeted DCs (111, 146). This suggests that optimal DC activation depends on synergistic triggering of several TLR ligands (101). Additionally, elimination of large tumor masses was achieved with PLGA MS co-encapsulating tumor lysates and TLR ligands. We could show, that PLGA MS co-encapsulated with TRAMP-prostate derived tumor lysate and CpG ODN in combination with PLGA MS containing polyI:C shows detectable ex vivo cytotoxic T lymphocyte responses and antitumor activity in tumor bearing mice (147). The antitumor efficacy of tumor lysate co-encapsulated with CpG ODNs in PLGA MS was also shown by Goforth et al. in a mouse model for melanoma (148). In relation to a potential use of PLGA MS in clinical application, sterilization of PLGA-MS by -irradiation does not negatively affect T cell responses (147). Recently, we could further expand the efficacy of the PLGA MS system by peptide specific vaccination. Encapsulation of poorly immunogenic peptides of prostate-specific antigens was able to induce potent CD8+ T cell activation (149). Moreover, immunization with PLGA MS encapsulated Influenza virus matrix M1 peptide could induce potent T cell-mediated anti-viral immune responses in influenza infection (150). Besides micro/nanoparticle polymers, various other particulate systems have been employed such as liposomes, archaeosomes, immune-stimulating complexes (ISCOMs) and virus like particles (VLP) (151), but should be of no further interest in this thesis. Antigen processing and presentation CD8+ T lymphocytes recognize antigens as short peptide fragments composed of 8-11 amino acids in length which are bound to MHC class I molecules via non covalent interactions on the surface of APCs, notably DCs. In contrast to MHC class I, which is expressed on every nucleated cell, MHC class II is expressed more selectively on immune cells that are involved in antigen presentation and stimulation of T cell responses. Besides induction of immune responses, T cells that recognize MHC class I in complex with a self-protein (e.g. derived from host proteins) with high avidity are deleted in negative T-cell selection in the thymus, whereas about 1-2% of all T-cells survive this procedure and are released into the periphery as auto-reactive T cells. The recognition of the complex of peptide antigens bound to an MHC molecule by the TCR of CTLs is selective and extremely sensitive, as the length of the peptide is restricted by the closed ends of the peptide binding groove (152), limiting binding of peptides of eight to eleven amino acids in length (153). These peptides contain specific anchor amino acids, which ensure exact binding into the MHC class I pocket. The ends of the MHC class I peptide binding groove interact with free N- and C- termini of peptides. Even a change in a single amino acid can turn a predominantly agonistic epitope into a non-stimulating peptide (154). However, an alteration in the sequence of a self- peptide into “non-self” may initiate responses against normal tissue resulting in autoimmunity (155). Due to interactions of the peptide backbone or various amino acid side chains with the MHC binding groove peptides are stabilized. Even more, with the help of conserved amino acids docking into the cleft 102 of MHC molecules, peptides are ideally positioned and support stable interaction with the help of these so-called anchor residues. Primary anchor residues at position two and at the C-terminus, as well as secondary anchor residues are required for efficient binding into the peptide binding groove (156). The peptides recognized by CD4+ helper T cells presented in association with MHC class II complexes are apparently not restrictive to a defined peptide length, as N- and C-termini can extend from both sides of the peptide binding groove. Owing to proteases which process the peptide’s end hanging out of the pocket, the size is usually limited to 10-12 amino acids.

Within a cell, endogenous nuclear and cytosolic proteins are constantly degraded by the ubiquitin- proteasome system (UPS) or other cellular proteases. In addition, newly synthesized misfolded proteins and peptides that are prematurely terminated, so called DRiPs (defective ribosomal products), are immediately subjected to proteasomal degradation (157). These intermediate peptides resulting from different digestion processes are fragments of about 3–22 amino acids in length. The majority of these peptides are rapidly processed by cytosolic proteolytic aminopeptidases (158). The small fraction of remaining peptide fragments are translocated across the ER membrane via the peptide transporter associated with antigen processing (TAP) to get access to ER-resident empty MHC class I complexes. TAP transporters do not cargo peptides smaller than 8 amino acids to ensure fitting into MHC class I peptide binding groove. Longer peptides are further processed in the ER lumen by the IFN- inducible aminopeptidases ERAP1 and ERAP2, which therefore yield optimal properties for binding onto MHC class I complexes (159, 160). Ideal peptides now can associate as third essential subunit with MHC class I complexes which comprise of a heavy and a light chain (βm). Proper folding is supported by the macromolecular peptide-loading complex, including TAP, and the chaperone proteins tapasin, calreticulin and ERp57. The fully assembled MHC class I complex with the loaded peptide is released from its chaperons and transported to the cell surface for presentation to CTLs via the secretory pathway using the Golgi-complex (161). Peptides and MHC class I complexes that fail binding are transported back to the cytosol and ultimately degraded by ER-associated protein degradation (ERAD) (162) (Figure 3, top). MHC class II molecules bind peptides, which stem from exogenous proteins that entered the cell via endocytosis, macropinocytosis or receptor-mediated uptake (58). In the ER, two transmembrane proteins, MHC class II α- and -chain, assemble to a molecule similar to the structure of MHC class I. Instead of binding a peptide, the MHC class II complex binds a specialized type II transmembrane chaperone protein: the invariant chain (Ii), which temporary fills the peptide binding groove. The resulting stable complex is released from the ER into a late endosome, called the MHC class II compartment (MIIC) (163). Within this compartment, Ii is cleaved by various proteases, notably the cathepsin family, leaving a short peptide fragment, the so called MHC class II associated invariant chain peptide (CLIP). CLIP remains in its high affinity MHC class II binding pocket due to its inaccessibility to proteases. The MIIC then fuses with late endosome which carries a reservoir of exogenous proteins and antigenic peptides that are fueled from the endosomal pathway and are also degraded by cathepsins and other late endosomal proteases and peptidases. The chaperone H2-M (HLA-DM in humans) which generally stabilizes MHC class II molecules now alters MHC class II structure and enables the exchange of the CLIP fragment for high-affinity binding peptides (164). Subsequently, peptide/MHC class II complexes are transported to the cell surface (Figure 3, bottom).

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Figure 3. Basic pathways of antigen processing and presentation. Top. Illustration of MHC class I biosynthesis and antigenic peptide binding. Cytosolic proteins are processed by the proteasome. The resulting peptides are TAP-dependent transported into the ER and subsequently assembly with MHC-I molecules. Bottom. Illustration of MHC class II biosynthesis and antigenic peptide binding. Exogenous antigens can also be fed into MHC class I pathway by cross-presentation but primarily loaded onto MHC class II molecules in mature or late endosomal compartment. Cytoplasmic antigens can also be trafficked into the endosomal network via autophagy. For further details refer to the text. Images taken from (165). 104

Molecular mechanisms of autophagy Besides the Ubiquitin-Proteasome system (UPS), autophagy (AP) represents the second major intracellular degradation pathway. It is a highly dynamic and genetically-conserved homeostatic degradation process by employing lysosomal hydrolysis and plays essential roles in cellular turnover of multiple cytoplasmic constituents such as protein aggregates and even entire organelles (166–168). In contrast to the proteasome dependent degradation which favors short-lived protein substrates, autophagy is responsible for the degradation of long-lived, aggregated or defective proteins and maintains multiple aspects of steady-state cellular homeostasis, while enabling adaptation to stressful conditions.

Metabolic stress such as hypoxia, oxidative stress, ER-stress or nutrient and growth factor deprivation causes upregulation of AP in order to recycle intracellular components to replenish metabolic needs including amino acids, nucleotides, fatty acids, sugars and ATP, thereby creating nutrient rich conditions (167, 169, 170). As well, AP also participates in protein quality control by eliminating protein aggregates, toxic waste, redundant or non-functional proteins and damaged organelles thereby preventing excessive ROS levels, suppressing DNA damage and by maintaining genomic stability (171). AP can be distinguished by three different processes, which are macroautophagy (hereafter referred to as autophagy), microautophagy and chaperon-mediated autophagy (168). Of that, macroautophagy seems to be responsible for degradation of the majority of intracellular proteins in mammalian cells. AP can also act in a tissue-dependent and complex selective manner by targeting specific cellular components including mitochondria (mitophagy), peroxisomes (pexophagy), ER (reticulophagy) or ribosomes (ribophagy) (172–175). AP is also involved in targeting intracellular pathogens for autophagosomal sequestration and subsequent lysosomal degradation (xenophagy) (176–178). The role of AP in tumorigenesis is somewhat controversial. It may act as a tumor suppression mechanism by removing damaged proteins thus limiting cell growth and genetic instability, while cancer cells may utilize autophagy to maintain cancer cell survival under stress conditions (179). Meanwhile, chemical inhibitors of autophagy (i.e. chloroquine) have been used as co-treatment with chemotherapeutic drugs, which was shown to increase tumor cell killing (180).

Regulated by a number of autophagy-related genes (Atg), cytosolic cargo is sequestered into a double membrane vesicle termed autophagosome. The autophagosome then fuses with the late endosomes or lysosome to generate the now single membrane autolysosome with consequent breakdown of cellular content together with its membrane (181). Formation of the autophagosome is a highly conserved process which involves a series of distinct events of initiation, nucleation, elongation, lysosomal fusion and degradation or recycling of the cargo. Several intracellular signals participate in the initiation of AP. ER-mitochondrial contact sites have been suggested to act as potential starting sites of autophagosome formation with additional contributions from endosomes, the plasma membrane or the Golgi. However, the origin of the autophagosomal membrane (phagophore) is still undefined, but most likely stemming from organelles such as mitochondria, the ER, the Golgi complex or endosomes. Also, de novo synthesis of the membrane could be plausible (182, 183).

During the pre-initiation step, two protein complexes (ULK and Beclin-1/class III PI3K) are formed (184). The ULK complex consists of ULK1/2 (uncoordinated-51-like kinase 1, 2), Atg13, Atg101 and FIP200 (focal adhesion kinase FAK family interacting protein of 200kDa) and is associated with pre- autophagosomal structures (PAS) (185). This complex further induces assembly of the second nucleation complex constituting of Atg6/Beclin-1 (BCL-2 interacting protein), ATG14L, the PI3K VPS34 and VPS15 and AMBRA1 (activating molecule in Beclin-1 regulated autophagy protein 1). The second complex generates a pool of PI3P (phosphatidyl-inositol-3-phosphate) on the isolation membrane in order to promote nucleation of the autophagosomal membrane. Formation of this membrane further recruits additional Atgs and PI3P effector proteins. Moreover, Atg9, WIPIs (WD-

105 repeat protein interacting with phosphoinositides) and VMP1 (vacuole membrane protein 1) have been shown to participate in membrane initiation (171).

Elongation, maturation and closure of the autophagosome membrane is orchestrated by two ubiquitin- like conjugation system. At first, Atg12 is coupled to Atg5 via E1-like ubiquitin activating enzyme Atg7 and the E2 ubiquitin-conjugating enzyme. After that the Atg16L1/L2 homodimer is recruited to stabilize this conjugate, thereby forming the trimeric complex Atg5-Atg12/Atg16L1 which facilitates membrane elongation. The second step is Atg5-Atg12/Atg16L1-mediated conjugation of Atg8/LC3 (microtubule associated protein 1A/1B light chain 3) to phosphotidylethanolamine (PE) lipid at the outer and inner surface of the isolation membrane. The second ubiquitin-like conjugating system is comprised of LC3, the cysteine protease Atg4, Atg7 and Atg3. Atg4 cleaves off five amino acids (MKLSV) at the C- terminal region of proLC3 to generate an accessible C-terminal glycine residue and to form the cytosolic form LC3-I for binding to PE of the autophagosomal vesicle. LC3-I is activated by the E1-like enzyme Atg7 and is transferred to PE by the E2-like Atg3. Eventually, LC3-I dissociates from Atg3 and covalently associates to the amino-group of PE via the formation of an amide bond (LC3-II, PE- conjugated LC3, ubiquitin like, membrane bound). After completion of the mature autophagosome, LC3 at the outer membrane is released via cleavage by Atg4 while LC3 bound to the inner membrane stays attached until complete degradation by the lysosome. These two systems are interconnected to each other. For instance, the Atg12-Atg5-Atg16 complex acts as an E3 ligase during LC3 lipidation, while LC3 conjugation is indispensable for the mentioned complex and responsible for the fusion of membrane vesicles. Furthermore, LC3 was also reported as a receptor or adaptor protein for selective cargo incorporation (184, 186, 187). Mature autophagosomes travel along microtubules and finally fuse with either late endocytic compartments such as multivesicular bodies (MVBs) to form so-called amphisomes or with lysosomes, thereby generating single-membraned autolysosome. Eventually, cellular material, the inner membrane and associated LC3-II is rapidly degraded and recycled via lysosomal proteases in the “autophagic flux” (188). LC3-II on the outer membrane is lastly cleaved by Atg4 and restored to LC3-I for repeated autophagosome formation. The enzymes involved in this process are the small GTP- binding protein Rab7, as well as lysosomal associated membrane proteins LAMP1 and 2 (181).

Figure 4. Key steps of autophagosome formation. Left. Upon deprivation of nutrients or growth factors, activation of AMPK and/or inhibition of mTOR leads to nucleation of an isolation membrane (phagophore) at preautophagosomal structures (PAS) which matures to an autophagosome. Cellular cargo is entrapped into a fully formed autophagosome prior to fusion with the endocytic compartment (amphisome) or with lysosomes (autolysome) for substrate degradation and recycling of nutrients and metabolites. Right. Autophagasome formation is initiated by assembling of the ULK complex at pre-autophagosomal structures (PAS), which phosphorylates Beclin-1, leading to VPS34 activation and phagophore formation. ULK functions in a complex with FIP200 and ATG13, whereas VPS34 n requires the regulatory subunit VPS15 (p150) and Beclin-1. The latter further mediates the association of other regulatory factors such as AMBRA, ATG14, UVRAG, and BIF-1. Two ubiquitin-like conjugation systems ATG16L1-ATG5-ATG12 and LC3-II which catalyze further autophagosome formation via the E1-like (autophagy gene 7 (ATG7)), E2-like (ATG3 and ATG10) and finally E3-like molecules (ATG5–ATG12–ATG16 complex). This allows proper incorporation of phosphatidylethanolamine (PE)-conjugated Atg8 LC3 (LC3-II) into the phagophore membrane. There, LC3 serves as a docking site for adaptor proteins and bound cargos. Images combined of (189, 190). 106

In general, autophagy is responsible for random uptake of cytosolic constituents. In contrast to that, selective autophagy requires the assistance of several autophagic adapter proteins which simultaneously interact with LC3 on nascent autophagosomes and the target substrate. Selective substrate recognition and directed targeting of ubiquitinated proteins or organelles to autophagosomes is facilitated by autophagy cargo receptors. These adapter proteins are characterized by a conserved LC3-interacting domain (LIR) and other specific domains, such as the UBA (ubiquitin-binding association) domain. Examples for adapter proteins include the ubiquitin binding protein p62/SQSTM1 (sequestosome 1 for polyubiquitinated substrates), the pro-apoptotic factor NIX (used by mitochondria), NBR1 (neighbor of BRCA1 gene 1), as well as NDP52 (nuclear dot protein 52) for sequestering invading pathogens and BNIP3/BNIP3L that are important for mitophagy (191, 192).

AP is furthermore regulated via multiple mechanisms throughout the autophagic pathway. The cellular energy sensor AMPK (AMP activated protein kinase) is involved in AP induction, for example via starvation-mediated dissociation of mTORC1 (mammalian target of rapamycin multi protein complex 1). On the other hand, mTORC1 up-regulates nutrient-consuming anabolic cellular mechanisms and thus suppresses autophagy via inhibition of the ULK complex (193–195). In addition, pattern- recognition receptors such as TLRs, as well as or necrotic cell products or ROS stimulate MAPK (mitogen-activated protein kinase) and NFκB ultimately initiating autophagy (196). Furthermore, ROS signaling and its underlying downstream effects such as mitochondrial damage, DNA damage and p53 activation further increase protein degradation via the autophagy pathway. Finally, hypoxia (197), oxidative stress (198) or ER stress that is accompanied by accumulation of unfolded protein aggregates may also cause autophagy induction via HIF-1α (hypoxia inducible factor 1 alpha), AMPK (Adenosine monophosphate-activated protein kinase) or the unfolded protein response (UPR) (199). Further regulation includes post-translation, transcriptional or epigenetic regulation as wells as the action of miRNAs (micro RNAs). Noticeably, Beclin-1 associates with several cellular intracellular mediators, for example the anti-apoptotic proteins Bcl-2 (B cell lymphoma 2) and Bcl-XL (B cell lymphoma, extra- large). This interaction was shown to lead to autophagy inhibition (200, 201). Interestingly, AP has established as an important regulator of tumorigenesis, possessing both tumor suppressive and pro- tumorigenic activity (202). The role of autophagy in immunity and antigen presentation Besides its appreciated importance for cell survival, intracellular trafficking and apoptosis, the importance of autophagy has been characterized as essential at the interface of innate and adaptive immunity (168, 203, 204). The role of autophagy in immunity is not only implicated in sensing and elimination of intracellular pathogens or the restriction of viral infections, but also takes part in antigen presentation. It has been shown that autophagosomes efficiently fuse with a subset of the endocytic vesicles, called MHC class II loading compartments. Thus, AP facilitates the degradation of endogenous, cytoplasmic antigens which results in increased antigen processing for MHC class II presentation to CD4+ T cells (203). Some of these antigens also include tumor antigens. For instance, the presentation of the tumor associated, MHC II-restricted peptide Mucin gene 1 was inhibited by using pharmacological inhibitors of class III PI3 kinase, 3-Methyladenine (3-MA) and wortmannin (WM) (205, 206). In addition, MHC class II presentation of peptides derived from the complement associated protein C5 was impaired in mouse macrophages and B cells after 3-MA and WM treatment (207). The Paludan group demonstrated that class II presentation of EBVNA (nuclear antigen 1 of EBV) was reduced after siRNA (short interfering RNA)-mediated knock-down of Atg12 and could show that influenza matrix protein M1 presentation on MHC class II to M1-specific CD4+ T cells was enhanced upon fusion of M1 to LC3 (208). Other reports show that MHC class II presentation of viral and bacterial antigens was perturbed in APCs deficient in macroautophagy (165, 209, 210). Noticeably, pro- inflammatory cytokines and TLR activation also leads to upregulation of autophagy-mediated antigen 107 processing to mediate MHC II presentation in both professional and nonprofessional APCs as shown by enhanced delivery of phagocytosed material to lysosomes and subsequent loading onto MHC class II molecules by autophagy after TLR2 stimulation of macrophages (211). Furthermore, priming CD4+ T cell responses after HSV infection and processing of extracellular ovalbumin was perturbed in Atg5-deficient DCs (212). Another study by Blanchet et. al. (213) could show that AP induction leads to enhancement of HIV antigen presentation on MHC class II, whereas siRNA-mediated silencing of Atg5 and Atg8 decreased this response.

While the impact of macroautophagy on MHC class II presentation is clearly defined, the role of autophagy in antigen processing and presentation on MHC class I is not well characterized to date. Given that the classical MHC class I antigen presentation pathway does not intersect with the autophagosomal route, a prominent role of autophagy in participating in the endogenous MHC class I pathway is still controversial. Several reports indicate that for most antigens autophagy plays either little or even no role for MHC class I presentation. For example, siRNA-mediated knockdown of Atg12 in an EBV (Epstein-Barr virus)-transformed bone marrow-derived lymphoblastoid cell line does not impair antigen-specific CD8+ T cell responses (208). Furthermore, inhibition of autophagy with 3-MA or wortmannin has no effect on HLA antigen presentation of tyrosinase (206). In addition, Schmid et al. did not observe increased IFN –positive CTL responses after selective targeting of the influenza matrix protein 1 (M1) to autophagosomes (214).

However, there are some reports indicating that autophagy-mediated MHC class I presentation is relevant for several viral antigens. As an example, treatment of HSV-1 (herpes simplex virus 1) infected macrophages with bafilomycin A1, 3-MA or knockdown of Atg5, impairs HSV-1 glycoprotein B (gB)- specific CD8+ T cell activation. Using fluorescence microscopy, the authors could further show that LC3+ autophagosomes are possibly originating from viral particles. This suggests that gB processing might start in the autophagosome after which it will further enter into the classical MHC class I pathway via escape to the cytosol (215). A similar pathway of MHC class I antigen presentation has been described for an epitope derived from respiratory syncytial virus. As shown in a similar experiment before, antigen presentation onto MHC class I was abrogated upon 3-MA treatment (216).

In contrast, several studies clearly support the contribution of AP to MHC class I cross-presentation, which is pivotal for the induction of antigen specific T cells in anti-tumor responses (206, 208, 217– 219). Activation of macroautophagy in tumor or target cells was shown to enhance phagocytosis and subsequent MHC class I cross-presentation to CD8+ T cells (220–222). There are two options by which the autophagic machinery may contribute to cross-presentation. First, AP may modulate endosomes in DCs to facilitate optimal antigen trafficking to MHC class I and thus provide a packaging transport to pass the antigens to neighboring DCs (223–225). Secondly, autophagosomes can serve as immunogenic compartments since autophagosomal membrane formation was shown to be particularly pronounced in cross-presenting splenic mouse CD8+ DCs (226). Martins and colleagues demonstrate that activation of autophagy facilitates the activation of DCs, which is pivotal for their role in elicitation of effective IFN producing T cell responses (227). Collectively, the underlying studies suggest that autophagosomes serve as effective vehicles for the delivery of exogenous antigen into the cross-presentation pathway. These observations may be underlined by the finding that autophagy is required for the engulfment of apoptotic cells. Compared to autophagy efficient cells, Atg5 and Beclin 1-deficient embryos are not phagocytosed expeditiously probably due to reduced cell surface expression of membrane-bound phosphatidylserine (PS) or secreted lysophosphotidylcholine (L-PC) which otherwise would lure pAPCs for phagocytosis. Therefore, autophagy may enhance cross presentation by promoting efficient phagocytosis of exogenous antigen (228).

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The notion that autophagy facilitates cross-presentation is further supported by the observation that trafficking of Aspergillus conidia-associated antigen from the early endosome to a Rab14+ cross- presenting compartment is promoted by the autophagic machinery (229).

The role of AP for efficient cross-presentation of exogenous antigens in the antigen donor cells was initially reported for tumor antigens. Li et. al. could show that LC3+ autophagosomes purified from rapamycin treated OVA-expressing fibroblasts greatly increased the cross-presentation of ovalbumin (OVA) on MHC class I by co-cultured DCs (230). In addition, AP contributes to activation of CD8+ CTL mediated anti-viral immunity. Dying cells from apoptosis-deficient mouse embryonic fibroblasts (Bax/Bak-/- MEFs) display enhanced autophagy. Furthermore, influenza A virus infection enhanced cross-presentation, while siRNA-mediated silencing of Atg5 then abrogated cross-presentation of an influenza A virus-derived antigen in these cells (231). Additionally, rapamycin treatment of the human melanoma cell line FEMX enhances cross-presentation of the tumor antigen gp100, while this is abrogated by preventing autophagy with 3-MA, wortmannin, (short hairpin) shRNA-mediated knockdown of Beclin 1 or siRNA-associated abrogation of Atg12 (221). This hypothesis was verified by several recent studies showing that treatment with AP inducers enhances antigen cross-presentation, while the latter was conversely significantly reduced with AP inhibitors (232, 233). Previously, it was shown that TNF induces autophagy and enables processing and presentation of mitochondrial antigens by MHC class I molecules (220). Collectively, these data indicate a plausible role for autophagy in MHC class I (cross-)presentation.

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Results

Inhibition of autophagy reduces cross-presentation of PLGA MS encapsulated antigens To investigate the influence of autophagy in presentation of encapsulated protein antigens, we examined whether activation or inhibition of autophagy in antigen donor cells would affect cross-presentation of ovalbumin (OVA) antigen. Therefore, we pulsed cross-presenting MutuDC2114 cells with PLGA MS co-encapsulating OVA together with the TLR9 agonist CpG in the presence of either autophagy inhibitors or inducers.

Cells were treated with the mTOR1 inhibitor rapamycin or were subjected to starvation by incubation in HBSS to induce autophagy. To prevent initiation of the autophagic machinery by blocking pre- autophagosome formation, cells were treated either with the class III phosphatidylinositol 3 kinase (PtdIns3K) wortmannin (WM) or the class I phosphoinositide 3-kinase (PI3K) inhibitor 3-methyl- adenosine (3MA). Additionally, pharmacological inhibition of autophagy was accomplished with the inhibitor of endosomal acidification bafilomycin A1 (B) and chloroquine (CQ) that blocks autophagic degradation by inhibiting the vacuolar H+ ATPase activity hereby suppressing autolysosomal degradation (see also Figure 5A for better illustration of mode of action).

Antigen cross-presentation of the OVA-derived SIINFEKL peptide (OVA257-264) was detected using the CD8+ T cell hybridoma cell line BγZ, which expresses -galactosidase under the control of the IL-2 promoter. With the use of this LacZ antigen presentation assay, we measured T cell receptor (TCR) - mediated activation of T cell hybridomas induced by MutuDCs after internalization of PLGA MS. Remarkably, cross-presentation of OVA antigen encapsulated in PLGA MS was almost completely abolished upon autophagy inhibition (Figure 6B, left). MutuDCs induced B3Z stimulation in presence of autophagy inducers to similar extent as the relevant control of MutuDCs pulsed with MS OVA/CpG only (Figure 6B, left, MS-OVA). Thus, we could not further increase class I cross-presentation (XP) with autophagy-inducing compounds or treatments. In a second set of experiment we obtained similar results using ex vivo derived cross-presenting bone marrow-derived dendritic cells (BMDCs) as antigen presenting cells. In agreement with the results obtained from the DC cell line, inhibition of the autophagy machinery significantly inhibited cross-presentation of the encapsulated OVA antigen also in primary cells (Figure 5B, right).

To verify the specific activity of every compound on PLGA MS containing MutuDC cells, we assessed intracellular levels of the autophagy-related protein Atg8 (autophagy gene 8) by western blotting against LC3 (Figure 5C). LC3 is the classical autophagosomal marker in mammalian autophagy and relative LC3 levels have been shown to reflect the status of autophagy by directly correlating with the number of autophagosomes (234–236). During initial activation of autophagy, the cytosolic form of LC3 (LC3- I) is recruited to the autophagosome. LC3-I is then transformed to membrane-bound and PE-conjugated LC3-II by site-specific proteolysis and lipidation near its C-terminus. Indeed, rapamycin (R) treatment led to conversion of LC3-I to the LC3-II form compared to untreated cells (NT), suggesting that the autophagic cascade was activated. The lysosomal protease inhibitors bafilomycin A1 (B) and chloroquine (CQ) caused significantly increased LC3-II accumulation, which confirms the inhibition of autophagosomal degradation and blockage of autophagic flux. The inhibitors of the initial steps of autophagy, wortmannin (WM) and 3-MA, showed less intense expression of LC3-II levels, which confirms the blockage of autophagosome formation by inhibition of LC3-II conversion. Interestingly, it could be shown that PLGA MS did not induce autophagy signaling as visualized by absent LC3-II in the untreated sample. 110

Due to reported inconsistency of class III phosphatidylinositol 3 kinase (PI3K) inhibitors and potential off-target effects on other cellular processes and catabolic pathways (such as DC endocytosis, antigen uptake and lysosomal acidification) (170), the pharmacological inhibition of autophagy was only used as a first indicator to analyze its role in cross-presentation. In order to confirm previous proposed roles of AP in MHC class I presentation of PLGA MS encapsulated antigens in a genetic setup, we used BMDCs of ITGAX/CD11c (integrin αX)-Cre x Atg5 loxP/loxP or Atg7 loxP/loxP conditional KO mice (termed as Atg5-/- mice and Atg7-/- mice). These mice express Cre recombinase under the control of the CD11c promoter and therefore delete the loxP-flanked Atg5 or Atg7 genes in CD11c-positive cells. Compared to T cells, which heavily rely on autophagy for intracellular homeostasis (237), DCs do not undergo apoptosis in the absence of autophagy (212). Therefore, dendritic cells seem less dependent on autophagy despite the probable constitutive usage of autophagic mechanisms in these cells. Notably, Atg5 and Atg7 homozygous KO leads to early postnatal lethality (238, 239). The E3-like enzyme Atg5 and the E1-like enzyme Atg7 are part of the ubiquitin-like conjugation system in autophagy and are furthermore essential for initiation of autophagosome formation and for LC3-I lipidation of autophagosomes (240). Lee et al. previously demonstrated the importance of Atg5 as one of the key AP genes in antigen processing and presentation by DCs, however, mainly for the MHC class II pathway (212).

Deletion of Atg5 or Atg7 was confirmed in BMDCs on day 9 of the BMDC cultures by immunoblot analysis (data not shown; of note: at earlier time points of BMDCs culture the Atg genes are still present). Additionally, these BMDCs did not show significant alterations in the proportions of Flt3L-induced CD8+ or CD8- expression compared to BMDCs from littermate controls (data not shown), suggesting that autophagy is not required for central homeostasis in BMDCs in the absence of Atg5 or Atg7.

We addressed the contribution of AP to MHC class I restricted cross-presentation of encapsulated OVA antigen by measuring the presentation of SIINFEKL peptide on MHC class I by co-culture with the B3Z hybridoma cell line, as described above. Cross-presentation of SIINFEKL peptide was significantly impaired in Atg5- and Atg7-deficient BMDCs, with a more profound effect in Atg5-/- compared to Atg7- /- BMDCs (Figure 5D). Moreover, we also examined presentation of the MHC class II-restricted OVA peptide (OVA323-339) to DOBW hybridoma cells and observed reduced class II presentation of this peptide in Atg5- or Atg7-deficient BMDCs (Figure 5E).

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Figure 5: Cross-presentation of PLGA MS OVA/CpG is impaired in response to pharmacological or genetic inhibition of autophagy in vitro A. The image depicts pharmacological intervention into the autophagic pathway of used autophagy inducers (black squares) and autophagy inhibitors (blue squares). Image with minor modification taken from [http://www.invivogen.com/autophagy] B. MutuDC2114 (left) or BMDCs of naïve C57BL/6 mice (right) were pulsed with MS-OVA/CpG (20 particles/cell) for 24 h in the presence of autophagy inducer (black; rapamycin (0.5μg/ml; Sigma-Aldrich), starvation (HBSS medium, Gibco)) or inhibitors (blue; bafilomycin A1 (100nM, Sigma-Aldrich), chloroquine (3 mM, Sigma- Aldrich), wortmannin (100nM, Calbiochem), 3-MA (3mM, Sigma-Aldrich). As control for standard levels of cross- presentation, cells were treated with MS OVA/CpG (MS OVA, grey). After that, cells were incubated with the OVA-specific CD8+ T cell hybridoma B3Z for 18 hours. Activation of B3Z was detected in a colorimetric LacZ assay (absorbance at 570/620 nm). As a positive control, SIINFEKL peptide (10-6 M, Charité Berlin) was added to B3Z hybridoma (peptide, black-bordered grey column). Summary of 3 to 6 experiments ± SEM with similar outcome. Significance was calculated by unpaired student’s t-test with *p ≤ 0.05, ****p ≤ 0.0001. C. Western blot analysis of anti-LC-3 antibody in MutuDC2114 after 24 hours incubation with either autophagy inducer (R, rapamycin (0.5μg/ml), S, starvation (HBSS medium)) or autophagy inhibitors (B1, bafilomycin A1 (10 nM), bafilomycin A1 (100 nM), CQ, chloroquine (3 mM), WM, wortmannin (100nM), 3MA, 3- methylalanine (3mM)). As a comparison, basal levels of autophagy in untreated MutuDC2114 cells are shown (NT, not treated). -tubulin served as a loading control. D. BMDCs of mice genetically deficient for Atg5 & Atg 7 (Atg5-/-, Atg7-/-, blue) were pulsed in the presence of empty (MS empty) or OVA/CpG containing PLGA MS for 24 hours. After that, cells were incubated with the OVA-specific CD8+ T cell hybridoma B3Z. BMDCs of heterozygous littermates served as internal control (Atg5 fl/fl, Atg7fl/fl, black). Activation of B3Z cells was detected in a colorimetric LacZ assay (absorbance at 570/620nm). Summary of 4 experiments ± SEM with similar outcome. Significance was calculated by unpaired student’s t-test with **p ≤ 0.01, ***p ≤ 0.001 E. BMDCs of (C) were cultured in presence of MS OVA/CpG with DOBW hybridoma cells. As a positive control, -6 BMDCs were incubated with 10 M of OVA323-339 peptide (Invivogen). After 24 hours, supernatants were analyzed for IL-2 production by ELISA. Summary of 2 experiments ± SEM with similar outcome. Significance was calculated by unpaired student’s t-test with *p ≤ 0.05.

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Autophagy may enhance cross-presentation by promoting phagocytosis of particulate antigens Cross-presenting BMDCs of Atg-deficient mice showed no differences in upregulation of expression levels of MHC class I after pulse treatment with PLGA MS (Figure 6A). Additionally, flow cytometric analysis for upregulation of DC maturation markers (CD86, CD40, MHC class II) after PLGA MS treatment was comparable in conditional KO BMDCs, as well as in BMDCs of heterozygous littermates (data not shown).

Since it was recently published that internalization of nanoparticles is enhanced under starvation (241), we investigated uptake of fluorescently labeled Quantum Dot PLGA microspheres that are encapsulated together with the antigen and the TLR ligand (MS QD). However, neither activation nor inhibition of autophagy did show differences in the percentage of quantum dot-positive BMDCs assessed by flow cytometry (Figure 6B, left). Also, genetic ablation of Atg5 and Atg7 had no apparent influence on MS uptake (Figure 6B, right)

Previous findings could show that bafilomycin A1 or chloroquine–mediated inhibition of fusion of autophagsosomes to lysosomes decreased cross-presentation (226). Additionally, it was reported that autophagosomes may represent a compartment for sequestering, accumulation and subsequent degradation of antigens (232). Thus, we intended to visualize localization of PLGA MS in LC3-positive autophagolysosomes by confocal microscopy. Therefore, we pulsed either heterozygous or Atg7- deficient BMDCs with QD-positive PLGA MS for 6 hours and performed immunostaining of the endosomal marker LAMP1. As shown in Figure 6C (top panel), Atg7-/- DCs possessed less QD+ PLGA MS, while simultaneously exhibiting more intense staining of (potentially empty) LAMP1+ endocytic vesicles compared to AP-sufficient BMDCs (Figure 6C, lower panel). In these BMDCs derived from littermate control animals, co-localization of PLGA MS in endosomes could be observed, which is consistent to previous findings of our lab (123). These data suggest a reduced internalization of PLGA MS into the endocytic compartment in the absence of autophagy. Thus, the probably reduced antigen pool may not feed peptides into the MHC class I presentation pathway.

Collectively, these data indicate that autophagy (in CD8+ DCs) is at least partly required for cross- presentation of PLGA MS encapsulated antigens on MHC class I.

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Figure 6: Uptake of fluorescently labeled PLGA MS by BMDCs in presence of autophagy inhibition in vitro A. BMDCs were incubated with fluorescent quantum dot (QD583)-positive MS OVA/CpG (MS-QD+) for 6h. MHC I upregulation was analyzed by flow cytometry. B. MS uptake of BMDCs of either naïve C57BL/6 (left) or Atg5-/- and Atg7-/- mice was analyzed by co-staining of CD11c+QD+ cells and evaluation of the percentage of QD583-positive signals by flow cytometry. Total events were separated by scattering pattern of free MS and cell-associated events. C. Confocal laser scanning microscopy (LSM) images of fluorescent CdSe (cadmium selenide) QD-labelled MS (orange) after uptake by Atg7-/- or Atg7 fl/fl BMDCs and co-staining for lysosomal marker LAMP1 (green) and DAPI (blue). Graphs show representative images of 2 independent experiments with similar outcome. Significance was calculated by unpaired student’s t-test with ns, not significant.

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Discussion and future implications

Autophagy is a highly orchestrated homeostatic process to maintain the intracellular energy equilibrium by many different ways and allows the cell to respond to a variety of cell stressors such as nutrient deprivation or pathogen invasion. Autophagy’s role in the immune system has been recently identified by several reports (242). Among others, autophagy is able to remove intracellular microbial pathogens including bacteria, viruses and parasites (203).

One of the major novel roles discovered for autophagy in the immune system is the degradation of cytosolic material in endo-lysosomal compartments including plasma membrane proteins, endosomal components or extracellular proteins for binding to MHC class II and presentation of AP-dependent peptides to CD4+ T cells. Thus, AP enables the expansion of the antigen repertoire of MHC class II peptide ligands (219). Besides its role in chronic infections by increasing both innate and adaptive immunity, recent findings suggest that AP represents a relevant mechanism for antigen cross- presentation (XP) derived from tumor tissue or pathogens (210, 221, 243). XP plays an eminent role in activation of T cell immunity targeting against viruses or cancer. However, it is still controversial how AP traffics endosomal compartment to the cytosol, since AP mainly transports antigens in the opposite direction, namely from the cytosol to endosome. While some studies have clearly identified that AP takes part in antigen XP (222, 242, 244–246) by either increasing XP (226, 247) or inhibiting XP (248, 249), others have suggested that XP of exogenous antigens to CD8+ T cells is AP-independent (212). Our data nicely indicate a possible role of cross-presenting encapsulated antigens on MHC class I in CD8+ BMDCs.

Interestingly, we could not detect differences in cross-presentation after treatment with different inhibitors of autophagy. Thus, early phases (initiation and formation of autophagosomes) are required for XP to the same extent as later stages of AP when engulfed proteins are degraded. However, Li et al. observed different effects using pharmacological AP inhibition. The authors claim that the autophagy inhibitors 3-MA and wortmannin (WM) (which are essential in the initiation phase of autophagosome formation) inhibited XP of the endogenous tumor antigen gp100 by a human melanoma cell line. On the other hand, NH4Cl treatment, which prevents lysosomal acidification similar to CQ and bafilomycin A1, was shown to increase cross-presentation. Thus, they concluded that only early phases of AP are required for XP, while late phases of the autophagic process rather amplifies cross-presentation (227).

A lot of recent studies point against our findings of AP-mediated cross-presentation of exogenous antigens. For example, Lee et. al. (212) proposed that that autophagy does not affect MHC class I cross- presentation in murine DCs. Priming of CD8+ OVA-specific OT-I T cell responses and cross- presentation of either soluble or cell-associated OVA (lethally irradiated OVA-pulsed MHCI-deficient splenocytes) was not defective in Atg5-deficient DCs. In addition, WM or 3-MA treatment as well as Atg12 knockdown failed to affect MHC class I presentation of the EBV nuclear antigen EBNA1 (208). In contrast to that, a lot of other in vitro studies clearly demonstrate a role of AP in intracellular and extracellular antigen processing for MHC class I presentation. However, in vivo studies primarily state enhanced CD8+ T cell responses in mice with defective autophagy. For instance, Atg5 or Atg16L1 deficiency in DCs or tumor cells augmented CD8+ T cell responses, probably due to the increased availability of substrates for canonical MHC class I pathway (225, 250).

Compared to our results, surface levels of MHC class I molecules and co-stimulatory molecules were found to be increased on Atg5- and Atg16L1-deficient DCs which is mainly attributed to a decreased endocytosis and degradation capacity. The underlying study particularly showed that the adaptor- associated kinase 1 (AAK1) was inefficiently recruited to the cell surface and was therefore unable to 115 internalize MHC class I molecules (225). On the other hand, Mintern et. al. showed no major differences in MHC class I expression levels or DC activation markers in Atg7-deficient DCs which is consistent with our findings (251). However, we characterized aforementioned surface expression levels after incubation with PLGA MS to verify efficient DC maturation after PLGA MS uptake. Therefore, we probably masked a potential different phenotype of immature BMDCs. Thus, surface markers have to be analyzed again at steady-state as well as in response to PLGA MS stimulation. Additionally, Li et al. also report a possible participation of autophagy-mediated degradation in MHC class I surface turnover. Pharmacological inhibition of autophagy using 3-MA, bafilomycin A1, chloroquine or wortmannin, as well as siRNA-mediated knockdown of Beclin 1, Atg5 or Atg7 was shown to increase expression of MHC class I molecules on the surface of murine macrophages. In turn, autophagy activation with rapamycin promoted co-localization of MHC class I molecules with autophagosomes. This was accompanied by reduced surface expression of MHC class I on B16F10 melanoma cells. Furthermore, co-treatment with rapamycin and IFN prevented the observed localization within autophagosomes and further enhanced surface expression of MHC class I molecules as well as antigen-specific killing of tumor cells (227).

Unfortunately, the crucial in vivo experiments on this project are still missing but will be analyzed soon. Thus, we will provide definite evidence for a role of the autophagic machinery in antigen cross- presentation in vivo to both MHC class I and II using PLGA MS vaccination in Atg knockout mice.

The use of monocyte-derived DCs generated from bone marrow precursor cultures as a model for cross- presenting cells does not fully resemble the complex XP pathway specialization of their in vivo counterparts, which are defined as CD11c+ CD103+ MHC-II+ CD64+ FcεRI+ Siglec F- DCs (4). By using Flt3L-mediated BMDC differentiation, which enables the generation of cross-presenting CD8+ DCs, we could increase the amount of CD8α+ CD11c+ cells in the BMDC culture. Nevertheless, more than 30% of these cells still harbored the CD8- phenotype. Additionally, the BMDC culture possessed low levels of plasmacytoid DCs, which are both known to be poor antigen (cross-)presenters and might impair the experimental outcome. A possible enrichment of cells by positive CD8 MACS-sorting of the BMDC culture was impossible due to the low number of cells.

Only two subpopulations of mouse DCs efficiently cross-present antigens onto class I due to unique features of their endocytic compartments that favor cross-presentation through the cytosolic pathway. These cells are the lymphoid organ-resident CD8+ DCs as well as the migratory CD103+ DCs (252). After antigen uptake, protein degradation is limited to the endosomal compartment. The transfer of exogenous antigens back to the cytosol is another key step in cross-presentation that is more efficiently conducted in CD8+ DCs. As well, CD8-positive DCs overexpress molecules that are associated with the MHC class I pathway, as opposed to CD8- DCs. In contrast, inflammatory DCs and their in vitro equivalents (GM-CSF-mediated BMDCs) use the cytosolic and vacuolar pathways for cross- presentation. However, cross-presentation efficiency seems to be inferior in these cells (253, 254). Regarding antigen uptake, both CD8+ and CD8− DCs efficiently capture soluble and particulate antigens for presentation on MHC class II, while only CD8+ DCs can efficiently cross-present internalized particulate antigens on MHC class I molecules (10, 90, 134, 255). Since the XP pathway of BMDCs does not follow the canonical endosome-to-cytosol route (256), which is probably needed for efficient cross-presentation of antigens after PLGA MS immunization, we will analyze the contribution of AP in vivo upon PLGA MS immunization of AP-deficient mice. The presentation of encapsulated OVA antigen by MHC class I could be measured by analysis of T cell proliferation of Cell Trace Violet- labeled naïve OT-I CD8+ OVA-specific T cells in vivo after PLGA MS immunization of Atg5 and Atg7 KO mice (which are EGFP (enhanced green fluorescent protein) positive).

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In order to analyze cross-presentation in vivo, we want to analyze presentation of SIINFEKL by intracellular cytokine staining (ICS) or Enzyme Linked Immuno Spot Assay (ELISpot) for IFN by OVA-specific T cells in spleens of either Atg-/- mice or littermate controls on day 6 following MS OVA/CpG vaccination. Additionally, we already have established a FACS-based cell sorting protocol for primary CD8α+ splenic mouse DCs (CD11c+, CD205+, MHC class II+, CD4-, CD11b-). Then, we will further investigate cross-presentation of these cells in co-culture with wild type OT-I CD8+ OVA- specific T cells in vitro in order to enable a reliable comparison of the contribution of AP in MHC class I presentation in the same cell type in vivo. These CD8+ DCs are rather moderately present in spleen and LNs, constituting of 23% and 17% of all CD11c+ DCs, respectively (10, 257). Furthermore, we could analyze the phenotype of DCs derived from skin-draining lymph nodes after PLGA MS immunization in Atg-KO mice. These tissues contain the resident CD8+ and CD8- cDC populations and, additionally, the migrating DC equivalents characterized by ITGAE/CD103+ (integrin αε) and CD103- expression (33% of all CD11c+ DCs). These DCs have encountered the antigenic stimuli in the periphery (258).

Although we could not observe increased efficacy of XP-dependent particulate OVA antigen into PLGA MS upon autophagy induction in vitro (Figure 1B) – as well, microspheres did not induce autophagy by itself - it would be interesting to further investigate if CD8+ T cells responses can be enhanced in vivo via immunization of e.g. rapamycin-treated BMDCs loaded with MS OVA/CpG, which will be assessed by different antigen presentation assays in vitro and in vivo.

Finally, Mintern et al. also proposed the form of the antigen to matter in AP and related cross- presentation (226). They showed, that XP of a soluble protein was more efficient compared to (apoptotic) cell associated antigens or antigen delivered by DEC-205 receptor-mediated endocytosis on DCs. However, XP of soluble OVA was solely dependent on Atg7. Li et al. already could show that autophagy increases MHC class I cross-presentation of nanoparticle-associated OVA (αAl2O3-OVA) by bone marrow-derived DCs in vitro and in vivo compared to OVA alone. Inhibiting of autophagy using 3-MA, wortmannin or via siRNA-mediated knockdown of Beclin-1 prevented cross-presentation of αAl2O3-encapsulated OVA. Furthermore, application of αAl2O3-coated autophagosomes to tumor- bearing mice improved anti-tumor OT-I CTL responses unlike the administration of OVA with the widely used adjuvant Alum. Notably, these αAl2O3-OVA nanoparticles co-localized with LC3-positive autophagosomes in DCs suggesting that the autophagosome serves as an important compartment for cross-presenting antigens (259).

In relation to this, we further want to investigate this issue by comparing PLGA MS encapsulated antigens with the respective soluble form. This could be accomplished by immunization of AP-KO mice with either PLGA MS or the same amount of antigen in Complete Freund’s Adjuvans (CFA). Yet, we expect reduced CTL responses in the absence of autophagy regardless of the increased half-life of proteins and the available protected antigen load of encapsulated antigens which also critically determines cross-presentation efficacy. Furthermore, we want to identify the contribution of antigen processing of PLGA MS delivered antigens in relation to the efficiency to (cross-) present either OVA full length protein, a synthetic long peptide composed of 40 amino acids or SIINFEKL peptide encapsulated into MS following immunization of Atg5-/- mice or Atg7-/- mice.

Using fluorescently labeled PLGA MS, we first did not observe any differences in the uptake of microspheres by BMDCs. Since we could not rule out that free MS still adhere to the surface of BMDCs, we need to repeat these experiments with a minor modification as described in Waeckerle-Men et al. A short washing step using 100% DMSO should remove sticky MS on the outside of the cell (122). Despite these inconsistent results, we could show that BMDCs deficient for Atg7 exhibit reduced PLGA MS in LAMP1+ endosomal vesicles, which is probably attributed to the lack of autophagosomes that may fuse with lytic compartments. The work of Sanjuan et al. could show that Atg5 and Atg7 – dependent AP 117 in murine macrophages promoted rapid recruitment of the autophagosome markers LC3 and Beclin- 1 to the phagosome. In these phagosomes, TLR engagement of CpG derived from phagocytosed particulate antigens led to accelerated fusion with the lysosome (211). A more elegant way to analyze the involvement of LC3-associated phagocytosis (LAP) for recruitment of fluorescently labeled PLGA MS into autophagosomes would be the isolation of CD8+ splenic DCs of GFP-LC3 transgenic mice and subsequent analysis of GFP-LC3-positive vesicles by confocal microscopy. Additionally, we intend to use the option of spinning disk confocal microscopy to identify potential differences in MS endocytosis and rerouting using live cell imaging.

Moreover, we need to further analyze the decreased peptide presentation on MHC class II in Atg5-/- and Atg7-/- BMDCs upon incubation with the DOBW hybridoma (Figure 1E). With these experiments we could evaluate impaired uptake and delivery to MHC class II loading compartments. Furthermore, we might observe altered degradation within phagosomes in AP-deficient BMDCs after PLGA MS uptake, which could be rescued by protease inhibitors leupeptin or peptstatin. Paludan et al. initially showed that endogenous MHC class II antigen processing is blocked after inhibition of autophagy, which results in decreased antigen-specific CD4+ T cell responses (208). Additionally, many other studies demonstrate that steady-state autophagy continuously and efficiently delivers intracellular, cytosolic antigens into the MHC class II presentation pathway in professional APC (DCs, monocytes, macrophages) but also in nonprofessional APCs such as B cells or IFN-induced epithelial cell lines. Using GFP-LC3 transgenic mice, they observed that the autophagosome frequently fuses with MIIC (MHC class II compartments), indicating that autophagosomal cargo gains access into the class II antigen presentation pathway (214, 260).

In summary, we could show that either pharmacological intervention or genetic ablation of the macroautophagy pathway significantly reduced presentation of PLGA MS encapsulated protein antigens. These results indicate that AP is involved in processing of MS derived antigens for MHC class I and II and assists in cross-presentation of antigens onto MHC class I via the endolysosomal pathway. However, a comprehensive understanding of the contribution of macroautophagy in MHC class I restricted antigen presentation of PLGA MS encapsulated antigens is still needed to further optimize the outcome of therapeutic PLGA MS application to boost T cell mediated immunity against cancer.

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Material and Methods

Cell lines and cell culture methods All cell culture media and supplements were obtained from Gibco (Invitrogen, Thermo Fisher). The + B3Z CD8 T-cell hybridoma, specific for the SIINFEKL peptide of ovalbumin (OVA257-264) was cultured in IMDM (Iscove’s modified Dulbecco’s medium) supplemented with 10% heat inactivated FCS (fetal calf serum) and 100 units/mL penicillin and 100 µg/mL streptomycin. The CD4+ T-cell hybridoma

DOBW specific for the ovalbumin class II epitope ISQAVHAAHAEINEAGR (OVA323-339) was cultured in DMEM (Dulbecco’s modified minimal essential medium) supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 0.5 mM - mercapto-ethanol (according to (261)). Cell cultures were maintained at 37°C with 5% CO2 in a humidified atmosphere. Adherent cells were detached by incubation with 0.05% trypsin-EDTA for 5 minutes at 37°C. Animals and ethics standard Atg5flox/flox and Atg7flox/flox mice on a C57BL/6 (H2-Kb) background were obtained from Prof. Christian Münz, ETH, Zurich (originally obtained from Noboru Mizushima (University of Tokyo, Japan) and Masaaki Komatsu (University of Tokyo, Japan), respectively). Mice were further bred in the animal facilities of the University of Konstanz. To generate conditional knockout mice lacking macroautophagy in CD11c+ APCs, CD11c-Cre-EGFP+/- mice were bred with either Atg5flox/flox or Atg7flox/flox mice to generate Atg5flox/flox CD11c-Cre-EGFP+/- mice (designated as Atg5-/- mice) or Atg7flox/flox CD11c-Cre-EGFP+/- mice (designated Atg7-/- mice). Mice were housed under specific pathogen free conditions on a 12/12 hour light/dark cycle with lights on at 7 a.m. Animals were provided ad libitum access to autoclaved standard chow and water. Experimental animals were used at 8–12 weeks of age. All animal experiments were approved by the ethical committee of the Regierungspräsidium Freiburg.

Mouse genotyping Genotyping of mice was performed by genomic DNA extraction obtained from tail biopsies of 4-5 week- old mice. Tail biopsies were lysed in 600 µl of 50 mM NaOH at 95°C under constant agitation at 900 rpm for 1 hour. After that, the digestion was neutralized using 50 μl of 1 M Tris pH 8.0. After centrifugation for 5 minutes at maximum speed to pellet cell debris, β μl of the supernatant were used as DNA templates. Standard genotyping protocol was performed using GoTaq® G2 Flexi DNA polymerase (Promega) for the CD11c-Cre and Atg5flox/flox alleles, while LATaq TaKaRa DNA polymerase (Clonetech) was used for Atg7flox/flox genotyping. Expected PCR products of the Cre transgene is 100 bp and 324 bp for the internal positive control. 650 bp for the Atg5flox allele and 350 bp for the Atg5 wild type allele, while 500 bp identified the Atg7flox allele and 1.5 kb the respective Atg7 wild type allele. Displayed primer pairs were synthesized at Microsynth:

Primer Sequence (5’-3’) Atg5 wild type forward GAA TAT GAA GGC ACA CCC CTG AAA TG Atg5 wild type reverse GTA CTG CAT AAT GGT TTA ACT CTT GC Atg5flox/flox forward ACA ACG TCG AGC ACA GCT GCG CAA GG Atg5flox/flox reverse CAG GGA ATG GTG TCT CCC AC CD11c-Cre-GFP forward GCG GTC TGG CAG TAA AAA CTA TC CD11c-Cre-GFP reverse GTG AAA CAG CAT TGC TGT CAC TT CD11c-Cre-GFP (control) forward CTA GGC CAC AGA ATT GAA AGA TCT 119

CD11c-Cre-GFP (control) reverse GTA GGT GGA AAT TCT AGC ATC ATC C Atg7flox/flox forward TGG CTG CTA CTT CTG CAA TGA TGT Atg7flox/flox reverse CAG GAC AGA GAC CCA TCA GCT CCA C

PCR cycling conditions for Atg5flox/flox

Temperature Time 95°C 2 min 95°C 30 sec 57°C 30 sec X 35 72°C 30 sec 72°C 5 min 4°C ∞

PCR cycling conditions for Atg7flox/flox

Temperature Time 94°C 5 min 94°C 30 sec 65°C 30 sec X 30 68°C 90 sec 72°C 10 min 4°C ∞

PCR cycling conditions for CD11c-Cre

Temperature Time 95°C 2 min 95°C 30 sec 56°C 30 sec X 35 72°C 30 sec 72°C 5 min 4°C ∞

Agarose gel electrophoresis For visualization of PCR products, agarose (Premium Serva) was dissolved in 1x TAE-Buffer (0.4 M

Tris, 0.2 M acetic glacial acid, 0.01 M EDTAxNA2xdH2O) at 2% (w/v). Ethidium bromide (Sigma- Aldrich) was added to the agarose solution at a final concentration of 0.5 µg/ml. The agarose solution was poured into an electrophoresis chamber and was allowed to harden. PCR products were separated electrophoretically at 100 V in 1x TAE buffer for approximately 20 minutes. Smart Ladder or Smart Ladder SF (small fragments) (Eurogentec) were used as DNA ladder for size determination. Bands were visualized under UV-light with the Molecular Imager® Gel Doc™ system (Bio-Rad).

Antibodies and flow cytometry Surface staining with fluorescently labeled monoclonal antibodies was performed according to standard procedures at a density of approximately 1x106 per 100 μl. All stainings and flow cytometric analyses were performed in 1x FACS buffer (2% FCS, 1 mM EDTA, 0.1% NaN3 in 1x PBS) unless mentioned 120 differently. All centrifugation steps were performed for five minutes at 1200 rpm unless otherwise stated. Cells were carefully resuspended in 50 µl of Fc-Block (BD, Biosciences) for 10 minutes at 4°C to block non-specific Fc-receptor binding of the subsequently applied antibodies in FACS buffer at a final dilution of 1:150. The cell suspension was incubated for another 20 to 30 minutes at 4°C. Then, cells were washed twice with FACS buffer and centrifuged again. The supernatant was discarded and the cells were resuspended in respective amount of FACS buffer for subsequent analysis via flow cytometry on the BD LSR Fortessa™ (BD, Biosciences) provided at the FlowKon Facility, University of Konstanz. FACSDiva software (BD Biosciences) was used for data acquisition, while FlowJo software was used for data analysis. The following antibodies were used: Fluorescein isothiocyanate (FITC)–conjugated mAbs to H-2Kb (clone #AF6-88.5) and SIRPα (clone #P84). Allophycocyanin (APC)-conjugated mAbs to CD4 (clone #RM4-5), CD8 (clone #52-6.7), NK1.1 (clone #PK136), CD19 (clone #1D3), CD11b (clone # M1/70) and Gr-1 (clone #1A8); phycoerythrin (PE)-labeled mAb to CD24 (clone #M1/69), CD80 (clone #16-10A1), CD86 (clone # GL1), CD40 (clone #3/23), CCR7 (clone #4B12). Brilliant Violet (BV421)-conjugated mAb to CD11c (clone #N418) and PE-cyanine7 (PE-Cy7)-conjugated mAb against B220 (CD45R, clone #RA3-6B2). QD MS were detected in the PE channel. Antibodies were purchased from either BD Biosciences, eBiosciences or Biolegend U.K. with respect to the given antibody clone.

Generation of CD8a+ bone marrow-derived dendritic cells (BMDCs) using Flt3 Ligand (Flt3L) For isolation of progenitor DCs from the bone marrow, mice were euthanized and skin and muscles from pelvis, femur and tibia were removed. Bones were transferred into medium and stored on ice until further processing. Bones were incubated in 70% EtOH for 30 seconds and were immersed into PBS after that before cutting open both edges of bones using sterilized scissors. Bone marrow was flushed out with 1x PBS using a γ0 G ½ ’’ needle (BD Microlance™). The obtained cells were passed through a 70 μm cell strainer (BD Falcon™) and topped with large volumes of 1x PBS. Next, cells were centrifuged at 1200 rpm for five minutes at room temperature and the supernatant was discarded. The cell pellet was resuspended in 3 ml ACK lysis buffer (50mM ammonium chloride; 10mM potassium bicarbonate; 0.1mM EDTA) and incubated for two minutes at room temperature. Red blood cell lysis was stopped by adding large volumes of medium and cells were centrifuged again. The supernatant was discarded and the cell number was adjusted to a final cell concentration of 2×106 cells/ml in complete BMDC medium (IMDM-2 mM L-glutamine supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM -mercapto-ethanol). Cells were cultured in a total volume of 10 ml BMDC medium (2×107 total cells) containing 300 ng/mL rmFlt3L (CD135, Immunotools) and 300 pg/mL mGM-CSF (Peprotech) in 10 cm non-tissue culture petri dishes for eight to maximum ten days at 37°C. To increase the portion of fully differentiated, cross-presenting CD103+CD8α+ DC-like phenotypes, 5 ml of complete BMDC medium supplemented with mGM-CSF at a final concentration of 1 ng/ml was added the DC culture two days before harvesting the cells. Both, non-adherent as well as adherent cells are collected; the latter were removed by incubation with PBS/0.5mM EDTA for 10 minutes at 37°C and used for further experiments. The purity and maturation status of the cells was confirmed by flow cytometry via discrimination of CD8α+, CD8α- and pDCs subpopulations using antibodies against CD11c, CD11b, CD24, B220 (CD45RA) and SIRPα. CD8α+ DCs are identified by B220-, CD11c+, CD24+, CD11b+, CD11b-, SIRPα-, while CD8α- are B220-, CD11c+, CD24low, CD11b+, SIRPα+. pDCs equivalents confer the phenotype of B220+, CD11c+. Generally, a purity of 90% of cDCs could be achieved. Among them, approximately 60% were CD8α+ and 30% CD8α-.

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Preparation of PLGA microspheres PLGA MS were prepared from 14 kDa PLGA carrying 50% hydroxyl- and 50% carboxyl-end groups (Resomer RG502H, Evonik) by spray drying. For OVA/CpG co-encapsulation 50 mg ovalbumin and 5 mg CpG oligodeoxynucleotides with a phosphothioate backbone (CpG-ODN 1826, Microsynth) were co-dissolved in 0.5 ml 0.1 M NaHCO3 (aqueous phase) and subsequently mixed with 0.8 g of PLGA dissolved in 16 ml of dichloromethane (organic phase). CdSe (cadmium-selenide) quantum dots (QD) were prepared using one pot hot injection synthesis as described elsewhere (262). Fluorescently labeled quantum dots MS were generated by adding 500 µl QD583 (emission wavelength at 583 nm) into the organic phase. The two phases were then emulsified by ultrasonication (amplitude 40%, UP200 H, Hielscher) for 10 seconds on ice. The obtained water/organic phase dispersion was immediately spray dried with the Mini Spray-Dryer 290 (Büchi) at a flow rate of 1 ml/min and inlet/outlet temperatures of 40°C /37°C. The spray-dried MS were washed out of the spray-dryer’s cyclone with 0.05% poloxamer 188 (Synperonic®F68, Serva) and collected on a cellulose acetate membrane filter. PLGA MS were dried under reduced pressure (20 mbar) for at least 18 hours at room temperature in a desiccator. PLGA MS were stored under desiccation at 4°C. Before use, indicated amounts of MS were dispersed in medium by sonication (Transsonic DigitalS, Elma Schmiedbauer) for several seconds in order to obtain a homogenous MS suspension.

Detection of antigen presentation by LacZ T cell hybridoma assay Antigen cross-presentation of SIINFEKL peptide was assessed using B3Z hybridoma, which is a lacZ- + b inducible CD8 T-cell hybridoma specific for the OVA257-264 peptide presented on murine H2-K MHC class I molecules. The lacZ assay makes use of the reporter construct consisting of the bacterial - galactosidase gene (lacZ) under transcriptional control of the nuclear factor of activated T cells (NFAT) element of the human IL-2 enhancer. The presentation of the SIINFEKL epitope by murine Kb to B3Z cells results in intracellular accumulation of -galactosidase in B3Z cells, which can be measured by hydrolysis of the chromogenic substrate chlorophenolred--d-galactoside (CPRG, Calbiochem). The conversion to a red-violet dye is a direct indicator of the amount of SIINFEKL/Kb complexes presented on the cell surface (263). 1x105 hybridoma cells were incubated with 2x105 BMDCs per well of a U- bottom 96-well plate. Cells were co-incubated with β5 μg/well of either microspheres containing ovalbumin (MS OVA/CpG) or empty MS as control (MS empty). As a positive control, BMDCs were pulsed with SIINFEKL peptide (final concentration of 10-6 M). After 20 hours of incubation, cells were pelleted at 1500 pm for 2 minutes and washed once with 1x PBS. 100 μl of LacZ buffer (0.13% NP40, 9 mM MgCl2, 0.15mM CPRG in 1x PBS) was added for an additional 4 hour incubation step at 37°C. Absorbance was measured at 570 nm with background subtraction at 620 nm using a spectrofluorometer (Infinite Pro 200, Tecan). Presentation on MHC class II was similarly assessed as described above, except for using the OVA323-339-specific hybridoma cell line DOBW. After incubating the cells for 20 hours with this hybridoma cell line, IL-2 secretion was measured from cell supernatants using a mouse IL-β ELISA kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s protocol.

Confocal laser scanning microscopy (LSM)

Preparation of cells for immunofluorescence microscopy

To study the uptake of QD-positive PLGA MS into LC3+ autophagosomes, confocal laser scanning microscopy of BMDCs was performed. BMDCs were generated as described above. At day nine of

BMDC culture, cells were harvested and washed with D-PBS (1 mM MgCl2, 0.1 mM CaCl2 in 1x PBS) before being seeded onto poly-L-lysine (pLL)-coated glass coverslips in 24 well plates. pLL (stock: 100 122

µg/mL in 1x PBS) coating was performed for at least 30 minutes at 37°C before that. Cells were allowed to attach to coverslips for 4 to 6 hours. After that, BMDCs were incubated with CdSe quantum dot- labelled PLGA MS under various conditions for 6 hours at 37°C. Unbound microspheres were removed by extensively washing the cells with D-PBS after incubation. Afterwards, cells were fixed in 4% PFA for 20 minutes at RT. Fixing solution was removed and the cells were then washed three times with D-

PBS. Cells were treated with 50mM ammonium chloride solution (NH4Cl in D-PBS) for 10 minutes at 4°C. Cells were again washed three times before being permeabilized in 0.2% Triton-X 100 in D-PBS for 5 minutes at RT. After another washing step, blocking was performed in 1x RotiBlock™/D-PBS for 1 hour at room temperature. Primary antibody against LAMP1 was added at a dilution of 1:150 in blocking buffer and cells were incubated overnight at 4°C. On the next day, the primary antibody was removed and cells were washed three times with washing buffer. Secondary antibodies (anti-rat Ig conjugated to Alexa Fluor 488 or Alexa Fluor 647 (1:500; Invitrogen, Thermo Fisher Scientific)) were applied for 2 hours at room temperature. Coverslips were washed again and then mounted onto glass slides using 4, 6-diamidino-2-phenylindole (DAPI) containing mounting medium (DAPI fluromountG (Molecular Probes)) to counterstain cell nuclei. Fluorescence images were captured at the LSM 880 with AiryScan (Zeiss, BIC, University of Konstanz). Cadmium-selenide specific fluorescence was excited by the DPSS laser delivering light at 561 nm. DAPI fluorescence was excited with the 405 nm diode. Excitation of LAMP1 fluorescence was achieved by using the 488 nm Argon ion laser. Digital image acquisition and image analysis was performed with the Zen Application Imaging Software (Zeiss).

Molecular biological methods – western blotting

Protein extraction and quantification

In order to enable LC-3 detection, cells were immediately processed without intermediate freezing breakpoints at any stage of the immunoblotting procedure. The harvested single cell suspension was washed with ice-cold 1x PBS. After a centrifugation step at 1200 rpm for five minutes at 4°C, the supernatant was discarded and the cell pellet was resuspended in 1 ml of ice-cold PBS and transferred to chilled 1.5 ml tubes. Cells were centrifuged again using a table top centrifuge (6000 rpm, two minutes) and the supernatant was completely removed. The obtained cell pellet was immediately resuspended in ice-cold RIPA lysis buffer (approximately 50 μl lysis buffer per 1×106 cells; 50 mM TrisHCl, pH 7.5; 150 mM NaCl; 1mM EDTA, pH 8.0; 1% (v/v) NP-40; 0.5% (v/v) SDS; freshly supplemented with a cocktail of protease inhibitors (Complete, Roche Pharmaceuticals)). A homogenous suspension was achieved by vigorously pipetting the cells up and down. The homogenates were further incubated on ice for 30 minutes while enabling mixing of lysed cells every 10 minutes by a short vortexing step. After incubation, cells were shortly sonicated on ice for 5 seconds, after which lysed cells were centrifuged at 13000 rpm for 15 minutes at 4°C to separate cell lysate from cell debris. Supernatants were transferred to fresh, chilled 1.5 ml tubes and small aliquots of 2.5 µl were removed for protein quantification using Pierce ™ BCA assay (Thermo Fisher) according to the manufacturer’s protocol. Serial dilutions of BSA in RIPA buffer (1 mg/ml) were used as a standard. Following 30 minutes of incubation at approximately 37°C, the absorbance at 595 nm was measured and protein concentration was determined by the obtained standard curve. Samples were uniformly adjusted to enable protein concentrations of 20µg. Samples were boiled in 4x Laemmli buffer (300 mM Tris-HCl, pH 6.8; 10% (w/v) SDS; 50% (v/v) glycerol; 0.05% (w/v) bromphenole blue; 5% (v/v) -mercapto-ethanol) at 95°C for five minutes to denature proteins. Samples were either immediately used for SDS PAGE analysis or stored at -80°C until further processing.

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SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Electrophoretic resolving of proteins under reducing conditions and subsequent western blot analysis was performed using the Mini-Protean® 3 Cell system (Bio-Rad). Equal amounts of protein lysates were loaded onto the gel, together with a protein standard to determine the molecular size of the proteins (PageRuler Prestained, Thermo Fisher Scientific). The SDS gel consisted of an upper collection gel of 4% polyacrylamide and a lower separating gel of 15% polyacrylamide. Detailed components and composition of SDS gels are given elsewhere (264). Pre-prepared gels were stored at 4°C for a maximum of one week. Electrophoresis was carried out in SDS running buffer (25 mM Tris-HCL, 192 mM glycine, 0.1% (w/v) SDS) at an initial voltage of 50 V until the running front entered into the upper resolving gel. Then, the voltage was increased to 100 V until the protein dye front reached the estimated position for displaying the protein of interest.

Wet blotting procedure and protein detection

Protein transfer after SDS-PAGE was carried out by wet blotting in the Mini Trans-Blot Cell™ system (Bio-Rad). Prior to usage, PVDF membranes of 0.2µm pore size (GE Healthcare) were hydrophilized in 100% methanol for 20 seconds, followed by extensive washing in dH20 and further equilibration in pre-cooled blotting buffer (25 mM Tris-HCl, 192 mM glycine, 20% (v/v) methanol). SDS gel and PVDF membrane were then placed between two pieces of blotting buffer-soaked whatman paper and covered by two sponges. These layers were held by a lattice cassette and placed into the electrophoresis container which was filled with ice-cold blotting buffer. Western blotting was performed in ice-cold blotting buffer at 110V for 1.5 hours on ice. Successful protein transfer was identified by visible transfer of the pre-stained molecular weight marker on the membrane. After transfer, the membrane was shortly washed in dH2O and incubated in blocking buffer (1x RotiBlock™ (C. Roth) in TBS/0.05%Tween®-20) for 1 hour under constant shaking to saturate unspecific protein background. Blots were further incubated with primary antibody in 1x RotiBlock™/TBS-T against LC-3 (1:1000, PM036, MBL International) overnight at 4°C. After incubation with the primary antibody solution, membranes were washed three times in TBS-T for 10 minutes each on a shaker to remove remaining primary antibody that was not tightly bound to the membrane. After that, membranes were incubated with HRP-conjugated secondary anti-rabbit Ig antibodies (1:3000, Dako) for 1 hour at RT. The blot was then washed three times as described above. Specific bands were detected by chemiluminescence after incubation with ECL Super Signal West Chemiluminescent Substrate (Thermo Fisher Scientific) using the Gel-Doc system and Quantity One software (Bio-Rad). To ascertain equivalent loading of the lanes, -tubulin (1:3000 in blocking buffer, Sigma-Aldrich) was used as a control.

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Appendix

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List of Abbreviations

-/- knockout 3-MA 3-methyladenine AOM azoxymethane AP autophagy Apc adenomatous polyposis coli (p)APC (professional) antigen presenting cell Atg autophagy-related gene Bcl-2 B cell lymphoma 2 BMDC bone marrow-derived dendritic cell bw body weight BZ Bortezomib CAC colitis-associated carcinogenesis CCL CC chemokine ligand CD cluster of differentiation CD Crohn's Disease cDC conventional dendritic cell CFSE Carboxyfluorescein succinimidyl ester CFZ Carfilzomib CIN chromosomal instability CP constitutive proteasome CpG-ODN cytosin-phosphatidyl-guanosin oligodeoxynucleotides CQ chloroquine CRC colorectal cancer CTL cytotoxic T lymphocyte CXCL CXC ligand DC dendritic cell DRiP defective ribosomal product DSS dextran sodium sulfate salt ER endoplasmatic reticulum FACS fluorescence activated cell sorting FAP familial adenomatous polyposis FDA U.S. Food and Drug Administration Flt3L FMS-related tyrosine kinase 3 Ligand Foxp3 forkhead box P3 GM-CSF granulocyte-macrophage colony stimulating factor GSK3 Glycogen synthase kinase 3 HLA human leukocyte antigen HNPCC hereditary non-polyposis colon cancer HRP horse radish peroxidase HSP heat-shock protein i.p. intraperitoneal i.v. intravenous IBD inflammatory bowel disease iDC immature dendritic cell IFN interferon IL interleukin 137

IP immunoproteasome IκB inhibitor of NFκB kDa kilo dalton KO knockout LMP low molecular mass polypeptide (d)LN (draining) lymph node mAb monoclonal antibody mDC myeloid dendritic cell MDSC myeloid-derived suppressor cell MECL-1 multi-catalytic endopeptidase complex-like 1 MHC major histocompatibility complex Min multiple intestinal neoplasia MM multiple myeloma MMP matrix metalloproteinase moDC monocyte derived dendritic cell MS microsphere MSI microsatellite instability mTOR mammalian target of rapamycin MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium NFκB nuclear factor κ-light chain enhancer in B cells NK(T) natural killer (T) cell NO nitric oxide OVA ovalbumin PBMC peripheral blood mononuclear cells pDC plasmacytoid dendritic cell PI proteasome inhibitor PI3K phoshoinositide-3-kinase PLGA poly (D,L)-lactide-co-glycolide polyI:C polymer of inosinic & cytidylic acid PRR pattern recognition receptors PSMA proteasome subunit type alpha PSMB proteasome subunit type beta Ras rat sarcoma RONS reactive oxygen and nitrogen species ROS reactive oxygen species RP regulatory particle RT room temperature s.c. subcutaneous S1 substrate specificity pocket STAT signal transducers and activators of transcription TAP transporter associated with antigen processing TBS Tris-buffered saline TCR T cell receptor TGFβ transforming growth factor beta Th T helper TIL tumor-infiltrating lymphocytes TLR toll-like receptor TME tumor microenvironment TNF tumor necrosis factor 138

Treg regulatory T cell UC Ulcerative Colitis

UPR unfolded protein response UPS Ubiquitin proteasome system V Volt v/v volume per volume w/v weight per volume WM wortmannin WT wild type XP cross-presentation

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Record of Contribution

I designed and I performed all experiments by myself except for the results depicted in Chapter I, Figures B, D, E and F which were obtained in the context of the Bachelor thesis of Leonie Schnell that was completely performed under my supervision.

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List of Publications

Parts of this dissertation have already been published. Koerner, J., T. Brunner, and M. Groettrup. 2017. Inhibition and deficiency of the immunoproteasome subunit LMP7 suppress the development and progression of colorectal carcinoma in mice. Oncotarget 8: 50873–50888.

Publications not highlighted in this thesis. Basler, M., E. Maurits, G. de Bruin, J. Koerner, H. S. Overkleeft, and M. Groettrup. 2018. Amelioration of autoimmunity with an inhibitor selectively targeting all active centres of the immunoproteasome. Br. J. Pharmacol. 175: 38–52. Sommershof, A., L. Scheuermann, J. Koerner, and M. Groettrup. 2017. Chronic stress suppresses anti-tumor T CD8 + responses and tumor regression following cancer immunotherapy in a mouse model of melanoma. Brain Behav. Immun. 65: 140–149. Mueller, M., W. Reichardt, J. Koerner, and M. Groettrup. 2012. Coencapsulation of tumor lysate and CpG-ODN in PLGA-microspheres enables successful immunotherapy of prostate carcinoma in TRAMP mice. J. Control. Release 162: 159–166.

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Acknowledgments

First of all, I would express my outermost thanks to Prof. Marcus Groettrup for giving me the opportunity to perform my PhD thesis in his lab. Thank your for your trust and believe in me, for all your support and the great scientific guidance. Now, I am a part of your group for a really long time - seems like there is no other place like it. I also want to cordially thank Prof. Thomi Brunner for his kind agreement to serve as reviewer of this thesis, for which he contributed so much. Thank you for all the essential scientific advices and wonderful new impulses, as well as for everything else.

My sincere thanks also goes to Prof. Carl Fidgdor for being my reviewer and oral examiner and for accepting me as a member of the great <> consortium.

Not less, I would like to thank Prof. Christiane Ruedl, Prof. Klaus Karjalainen and of course Irene Soncin for scientific input and the wonderful time in Singapore.

Besides, I would like to express my sincere gratitude to Dr. Michael Basler and Dr. Annette Sommershof, for being a constant source of motivation, inspiration and entertainment. It is a pleasure to be with you in the lab and thank you as well for all the effort in proof-reading my thesis. Thank you, Dr. Gunter Schmitdke for patiently answering all my stupid questions and for all your humor.

An even bigger thanks goes to the old and new members of our Tussi-Box, especially to my “second Marc” Dr. Valerie Herrmann, Dr. Stefanie “Stef” Bürger and my super-fancy unicorn Richi Schregle. Thank you so much for your helpfulness and support – and for drying my tears in times of greatest desperation. Let alone, I need to mention all the other members of the P11 lab, especially my Western Baby Prof. Lili, Jun Li and my students Leonie, Franzi and Elena. I appreciate a lot that you survived my immense work-load during supervision. Furthermore, I would like to say thank you to my soul-mate Julia Malaika for your support and friendship. And I even have to extent my thanks to the whole Brunner lab, especially to Astrid. Thank you all a ton for making scientific work so much fun.

Additionally, I am in deep gratefulness for the whole TFA staff. Thank you so much Heidi, Birgitt, Andrea and Janine for all the kindness and help and for everything apart from work. Everyone would wish to have friends like you. One of the biggest gratitude I need to speak to the outmost supervisors I had and I will ever have. I cherish Dr. Marc Müller and Dr. Feodora Ivanova for ever-present support and brilliant guidance and advice – even when you are no longer around. Not only speaking of all the scientific knowledge I gained, but thank you for the best moments I had in my life.

My heartfelt thanks also goes to my BFF and my sister-of-another-mother Katrin. Thank you for never losing faith in me and for your big heart and trust in mine. #K= ♥.

And….Matthias: Just thank you for making our days together so wonderful and so much worth living it.

Finally, I sincerely thank my brother for his love, encouragement and everything he did for me. I would like to dedicate this thesis to you, since I would not be here right now without you.

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