Institute for Experimental Cancer Research

Ulm University

Chair: Prof. Dr. med. C. Buske

The role of MAPRE2 as a potential key gene in leukemic

stem cells

Thesis submitted for the doctoral degree in Medicine at the Faculty of Medicine

Ulm University, Germany

Sascha Winkler

Böblingen

2020 Sitting Dean: Prof. Dr. Thomas Wirth

First Examiner: Prof. Dr. med. Christian Buske

Second Examiner: Prof. Dr. med. Christian Sinzger

Date of oral examination: 03.07.2020

Table of Contents

Abbreviations ...... III

1 Introduction ...... 1

1.1 Acute myeloid leukemia ...... 1

1.2 The cancer stem cell model ...... 4

1.3 CALM-AF10 ...... 7

1.4 MAPRE2 ...... 10

1.5 RNA interference ...... 11

1.6 Aim of study ...... 16

2 Materials and methods ...... 18

2.1 Materials ...... 18

2.2 Methods ...... 22

3 Results ...... 28

3.1 Mapre2 expression in CALM/AF10 positive tertiary leukemic murine bone marrow ...... 28

3.2 In vitro knockdown in CALM/AF10 cell line ...... 29

3.3 In vitro knockdown in tertiary leukemic bone marrow cells ...... 30

3.4 FACS sorting for transduced B220+/Mac1- population ...... 31

3.5 Knockdown efficiency prior to injection ...... 32

3.6 Proliferation assay ...... 33

3.7 Colony forming cell assay ...... 34

3.8 Overall survival ...... 35

3.9 Transduction efficiency in ex vivo bone marrow...... 36

3.10 Mapre2 expression in ex vivo bone marrow ...... 37

4 Discussion ...... 38

4.1 Results ...... 38

4.2 Limitations ...... 40 I

5 Summary ...... 45

6 References ...... 47

7 Acknowledgement ...... 55

8 Curriculum vitae ...... 56

II

Abbreviations

ABL1 ABL proto-oncogene 1

AF10 Also MLLT10, myeloid/lymphoid or

mixed-lineage leukemia; translocated to

chromosome 10

ALL Acute lymphoblastic leukemia

AML Acute Myeloid Leukemia

APC Allophycocyanin

APC Adenomatous polyposis coli

APL Acute Promyelocytic Leukemia

As2O3 Arsenic trioxide

ATRA All-Trans Retinoic Acid

BCR RhoGEF and GTPase activating protein

BFP Blue fluorescing protein

CaCl2 Calcium chloride

CALM Also PICALM, phosphatidylinositol

binding clathrin assembly protein

CBF Core Binding Factor

Cdk1 Cyclin dependent kinase 1

CEBPA CCAAT/enhancer-binding protein alpha

CFC Colony Forming Cell

CLASP CLIP associated protein

CLIP Cytoplasmic linker protein

CLP Common lymphoid progenitor

III

CMV Cytomegalovirus cPPT Central polypurine tract

CSC Cancer Stem Cell

DMEM Dulbecco’s Modified Eagle’s Medium

DNA Deoxyribonucelic acid

FACS Flow Activated Cell Sorting

FBS Fetal bovine serum

FLT3-ITD Fms-like tyrosine kinase 3 internal tandem

duplication

FSC Forward scatter

GFP Green fluorescing protein

Gr-1 Also Ly6G (lymphocyte antigen 6)

H3K79 Histone H3 lysine 79

HEPES 2-[4-(2-hydroxyethyl)piperazin-1-

yl]ethanesulfonic acid

HiDAC High dose cytarabine

HIV-1 Human immunodeficiency virus type 1

HOXA Homeobox A

HSC Hematopoietic stem cell

HSCT Hematopoietic stem cell transplantation

IDH Isocitrate dehydrogenase

IG Immunoglobulin

KIT KIT proto-oncogene, receptor tyrosine

kinase

LSC Leukemic stem cell

IV

LTR Long terminal repeats

Mac-1 Also ITGAM (integrin subunit alpha M) or

CD11b

Mapre2 Microtubule associated protein RP/EB

family member 2

MDS Myelodysplastic syndrome

Meis1 myeloid ecotropic viral integration site 1

homolog miRNA Micro RNA

MLL Mixed lineage leukemia

MM Myeloid marker

MPO Myeloperoxidase

NOD/SCID Non-obese diabetic severe combined

immunodeficiency

NPM1 Nucleophosmin 1

NSG NOD-SCID with mutation of interleukin 2

receptor gamma chain

PARP1 Poly(ADP–ribose) polymerase 1

Pax5 Paired box 5

PE Phycoerythrin

PI Propidium iodide pmD2.G VSV-G envelope expressing plasmid

PML Promyelocytic leukemia

PRE Posttranscriptional regulatory element

PS Penicillin / streptomycin

V psPAX2 2nd generation lentiviral packaging plasmid qRT-PCR Quantitative real-time polymerase chain

reaction

Rag2 Recombination activating gene 2

RARA Retinoic acid receptor alpha

RISC RNA induced silencing complex rmIL-3 Recombinant murine interleukin 3 rmIL-6 Recombinant murine interleukin 6 rmSCF Recombinant murine stem cell factor

RNA Ribonucleic acid

RNAi RNA interference

Rpm Rotations per minute

RSV Rous sarcoma virus

RUNX1 Runt related transcription factor 1

SFFV Spleen focus-forming virus shRNA Short hairpin RNA siRNA Short interfering RNA

SSC Sideward scatter

TME Tumor microenvironment

TUBB Beta tubulin

VCM Virus containing medium

VpreB Pre-B lymphocyte gene

VSV Vesicular stomatitis virus

VI

1 Introduction

1.1 Acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignant disease of hematopoietic stem and progenitor cells where disruption of the balance between differentiation and proliferation leads to the expansion of dysregulated immature myeloid blasts that infiltrate the bone marrow and blood but may also affect central nervous system, internal organs or the skin.

While AML may manifest at any age, it predominantly affects the elderly with a median age of onset of 72 years in the industrialized world and highest incidences between the ages of

80 and 85 years (33). After induction therapy with standard chemotherapeutic regimens consisting of anthracyclines and cytarabine, overall survival for patients younger than 60 years of age is approximately 40-60 % after first line treatment. In the group of patients over

60 years of age, overall survival is reduced to 10-20 %. Furthermore, depending on individual constitution and comorbidities, patients may not be eligible for intensive induction therapy. For this subgroup of people median survival is a mere 3-6 months (16).

Overall survival also varies among subgroups of AML. According to the 2016 World Health

Organization classification of myeloid neoplasms and acute leukemia, it is divided into different categories according to the presence of recurrent genetic abnormalities, such as cytogenetic abnormalities (e. g. balanced translocations as in PML-RARA or BCR-ABL1) and single gene mutations (e. g. NPM1, RUNX1 and CEBPA), as well as prognostic significance of these changes. Further classification also comprises the etiology, such as association with

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myelodysplasia-related changes, chemotherapy-related AML or AML related to Down syndrome (2).

Tab. 1: 2016 revision of the WHO Classification of acute myeloid leukemia (2)

AML with recurrent genetic abnormalities

AML with t(8;21)(q22;q22.1);RUNX1-RUNX1T1

AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11

APL with PML-RARA

AML with t(9;11)(p21.3;q23.3);MLLT3-KMT2A

AML with t(6;9)(p23;q34.1);DEK-NUP214

AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2, MECOM

AML (megakaryoblastic) with t(1;22)(p13.3;q13.3);RBM15-MKL1

Provisional entity: AML with BCR-ABL1

AML with mutated NPM1

AML with biallelic mutations of CEBPA

Provisional entity: AML with mutated RUNX1

AML with myelodysplasia-related changes

Therapy-related myeloid neoplasms

AML, not otherwise specified (NOS)

AML with minimal differentiation

AML without maturation

AML with maturation

Acute myelomonocytic leukemia

Acute monoblastic/monocytic leukemia

Pure erythroid leukemia

Acute megakaryoblastic leukemia

Acute basophilic leukemia

Acute panmyelosis with myelofibrosis

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Myeloid sarcoma

Myeloid proliferations related to Down syndrome

Transient abnormal myelopoiesis (TAM)

Myeloid leukemia associated with Down syndrome

Although a lot of progress has been made in classifying subgroups in AML, therapy largely relies on classic cytotoxic therapy and hematopoietic stem cell transplantation (HSCT).

Therapy consists of an induction therapy, most commonly a “3+7” regimen with administration of anthracyclines for three days and cytarabine for seven days. Once complete remission is achieved, postremission treatment or consolidation therapy is initiated, either by administration of high dose cytarabine (HiDAC) or HSCT depending on disease characteristics, relapse risk and patient morbidity (17). Only for a small subset of entities, e. g. Acute Promyelocytic Leukemia (APL), is there an established targeted therapy available. Regimens containing all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) lead to a degradation of the PML-RARA fusion protein responsible for this certain subtype of AML and thereby offer a causative approach of therapy (12). Other subtype-specific agents such as midostaurin for FLT3-ITD positive AML, dasatinib for Core Binding Factor

(CBF)-AML with KIT mutations or specific inhibitors for mutated isocitrate dehydrogenase

(IDH) and many more are in the stages of clinical trials (16, 17).

Another therapeutic difficulty with AML lies in the high rate of relapse after attaining first complete remission, which may be as high as 50% of patients. In these cases, response rates to salvage therapies have been shown to be decreased (6, 65). This is partly due to the existence of so called Leukemic Stem Cells (LSCs), which have an innate resilience to cytotoxic agents as well as the ability to attain new mutations by clonal differentiation and 3

selection processes, resulting in a more aggressive leukemic phenotype at the time of relapse

(38, 48).

1.2 The cancer stem cell model

1.2.1 General concepts

The recent understanding of cancer is that it consists of phenotypically and genetically heterogeneous subpopulations of cells rather than being a homogeneous mass of cells (29,

38), with evidence for clonal evolution during tumorigenesis (31, 61). A putatively functional subpopulation of cells with so called “stemness properties”, most commonly referred to as Cancer Stem Cells (CSC), resides at the top of the tumor cell hierarchy.

Stemness is defined as the entirety of molecular and functional properties that maintain the stem cell state, including self-renewal and long term clonal repopulation (38). In most cancers, CSCs are a rare subpopulation accounting for only up to 2 % of cells (47, 54, 57), although this number may be underestimated due to a bias for aggressive growth behavior in most assays (49). Non-CSCs may (re)acquire self-renewal capacities hinting at CSCs being a functional population with a high degree of plasticity rather than a distinct subpopulation of cells (9, 28, 55). Although there have been attempts to define cell surface marker profiles to isolate CSCs in different types of tumors, as of yet the goldstandard in identification of CSCs remains a functional assay, i. e. a serial xeno-transplantation assay in animal models (29, 38). Different strains of immunodeficient mice, such as NOD/SCID or

NSG, are transplanted or injected with tumor cells to test for their ability to induce tumors and to give rise to phenotypically distinct subpopulations. In case of a CSC, the new-formed tumor will share similarities with its parent tumor and consists of a varying subset of new

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CSCs as well as different subpopulations of bulk tumor cells. This holds true not only for leukemias but also for different solid tumors, as has been demonstrated in breast, prostate, colorectal and brain tumors (1, 32, 47, 57). Newer animal models are further engineered to recapitulate human tumor microenvironment (TME), a specific cellular and metabolic niche hosting CSCs. As of yet the precise origin of CSCs is unclear and may differ between different types of cancer. Major hypotheses include firstly the transformation of normal stem cells, secondly the dedifferentiation of more mature cancer cells with epithelial- mesenchymal transition and thirdly induced pluripotent cancer cells (29).

Fig. 1: Model of Cancer Stem Cells and clonal evolution. Different colors represent different genetic subclones.

From left to right broadening of the graph illustrates expansion of the founder clone (green). By obtaining further mutations, different subclones (blue, purple, grey, black) evolve in parallel. On the bottom, different levels of hierarchy within the subclones is represented. Within the green subclone a steep developmental hierarchy is depicted with a low CSC frequency. In contrast, in the black subclone on the right, almost all cells

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have CSC properties. Reprinted from: Kreso A, Dick JE: Evolution of the cancer stem cell model. Cell stem cell 14: 275–291 (2014) with permission from Elsevier (38).

1.2.2 Clinical relevance

In recent years, more and more evidence is pointing at CSCs being an important factor for chemotherapeutic resistance of tumors, disease relapse and metastatic disease (4, 22, 26, 53,

63, 64). CSCs have been shown to have upregulated ATP-binding cassette proteins (ABC transporters), including P-glycoprotein (MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), thereby increasing efflux of cytotoxic agents and thus rendering chemotherapy less effective against this subpopulation of cells (26, 56). Other mechanisms of drug resistance in cancer cells include the epigenetic inactivation of enzymes necessary to convert pro-drugs into their respective active components, e. g. capecitabine resistance by inactivation of thymidine phosphorylase (37). Mutations in proteins governing cell cycling and DNA damage repair mechanisms are another possible resistance mechanism, as many cytotoxic agents such as anthracyclines or topoisomerase-inhibitors induce DNA damage and thus cell cycle arrest in affected cells. Interestingly, cancers may exhibit a functional dependence on a certain DNA damage repair pathway due to lacking or dysfunctional alternative pathways, termed “synthetic lethality” (34). This is exploited in breast and ovarian tumors harboring the BRCA1/BRCA2 mutation by inhibition of single stranded

DNA repair pathways via PARP1 (poly(ADP–ribose) polymerase 1)-inhibitors (21). As a result of tumor heterogeneity, all of the above mentioned resistance mechanisms may be innate in cancer cells or may be selected for by cytotoxic therapies (26). Additionally, due to the slow cell cycling of CSCs, these cells possess an innate resilience against many classes of chemotherapeutic agents aimed at rapidly proliferating cells. Furthermore, properties of

CSCs can influence overall survival as has been shown by Eppert et al.: After establishing a 6

gene expression signature from functionally validated LSCs as well as HSCs and showing an overlap in these signatures responsible for the “stemness” properties of both cell populations, Eppert et al. showed that these signatures were able to predict the clinical outcome in patients with cytogenetically normal AML. High expression of the LSC signature could identify poor patient outcome and patient populations who might profit from HSCT despite the favorable prognosis associated with cytogenetically normal AML (20).

Considering these features of CSCs, future therapies must be designed to eradicate this subpopulation of cells, that is most likely contributing to poor patient survival, disease relapse and treatment failure. Hence, more studies to elucidate the biology of these cells are needed to design adequate targeted therapies.

1.3 CALM-AF10

1.3.1 Biochemistry

The CALM-AF10 fusion protein results from the chromosomal translocation t(10;11)(p13-

14;q14–21) and is a rare but recurring chromosomal rearrangement in AML, ALL and malignant lymphoma. The leucine zipper motif (OM-LZ) on the C-terminal end of AF10 and the clathrin-binding domain of CALM together exert a leukemogenic effect by interacting with Dot1l, a histone H3 lysine 79 methyltransferase, leading to global hypomethylation of histone H3K79 and subsequent chromosomal instability, as well as local hypermethylation of H3K79 at HOXA gene clusters (11, 14, 40) . Further effects of CALM-

AF10 include interaction with cellular transcription factors such as Ikaros (24), as well as upregulation of genes involved in chromatin assembly and DNA repair processes.

Interestingly, CALM-AF10 positive leukemias show similar patterns of differential gene

7

expression with MLL (mixed lineage leukemia) fusion protein positive leukemias, which also have upregulated HOXA cluster genes, as well as upregulation of e. g. MEIS1 (myeloid ecotropic viral integration site 1 homolog) (46). HOX cluster genes A-C are important factors in physiological hematopoiesis.

Clinical characteristics of CALM-AF10 positive leukemias include younger age of onset with a mean age of 20,7 years (3 to 43 years) in a patient collective of T-ALL (8), and 25,6 years (4 to 47 years) in AML (13), as well as poor clinical outcome with overall survival of

11 ± 7,2 months (18). In AML, it is associated with hepatosplenomegaly, mediastinal masses and central nervous system leukemia (8). Of interest, CALM-AF10 positive AML show clonal rearrangements in IGH and TCRG/TCRD genes, suggesting that these cases originate from a multipotent hematopoietic precursor cell (3, 8).

Fig. 2: Schematic depiction of the CALM-AF10 fusion protein with its different domains. ENTH: Epsin N- terminal homology, TAD: potential trans-activating domain, NES: nuclear export signal, PHD: plant homeodomain, ePHD: extended PHD, NLS: nuclear localization signal, OM-LZ: octapeptide motif and leucine zipper. Reprinted by permission from Springer Nature Customer Service Centre GmbH: Springer Nature.

Leukemia. The clathrin-binding domain of CALM and the OM-LZ domain of AF10 are sufficient to induce acute myeloid leukemia in mice, Deshpande AJ et al. [2011] (14).

1.3.2 Mouse model

Deshpande et al. established a murine model of CALM-AF10 positive leukemia by constitutively expressing CALM-AF10 in hematopoietic progenitor cells via retroviral gene 8

transfer. While nearly all cells expressed myeloid markers (MM), e. g. Gr1 and Mac1, a subpopulation of cells expressing B220, a lymphoid marker and murine homolog of human

CD45RA, without MM could be identified. Other molecular characteristics of this subpopulation include IG DH-JH rearrangements, and expression of lymphoid factors such as EBF, VpreB and Rag2, while simultaneously expressing myeloid markers such as MPO.

Collectively, this subpopulation is classified as a lymphoid progenitor. It does not qualify as a common lymphoid progenitor (CLP), which do not express B220, nor does it qualify as an early B-cell due to the lack of Pax5 and CD19 expression. Interestingly, in limiting dilution assays this B220+/MM- population showed to have the highest frequency of LSC at around

1/36 cells. These cells were capable of giving rise not only to the B220+/MM- populations, but to the MM+ subpopulations as well and thereby recapitulating the original disease after transplantation into irradiated mice. While the precise cell of origin for this CALM-AF10 leukemia is unknown, Dutta et al. have shown that it must be either a hematopoetic stem cell or multilineage progenitor cell, by employing panhematopoietic expression of CALM-AF10 in a more physiological model of hematopoiesis (19). In the mouse model of Deshpande et al., upon serial transplantation, disease aggressiveness increased from a median overall survival of around 110 days post transplantation in primary transplants to a median of 22 days in secondary transplants. Furthermore, the authors showed that this murine model well depicts the human CALM-AF10 positive leukemias. Both are characterized by myeloid disease with lineage-promiscuous expression of B220/CD45RA and IG DH-JH rearrangements (13).

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1.4 MAPRE2

1.4.1 Biochemistry

The human MAPRE2 gene (microtubule associated protein end binding 2) codes for the RP1 protein from the EB1 family of proteins, also termed EB2 (see Fig. 3). The two other members of this gene family are MAPRE1, coding for EB1, and MAPRE3, coding for EB3.

The EB1 protein family are microtubule binding proteins with different functions in mitosis and cell adhesion. The MAPRE2 gene is located on Chromosome 18 and codes for two different isoforms of RP1 depending on the start codon. While EB1 and EB3 are known to interact with APC (Adenomatous polyposis coli) and other plus-end tracking proteins, such as CLIPs (cytoplasmic linker proteins), CLASPs (CLIP associated proteins), and others via its C-terminal domain, RP1 does so to a lesser extent (23, 60). It binds to microtubules plus ends with its N-terminal caponin homology domain. It acts as a key regulator in initial microtubule organization during apico-basal epithelial differentiation by enhancing microtubule dynamics, especially in early stages of differentiation. In later stages, it seems to be downregulated leading to more stable microtubule structures. Accordingly, low RP1 expression in early stages inhibits microtubule reorganization (23). Iimori et al. discovered the role of RP1 in mitotic progression. Phosphorylation by CDK1 (cyclin dependent kinase

1) and Aurora B is required for regular mitosis, otherwise lengthened duration of NEBD

(nuclear envelope breakdown) to metaphase alignment and metaphase to anaphase onset ensue, resulting in mitotic delay as well as chromosomal instability (27). Additionally, RP1 is involved in cell adhesion as shown by Stenner and colleagues. They showed increased resistance to shear stress by phosphorylation and subsequent degradation of RP1, accordingly high expression of RP1 meant reduced cell adhesion (59). These findings verify

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the assumptions of Renner et al., who discovered that activation of T-lymphocytes leads to rapid upregulation of MAPRE2, and postulated that RP1 plays a role in cell cycling (52).

1.4.2 Clinical relevance

Clinically, MAPRE2 has been involved in a number of different entities. Firstly, it is one of two main genes that leads to the so-called “Michelin Tire Baby”, or Circumferential Skin

Creases Kunze Type disease. It is a congenital disease caused by mutations in either

MAPRE2 or TUBB (beta tubulin), thus classifying it as a microtubulopathy. The phenotype consists of intellectual disability, circumferential skin creases, facial dysmorpism (e. g. cleft palate) as well as cardiac and genital anomalies (30). Secondly, Kleeff et al. have shown

MAPRE2 to be upregulated in pancreatic cancer, especially in cases with high perineural invasion and thus a potential key player in disease relapse (35). Lastly, in a study by Tran et al. MAPRE2 was part of a gene signature with high positive prognostic value for NSCLC

(non-small cell lung carcinoma), underlining the potential role of MAPRE2 in carcinogenesis

(62).

Fig. 3: Schematic representation of the Mapre2 (RP1) protein with its functional domains, adapted with permission from: Bu W, Su L-K: Characterization of functional domains of human EB1 family proteins. The

Journal of biological chemistry 278: 49721–49731 [2003] (7).

1.5 RNA interference

In mammalian cells, regulation of gene translation may occur by RNA interference (RNAi).

Endogenous microRNA (miRNA) are expressed in the nucleus and, due to intramolecular complementarity, form hairpin structures. This Pri-miRNA is then processed by the RNAse 11

III Drosha and its dsRNA-binding partner DCGR8 to form a Pre-miRNA. Pre-miRNAs are exported via Exportin5 into the cytoplasm and further processed to approximately 20-25 nucleotide long active miRNA by the RNAse III Dicer. These double-stranded RNA molecules are loaded onto the RNA induced silencing complex (RISC) and subsequently unwinded. Now RISC can bind homologous mRNA and in case of complete complementarity between the miRNA and the mRNA lead to cleavage of the target mRNA.

If mismatches between the miRNA and target mRNA are present, instead of cleaving the mRNA, the binding leads to translational repression (39).

1.5.1 shRNA mediated RNA interference

Short hairpin RNA (shRNA) are artificial RNA molecules that make use of the RNA interference pathway. A DNA coding for a shRNA molecule is integrated into the target cell via plasmids or viral vectors leading to promoter dependent expression of the shRNA within the nucleus. The secondary structure of a shRNA consists of a stem region (paired sense and antisense strand) and a loop region (unpaired nucleotides) connecting the 3’ end of the shRNA sense strand and the 5’ end of the antisense strand. The shRNA then is exported to the cytoplasm by Exportin5, similarly to pre-miRNA, and further processed by the same pathway leading to mRNA degradation or translational repression via RISC. Compared to

RNA interference using synthetic siRNA, which are introduced into the cytoplasm and after being processed by Dicer lead to RISC formation, shRNA lead to a more effective and sustained gene knockdown (39, 58), as well as less toxic off-site effects (36). Furthermore, employing shRNA modelled after the endogenous miRNA, e. g. by introducing miRNA loop sequences or imperfect base pairing within the stem region, may lead to increased efficacy and reduction of side effects (39).

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Fig. 4: shRNA mediated RNA interference. (1) Adhesion of lentiviral particle and release of vectors into the cytosol, (2) integration of shRNA vectors into target cell genome, (3) expression of pri- shRNA, (4) processing to pre-shRNA by Drosha/DGCR8 (light grey), (5) export of pre-shRNA into the cytoplasm via Exportin5 (blue),

(6) further processing by Dicer (dark grey) and loading onto RISC (light grey), (7) unwinding of double- stranded shRNA to single strands, (8) binding of homologous mRNA by RISC, (9) cleavage of target mRNA

(right) and translational repression by formation of p-bodies in case of mismatches between shRNA and target mRNA (left). Adapted from Rao DD, Vorhies JS, Senzer N, Nemunaitis J: siRNA vs. shRNA. Similarities and differences. Advanced drug delivery reviews 61: 746–759 (2009) with permission from Elsevier (50).

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1.5.2 Lentiviral vectors

Retroviral vectors are an established route of delivering transgenes into target cells for research purposes because of their ability to stably integrate their DNA into the host genome.

But since simple retroviral vectors can only integrate into actively dividing cells, lentiviral vectors derived from HIV-1 (human immunodeficiency virus type 1) came into focus due to their ability to target both dividing and non-dividing cells (41). The HIV-1 genome consists of 9 genes: gag, pol, env, tat, rev, vif, vpr, vpu and nef. Gag codes for the viral matrix, the capsid, the nucleocapsid and the p6 protein. Pol codes for the enzymes reverse transcriptase, integrase and protease and is thus another key element of HIV-1. Env, the third gene found across all retroviruses, codes for the viral envelope glycoprotein gp160, which is processed into the gp120 and gp41 proteins relevant for cell adhesion of the virus. Tat codes for a transcriptional activator protein that induces viral transcription from the LTR (long terminal repeats) promoter. The Rev protein plays a role in nuclear export of virus transcripts through

RRE (rev response element). The four latter genes code for accessory proteins that play a role in infectivity and pathogenicity of native HIV-1. For research and laboratory safety, the lentiviral genome is engineered to reduce the risk of creation of replication competent lentiviruses. The genes are spread across multiple different vectors containing the transgene bearing transfer vector and separate vectors coding for the packaging proteins (e. g. pmD2.G or psPAX2). Self-inactivating vectors were engineered to further reduce replicative potential of the lentiviral constructs by deletion of enhancer and promoter sequences in the 3’ LTR, resulting in transcriptional deactivation of the LTR promoter after transduction of target cells

(44, 66). In so-called third generation lentiviral vectors, the LTR promoter is exchanged for a constitutive promoter derived from Rous Sarcoma Virus (RSV) or Cytomegalovirus

(CMV), thereby enabling transcription independent of Tat (44). Further additions, like 14

inclusion of a central polypurine tract (cPPT) and a posttranscriptional regulatory element

(PRE), lead to increased transduction efficiency, deletion of the accessory HIV-1 genes lead to improved safety. Moreover, the packaging construct was also successively improved, by moving the env gene to a different vector, by deletion of the packaging signal ψ, thus preventing packaging into virions, as well as using a different envelope protein derived from the Vesicular Stomatitis Virus (VSV)-G envelope protein, broadening the cellular tropism of the viral constructs (41).

Fig. 5: Schematic depiction of an HIV-1 particle. gp120 (envelope glycoprotein 120), gp41 (envelope glycoprotein 41). Adapted by permission from Springer Nature Customer Service Center GmbH: Springer

Nature. Methods in Molecular Biology. HIV-1-based lentiviral vectors. Liu YP, Berkhout B, 2014 (41).

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Fig. 6: Schematic representation of a lentiviral vector system. (a) Third generation lentiviral transfer vector.

Deletion of enhancer and promoter sequences in the 3’ LTR U3 region leads to self-inactivation. The central polypurine tract (cPPT), as well as the posttranscriptional regulatory element (PRE) increase transcription and transgene expression. GFP is included as a marker for successful transduction. (b) Third generation lentiviral packaging system. The first vector contains the VSV envelope for improved cell tropism. The second vector codes for the matrix proteins as well as the reverse transcriptase, integrase and protease. Additionally, the accessory proteins vif, vpr, vpu and nef, as well as the packaging signal ψ are deleted. All 5’ LTRs are replaced by constitutive promoters derived from CMV or RSV resulting in a Tat-independent expression.

Adapted by permission from Springer Nature Customer Service Center GmbH: Springer Nature. Methods in

Molecular Biology. HIV-1-based lentiviral vectors. Liu YP, Berkhout B, 2014 (41).

1.6 Aim of study

This study aims to investigate the effect of single gene knockdown of MAPRE2 on the LSC phenotype both in vitro and in vivo in a model of CALM-AF10 positive AML by employing

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lentiviral RNA-interference. Eppert et al. established a LSC-specific gene expression signature prognostic of clinical outcome in cytogenetically normal AML patients (20).

Among these genes upregulated in LSC is MAPRE2, a microtubulus-associated protein with putative roles in cell differentiation and adhesion. Thus far, there has not been substantial data about MAPRE2 in the context of acute leukemia and stem cell biology. Therefore, in an effort to find new potential key genes in leukemogenesis and leukemic stem cell biology, we decided to further evaluate this specific gene. In more detail the project aimed first at testing the effect of single gene knockdown on in vitro properties, such as proliferation and colony cell formation; secondly, in vivo, by testing whether knockdown of the gene impacts leukemic engraftment and disease latency and with this survival in the CALM-AF10 AML model.

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2 Materials and methods

2.1 Materials

2.1.1 Consumables

Chloroform Appli Chem, Darmstadt, Germany

DEPC-treated water Applied Biosystems, Foster City, CA, USA

DMEM PAN Biotech, Aidenbach, Germany

DMSO Sigma-Aldrich, Taufkirchen, Germany

DPBS PAN Biotech, Aidenbach, Germany

Dynabeads-M280 strepavidin Invitrogen

Ethanol Sigma-Aldrich, Taufkirchen, Germany

FBS PAN Biotech, Aidenbach, Germany

HBSS Lonza

Histopaque 1083 Sigma-Aldrich, Taufkirchen, Germany

IMDM Gibco /Invitrogen, Carlsbad, CA, USA

Isopropanol Sigma-Aldrich, Taufkirchen,Germany

Methylcellulose M3434 Stem Cell Technologies, Vancouver, Canada

Penicillin/Streptomycin PAN Biotech, Aidenbach, Germany

Propidium Iodide Sigma-Aldrich

Sytox blue Gibco /Invitrogen, Carlsbad, CA, USA

TBS Appli Chem, Darmstadt, Germany

Trizol Gibco /Invitrogen, Carlsbad, CA, USA

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Trypan blue solution Sigma-Aldrich, Taufkirchen, Germany

Trypsin EDTA Gibco /Invitrogen, Carlsbad, CA, USA

2.1.2 Technical equipment

12-well culture plate Sarstedt, Nümbrecht, Germany

15 ml Falcon tube Sarstedt, Nümbrecht, Germany

24-well culture plate Sarstedt, Nümbrecht, Germany

50 ml Falcon tube Becton Dickinson, Franklin Lakes, NJ, USA

6-well culture plate Sarstedt, Nümbrecht, Germany

7900 HT Fast Real Time PCR System Applied Biosystems, Foster City, CA, USA

Cell incubator Binder, Tuftlingen, Germany

Centrifuge 5810, 5415R Eppendorf, Leipzig, Germany

Corning dish Corning, NY, USA

Cryo tube Nunc, Roskilde, Denmark

Culture flask (20 ml, 200 ml) Sarstedt, Newton, NC, USA

Cytospin 4 Centrifuge Thermo Fisher Scientific, Waltham, MA, USA

Cytospin cuvette Thermo Fisher Scientific, Waltham, MA, USA

Cytospin filter Thermo Fisher Scientific, Waltham, MA, USA

Eppendorf tube 1,5 ml Roth, Karlsruhe, Germany

FACS Aria III Sorter Becton Dickinson, Franklin Lakes, NJ, USA

FACS Calibur with Cellquest software Becton Dickinson, Franklin Lakes, NJ, USA

FACS tube 5 ml Becton Dickinson, Franklin Lakes, NJ, USA

Filter tip Sarstedt, Newton, NC, USA

Fridge Liebherr, Ochsenhausen, Germany

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Insulin needle Becton Dickinson, Franklin Lakes, NJ, USA

Liquid nitrogen Chart Biomed, GA, USA

Microscope Axiovert 40C Carl Zeiss AG, Jena, Germany

Needle Becton Dickinson, Franklin Lakes, NJ, USA

Neubauer counting chamber Laboroptik, Friedrichsdorf, Germany

PCR soft tube Biozym, Oldenburg, Germany

PCR Thermocycler PeqLAB Biotechnologie, Erlangen, Germany

Petri dish Becton Dickinson, Franklin Lakes, NJ, USA

Pipette Eppendorf, Leipzig, Germany

Precision balance Ohaus, Nänikon, Switzerland

Slide Roth, Karlsruhe, Germany

Spectrophotometer Nano Drop 1000 Thermo Fisher Scientific, Waltham, MA, USA

Syringe Becton Dickinson, Franklin Lakes, NJ, USA

Syringe filter (0,70 µl, 0,45 µl, 0,22 µl) Millipore, Billerica, MA, USA

Vortexer Scientific Industries, NY, USA

Water bath GFL, Burgwedel, Germany

2.1.3 Cytokines and antibodies rmIL-3 ImmunoTools, Friesoythe, Germany rmIL-6 ImmunoTools, Friesoythe, Germany rmSCF ImmunoTools, Friesoythe, Germany anti-CD16/32 ebioscience, Hatfield, United Kingdom anti-Mac1 BioLegend, San Diego, USA anti-B220 APC BD Biosciences, Heidelberg, Germany

20

2.1.4 Buffers and stock solutions

Freezing medium 90% FBS, 10% DMSO

Cell cultivating medium adherent cells 90% DMEM, 10% FBS

Cell cultivating medium murine cells 85% DMEM, 15% FBS, rmIL-3, rmIL-6, rmSCF

2.1.5 Kits

RNAzol Sigma-Aldrich, Taufkirchen, Germany

PrimeScript RT reagent kit Takara Bio, Shiga prefecture, Japan qPCR Sigma Aldrich, Taufkirchen, Germany

2.1.6 shRNA sequences

SFFV-shRNA-BFP-GIPZ KL Rudolph, Fritz Lipmann Institute, Jena

Anti-Mapre2-shRNA In house

2.1.7 Cell lines

LentiX 293T cell line Clontech, Mountain View, CA, USA

CALM-AF10 cell line B220-/MM+ population of primary leukemic

Calm/Af10 positive recipient mice expanded in

vitro

2.1.8 Animals

C57BL/6 x C3H/HeJ In house

All animal experiments were conducted according to the University Ulm animal facilitiy’s policies after obtaining an ethics approval (reference no. 35/9185.81-3 / Versuch-Nr. 996).

21

2.2 Methods

2.2.1 Cell cultivation

Adherent cell lines were cultivated in DMEM plus 10 % FBS and 1 % PS. Suspension cells, including murine bone marrow cells, were cultivated in DMEM plus 15 % FBS and 1 % PS.

For murine bone marrow cells, recombinant murine IL-3, IL-6 and Stem Cell Factor were added. The cells were split into 1:10 dilutions every 2 to 3 days due to their high doubling rate (approximately 24 hours) and to maintain log phase growth.

2.2.2 Thawing and freezing cells

Frozen cells were incubated in a water bath at 37°C while shaking gently and transferred to a Falcon tube directly after thawing. 10−20 ml of medium were added dropwise. After centrifugation at 1100 rpm for 5−10 minutes the cell pellet was washed twice with warm medium to remove residues of DMSO. The cells were then transferred to 10 cm Corning dishes or culture bottles.

If necessary for freezing, the cells were trypsinized and centrifuged under the conditions specified above. 1 ml of freezing medium per 5×105–5×107 cells was pipetted slowly onto the cell pellet. Aliquots of 1 ml were frozen in cryo tubes at −80 °C for short-term storage and subsequently to the liquid nitrogen tank for long-term storage.

2.2.3 Viability test and cell counting

Trypan blue dye was used to determine the number and proportion of live cells. Trypan blue is an anionic azo dye which binds to cell proteins. Due to the defective cell membranes of dead cells, trypan blue penetrates the cytosol of the cells and results in a dark blue stain. In contrast, live cells show a clear cytoplasm under the microscope at 10- to 40-fold

22

magnification. To count the cells, 10 µl of the cell suspension were diluted with 10 µl of trypan blue (0.4 %) and the cell number per ml was calculated in the Neubauer counting chamber as follows:

Mean value of 4 large quadrants × 2 (dilution factor) × 104 (chamber factor)

2.2.4 Lentiviral constructs

LentiX 293T cells at a confluency of 70% were incubated with CaCl2, pmD2.G, psPAX2, the shRNA vector and HEPES buffer for 24 hours. The medium was changed and the cells cultivated for another 48 hours. The supernatant, or virus containing medium (VCM), was then filtered through 45 µm cell strainers and subsequently ultra-centrifuged at 25.000 rpm.

The resulting pellet containing the viral constructs was then dissolved in 1 µl of DPBS per

1 ml of VCM thereby concentrating the original VCM 1000-fold.

2.2.5 Transduction of CALM-AF10 cell line

106 cells were incubated with 10 µl of 1000-fold concentrated VCM in 1 ml of medium for

24 hours. The cells were then centrifuged at 1100 rpm to remove the VCM and subsequently cultured for 48 hours in appropriate culture medium.

2.2.6 Transduction of murine CALM-AF10 bone marrow

B220+/Mac1- cells from murine CALM-AF10 positive tertiary leukemic bone marrow were inoculated with 30 µl of 1000-fold concentrated VCM for 24 hours. The cells were then centrifuged at 1100 rpm to remove the VCM and subsequently washed with DPBS and further cultivated in appropriate medium for 48 hours.

23

2.2.7 Transplantation of cells into mice

Recipient mice were lethally irradiated with 1200 cGy. After immobilizing recipient mice in an appropriate restrainer, the tail vein was identified by swabbing with gauze dampened in alcohol. The cells were suspended in 100 µl of sterile DPBS and injected into the tail vein with a 27 Gauge needle. After injection, recipient mice were monitored for 5-10 minutes to ensure appropriate hemostasis.

2.2.8 Bone marrow and organ harvesting from mice

Donor mice were anesthetized with CO2 and subsequently sacrificed by breaking their necks and bleeding out by puncturing the heart with a 27 Gauge needle and syringe. After dissecting the mice, liver and spleen were inspected for leukemic infiltration. The tibiae, femora and hip bones were cleaned and then crushed using a mortar and pestil and suspended in DPBS. Subsequent filtering through 45 µm cell strainers resulted in a cell suspension of bone marrow cells, which were washed again with DPBS.

2.2.9 Cell sorting of leukemic stem cells

Bone marrow cells from CALM-AF10 positive tertiary leukemic mice were stained with immunofluorescent antibodies directed against B220 and Mac1 according to standard protocols. PI was added in a dilution of 1:10000 to distinguish between live and dead cells.

By using Flow Assisted Cell Sorting (FACS), the B220+/Mac1- population was sorted out for further experiments.

2.2.10 Immunophenotyping of transduced bone marrow cells

72 hours after transduction the B220+/Mac1- were sorted by FACS according to standard protocols for the GFP+/BFP+ population. The cells transduced with our vector containing our

24

shRNA constructs also expressed BFP as a sign of successful transduction in addition to the canonical expression of GFP of the CALM-AF10 positive cells.

2.2.11 Colony forming cell assay

In order to test the effect of MAPRE2 knockdown on clonogenicity, 1000 B220+/Mac1- cells transduced with MAPRE2 shRNA or with the empty control vector were seeded in methylcellulose and incubated for 7 days. The number of cell colonies was then counted on a gridded scoring dish under the microscope.

MethoCult® medium was thawed at room temperature and 0.3 ml of diluted cells were added to 3 ml of methylcellulose. After vigorous shaking to allow for even spread of cells, it was let stand still for 5 minutes to remove any bubbles. An aliquot of 1.1 ml was then dispensed into a 35 mm culture dish with a blunt needle and evenly spread out by gently tilting the dish.

Cultures were incubated for 7 days in 5% CO2 with >95% humidity. Colonies were counted under an inverted microscope.

2.2.12 RNA extraction

After centrifugation at 1100 rpm the cells were lysed in 1 ml of Trizol and incubated at room temperature for 5−10 minutes to allow nucleoprotein complexes to dissociate completely.

After adding 200 µl of chloroform/ml Trizol and vortexing for 10 seconds, further incubation was carried out for 2−3 minutes. Subsequently, centrifugation was carried out at 12000 rpm for 15 minutes at 4°C. Phase separation of the upper phase containing RNA from the lower phase containing phenol was carried out by careful removal and transfer of the upper phase to a new 1.5 ml Eppendorf tube. For the precipitation of RNA 500 µl of isopropanol/ml

Trizol were added and the tube inverted several times. 10-minute incubation at room temperature and repeated centrifugation at 12000 rpm and 4°C followed. The supernatant 25

was discarded and the RNA pellet was washed with 1 ml of 75 % ethanol/ml Trizol and vortexed. After centrifugation at 7500 rpm and 4°C for 10 minutes at room temperature the supernatant ethanol was removed. Residual ethanol was removed by drying the pellet for

5−10 minutes at room temperature. The RNA pellet was dissolved in 20−50 µl of DEPC- treated water depending on the size of the pellet. The dissolved RNA was stored at −80°C.

2.2.13 Measurement of RNA and DNA concentration

Concentrations of RNA and DNA samples were measured in ng/µl using a Nanodrop 1000 spectrophotometer at a wavelength of 260 nm, which represents the absorption peak of nucleic acids. In order to assess the purity of the samples, the ratio of 260/280 nm signal intensity was calculated by simultaneous measurement of the absorption peak of proteins at

280 nm. The RNA sample was regarded as pure with a ratio of 1.7 to 2.0. Significantly lower ratios indicated the presence of proteins or other contaminants.

2.2.14 cDNA synthesis cDNA was synthesized from extracted RNA by using the Takara PrimeScript 1st strand cDNA synthesis kit according to the manufacturer’s instructions.

2.2.15 Quantitative real time PCR (qRT-PCR)

To assess levels of gene expression, the TaqMan system for quantitative real time PCR was used. During each cycle, a specific primer as well as a probe labelled with a fluorescent dye

(e. g. VIC or FAM) anneals to the cDNA template and during the process of synthesizing a new DNA strand, the 5’ nuclease activity of the TaqPolymerase cleaves the probe, releasing the dye. With each PCR cycle the fluorescence intensity increases proportional to the number of amplicons and thereby proportional to the initial amount of template cDNA. The number

26

of cycles to reach a certain threshold fluorescence is termed CT. By comparing the CT values for the gene of interest and housekeeping genes, relative expression can be expressed as ΔCT.

The relative expression of two genes of interest to one another can be calculated by the

2−∆∆퐶푇 method proposed by Livak et al. (42).

2.2.16 Statistical analysis

Data were evaluated with GraphPad Prism 6 using Student’s t-test. P values of < 0.05 were considered to be statistically significant.

Kaplan-Meier plots were designed with GraphPad Prism 6.

27

3 Results

3.1 Mapre2 expression in CALM/AF10 positive tertiary leukemic murine

bone marrow

Mapre2 expression was measured in bulk CALM/AF10 positive tertiary leukemic murine bone marrow as well as in four populations sorted according to surface B220 and Mac1 expression and CALM/AF10 cell line (see 2.1.7) by qRT-PCR (Fig. 7). Beta actin was used as endogenous murine control. A statistically significant two-fold increase in expression of

MAPRE2 relative to bulk bone marrow was observed in the B220+/Mac1- population and the

CALM-AF10 cell line (P < 0.01).

p = 0 .0 0 0 7

3

n

o

i s

s 2

e

r

p

x

E

e

v 1

i

t

a

l

e R

0 + - + - s ll 1 1 1 1 e e c c c c in a a l c a a ll k M - M l + /M + / - /M / e u 0 c b 0 0 0 2 2 2 /A 2 2 2 2 2 B C B B B

Fig. 7: Columns represent relative expression (average ± standard deviation) of Mapre2 in CALM/AF10 positive tertiary leukemic murine bone marrow, further subdivided into four populations according to surface

B220 and Mac1 expression, and a CALM/AF10 cell line. 28

3.2 In vitro knockdown in CALM/AF10 cell line

Four different shRNA vectors against Mapre2 were evaluated for their knockdown efficiency in the CALM/AF10 cell line by qRT-PCR (Fig. 8). Construct E4-1 induced the lowest relative expression with 0.6 ± 0.6 % (P < 0.0001). Relative expression in the scramble control was reduced to 21 ± 12.3 % compared to untreated cells (100.0 ± 12.6 %).

p < 0 .0 0 0 1

1 .5

n o

i p = 0 .0 0 7 7

s s

e 1 .0

r

p

x

E

e 0 .5

v

i

t

a

l

e R 0 .0

6 4 3 1 r - - - - c d 1 2 3 4 S te E E E E a e tr n u

Fig. 8: Columns represent relative expression (average ± standard deviation) of Mapre2 in CALM/AF10 cell line after knockdown with four shRNA constructs, a scramble control and untreated cells.

29

3.3 In vitro knockdown in tertiary leukemic bone marrow cells

Four different shRNA vectors against Mapre2 were evaluated for their knockdown efficiency in CALM/AF10 positive murine tertiary leukemic bone marrow cells by qRT-PCR.

The shRNA constructs expressed BFP as a marker for successful integration. After sorting for BFP-positive cells by FACS, relative expression of MAPRE2 was evaluated by qRT-

PCR (Fig. 9). Construct E4-1 induced the lowest relative expression at 0.5 ± 0.6 % (P <

0.0001). Relative expression in the scramble control was reduced to 17.9 ± 14.2 % compared to untreated cells (100.0% ± 14.6 %).

p < 0 .0 0 0 1 1 .5

n p = 0 .0 4 3 4

o

i

s s

e 1 .0

r

p

x

E

e v

i 0 .5

t

a

l

e R

0 .0

6 4 3 1 e - - - - l /A 1 2 3 4 b C E E E E m a d r te c a S e tr n u

Fig. 9: Columns represent relative expression (average ± standard deviation) of Mapre2 in CALM/AF10 positive tertiary leukemic murine bone marrow cells after treatment with different shRNA 48 hours post transduction

30

3.4 FACS sorting for transduced B220+/Mac1- population Bulk CALM/AF10 positive tertiary leukemic murine bone marrow cells were transduced and after 48 hours stained with monoclonal antibodies directed against surface B220 and Mac1 and sorted for the B220+/Mac1- population by FACS. A representative FACS plot is shown

(Fig. 10).

Fig. 10 Representative FACS plot of tertiary leukemic bone marrow cells sorted for the B220+/Mac1- population after transduction with BFP expressing shRNA and scramble vectors. B220 expression is represented by APC and Mac1 by PE. B220+/Mac1-(Q1), B220+/Mac1+(Q2), B220-/Mac1-(Q3), B220-

/Mac1+(Q4). BFP+/GFP+ gate (purple), GFP+ single positive gate (orange), non vital cells (green), debris

(black).

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3.5 Knockdown efficiency prior to injection

As the shRNA construct E4-1 yielded the highest knockdown efficiency (as shown in 3.2,

3.3) all further knockdown experiments were conducted with this construct. 48h after transduction of B220+/Mac1- cells with shRNA and scramble shRNA, cells were sorted into

BFP+ and BFP- populations by FACS, with a positive BFP signal being indicative of vector insertion into the target genome and serving as a surrogate for intracellular shRNA or scramble expression (Fig. 11). Relative expression of Mapre2 before injection into mice was assessed by qRT-PCR. As expected, relative expression of Mapre2 was lowest in the BFP+ shRNA population (16,5 ± 6,4 %), followed by the BFP- shRNA population (58,4 ± 12,9 %).

p = 0 .0 0 0 1 p = 0 .0 0 0 3

3 .0

n o

i 2 .5

s

s e

r 2 .0

p x

E 1 .5

p = 0 .0 0 0 1

e v

i 1 .0

t

a l

e 0 .5 R

0 .0 + - + - d P P P P e F F F F t B B a B B e A e r A le l t N N b b n R m u R h m h a a s s r r c c s s

Fig. 11: Columns represent relative expression of Mapre2 (average ± standard deviation) in B220+/Mac1- cells from CALM/AF10 positive tertiary leukemic murine bone marrow 48 hours after transduction. Cells were sorted by FACS according to their expression of BFP to show that knockdown of Mapre2 was present in cells before transplantation.

32

3.6 Proliferation assay

FACS sorted B220+/Mac1- cells were transduced with shRNA and the scramble control and seeded at a starting cell number of 10.000 cells in technical triplicates. Proliferation was measured by manual counting in a Neubauer counting chamber every 48 hours (Fig. 12). No significant differences in cell proliferation could be detected between groups.

1 0 7 u n tre a te d s c ra m b le 1 0 6

r s h R N A

e b

m 5

u 1 0

n

l

l e

c 1 0 4

1 0 1

1 0 0 0 2 4 6 8 d a y s

Fig. 12: Graph shows cell number (average ± standard deviation) over time on a logarithmic scale. Transduced

CALM/AF10 positive tertiary leukemic murine bone marrow cells were cultured with a starting cell number of

10.000 cells and counted manually in a Neubauer counting chamber every 48 hours.

33

3.7 Colony forming cell assay

After transduction of FACS sorted B220+/Mac1- cells from CALM/AF10 positive tertiary leukemic bone marrow with shRNA and the scrambled control, their in vitro colony forming properties were evaluated in the Colony Forming Cell Assay (Fig. 13). No significant differences in colony forming capabilities were found between scramble and shRNA groups.

The number of colonies was significantly higher in untreated samples (730 ± 10 colonies,

P < 0.05).

p < 0 .0 5

8 0 0

s e

i 6 0 0

n

o

l

o

c

f 4 0 0

o

r

e b

m 2 0 0

u N

0

+ - + - d P P P P e F F F F t B B a + /B + / + /B + / e r P t P P P n F F F F G U G G G e 2 2 le l e e r r b b p p m m a a a a M r r M c c h h s s s s

Fig. 13: Columns represent number of colonies counted (average ± standard deviation). 1000 cells were seeded in a three-dimensional methylcellulose matrix and colonies were counted after 7 days of cultivation.

34

3.8 Overall survival

To assess whether knockdown of Mapre2 had any influence on disease aggressiveness and overall survival in vivo, a murine transplantation experiment was conducted. A total of n = 16 lethally irradiated mice were injected with 10.000 untreated (n = 3), scramble (n = 4) and shRNA (n = 9) transduced CALM/AF10 positive tertiary leukemic murine bone marrow cells as well as 2.000.000 helper cells (regular murine bone marrow cells). Overall survival is shown in a Kaplan-Meier plot (Fig. 14). Median survival was 21 days for all groups, no significant differences in overall survival were found between groups.

1 0 0 u n tre a te d ]

% s c ra m b le

[

l

a sh R N A

v

i

v r

u 5 0

S

l

l

a

r

e

v O

0 0 1 0 2 0 3 0 4 0 5 0 d a y s p o s t tra n s p la n ta tio n

Fig. 14: Kaplan-Meier plot showing overall survival of lethally irradiated mice injected with either untreated, scramble or shRNA treated CALM/AF10 positive tertiary leukemic bone marrow cells.

35

3.9 Transduction efficiency in ex vivo bone marrow

Transduction efficiency in harvested bone marrow determined by percentage of BFP expressing cells ranged from 49.2 % to 91.8 % (mean 72.5 ± 16.9 %) for shRNA vector samples and 74.7 % to 90.1 % (mean 84.8 ± 7.1 %) for control scramble vector samples. A representative FACS plot is shown (Fig. 15).

Fig. 15: Representative FACS plot of harvested bone marrow after transplantation of transduced LSC. B220 expression is represented by APC and Mac1 by PE. B220+/Mac1-(Q1), B220+/Mac1+(Q2), B220-/Mac1-(Q3),

B220-/Mac1+(Q4). BFP+/GFP+ gate (purple, in this instance 49.2% of live cells), GFP+ single positive gate

(orange), non vital cells (green), debris (black).

36

3.10 Mapre2 expression in ex vivo bone marrow

To ascertain that knockdown of Mapre2 was still present in the deceased mice, relative expression was evaluated in their bone marrow for shRNA (n = 8), scramble (n = 3) and untreated (n = 2) in the BFP+ and BFP- populations, respectively (Fig. 16). Relative expression was lowest in the shRNA BFP+ population with 13.0 ± 6.8 %.

p < 0 .0 0 0 1

1 .5

n

o

i

s s

e 1 .0

r

p

x

E

e

v i

t 0 .5

a

l

e R

0 .0 + - + - d P P P e F P F t F F a B B B B e r 2 2 e le t e e l r r b b n p p m u a a m a a r M M r c h h c s s s s

Fig. 16: Columns represent relative expression of Mapre2 (average ± standard deviation) in bone marrow cells extracted from mice of the in vivo experiment.

37

4 Discussion

4.1 Results

4.1.1 MAPRE2 expression

To elucidate the role of MAPRE2 as a potential key gene in leukemic stem cell biology, we planned this gene knockdown study. After repressing Mapre2 expression in our CALM/AF10 mouse model, for the in vitro part of this study, we examined whether gene knockdown leads to differences in cell proliferation or colony formation capabilities. For the in vivo part of the study, we transplanted transduced tertiary leukemic murine CALM/AF10 cells into lethally irradiated mice to assess the effect of gene knockdown on overall survival of recipient mice.

Firstly, we determined the expression levels of Mapre2 in a CALM/AF10 cell line as well as in different subpopulations of murine leukemia cells derived from the CALM/AF10 mouse model. We could show that expression of Mapre2 was highest in the cell line as well as the

B220+/Mac1- subpopulation (see 3.1), which is the compartment containing the highest concentration of LSC, as shown by Deshpande et al. (13). This finding is in line with the study published by Eppert et al., in which LSCs expressed a stem cell gene signature, among which MAPRE2 could also be found (20). Looking at normal hematopoiesis, MAPRE2 is also highest expressed in HSC in the data set provided by the study of Rapin et al. (51).

Assuming, that this stem cell gene signature governs “stemness properties” in LSCs, it is fair to assume that these genes would also be more highly expressed in cell compartments harboring more LSCs, such as the B220+/Mac1- cell compartment in the CALM/AF10 mouse model. Building on these results, we conducted RNA interference based gene knockdown 38

experiments aiming at the B220+/Mac1- population, expecting the most dominant phenotypic effect in the population with the highest gene expression.

4.1.2 Successful knockdown of Mapre2 expression

By employing a lentiviral BFP-tagged shRNA vector based on the SFFV promoter, we could attain stable gene repression with our shRNA sequences. Particularly sequence E4-1 showed the highest knockdown properties both in the CALM/AF10 cell line as well as in the mouse model (see 3.2, 3.3). We continued our studies with the shRNA sequence E4-1 showing a knockdown efficiency > 99%. It is of note that our scramble sequence, a random shRNA sequence with no specific target in the murine genome, showed a significant gene knockdown compared to our untreated samples but importantly significantly less than our shRNA sequence targeted at Mapre2. Interestingly, this phenomenon could be observed in other unrelated genes we examined as well (data not shown), suggesting that it is caused by unspecific off-target effects of the vector system, e. g. by gene silencing activity of the shRNA sense strand (45). This effect was reversed when we examined the gene expression in our subpopulation of interest, the B220+/Mac1- population (see 3.5). 48 hours after transduction of tertiary murine CALM/AF10 positive cells, the cells were sorted by BFP signal indicative of vector insertion, as well as the cell surface markers B220 and Mac1.

Transduction efficiency (percentage of BFP+/GFP+ cells) ranged from 49.2 % to 91.8 %

(see 3.9). As expected, the statistically significant lowest Mapre2 expression could be seen in the BFP+ population of cells transduced with the target shRNA with a slightly lower knockdown efficiency compared to our previous experiments. We could also observe a statistically significant reduction of Mapre2 expression in the BFP- population of shRNA transduced cells. This phenomenon may be explained by silencing of BFP in shRNA positive

39

cells. Another possible explanation might be contamination of the BFP- population with shRNA BFP low positive cells at the stage of gating during cell sorting.

4.1.3 Knockdown of Mapre2 did not have any phenotypic influence

Knockdown of Mapre2 did not have any observable influence on phenotype, neither in vitro nor in vivo. No differences between untreated samples or samples treated with either scramble vector or shRNA could be seen in proliferation speed, colony formation capabilities and overall survival of transplanted mice (see 3.6, 3.7, 3.8). Overall survival for transplanted mice was 21 days, which is in accordance with previously published data for tertiary leukemic transplants by Deshpande et al. (13).

4.2 Limitations

4.2.1 Knockdown on mRNA level may not equal knockdown on protein level

A possible explanation for the lack of phenotypic effect of our knockdown approach could be retained protein function. By a number of different possible ways, our transplanted cells could have retained Mapre2 function despite the shRNA induced knockdown. Firstly, the residual transcriptional activity could have maintained a sufficient protein level to allow for residual Mapre2 function, thereby mitigating the knockdown. Moreover, a low protein turnover rate could mean that despite translational repression, protein levels would remain relatively unaffected. These possibilities could be ruled out e. g. by assessing protein levels in knocked down cells by Western Blotting. Secondly, functional redundancies of the cell might be at play. Part of the Mapre2 activity could be substituted by protein isoforms, or functionally replaced by different molecular pathways altogether.

40

4.2.2 Vector silencing

A further possibility to explain our results would be partial silencing of the shRNA vectors.

Interestingly, when we examined the transduction efficiency in our mice ex vivo at the time of death, we could observe a transduction efficiency varying from 49.2 % to 91.2 % (see

3.9) although only BFP+/GFP+ B220+/Mac1- cells had been initially transplanted. This raised the question, whether the unexpectedly high proportion of BFP-/GFP+ cells were due to a contamination of the transplant, meaning that in the samples with low proportion of only

BFP+/GFP+, GFP single positive cells would have had a growth advantage over the

BFP+/GFP+ population. This hypothesis however is not supported by our in vitro experiments, which showed no difference in proliferation between the two groups (see 3.6).

Herbst et al. have found that the SFFV promoter, if used in lentiviral vector constructs for murine transgene expression, can be inactivated by promoter methylation in an in vivo setting. After examining different promoters used in lentiviral vectors, such as CMV, SFFV and PGK, the group found that complete transgene silencing occurred with the commonly used CMV and SFFV promoters during differentiation of cells by extensive CpG island methylation of the promoters. This was shown to hold true both for murine embryonic stem cells (ESC) differentiating into cardiomyocytes, as well as murine HSCs differentiating into the different myeloid and lymphocytic lineages. As early as 48 to 96 hours after transduction of ESCs, fluorescence of the reporter protein GFP could not be detected anymore, despite successful integration of the viral construct into the target genome. The silencing effect occurred independent of transduction efficiency. In case of murine HSCs, a successive reduction of GFP expression over time could be observed. These effects could not be observed when using endogenous promoters, such as PGK or when using γ-retroviral vector backbones instead of lentiviral backbones (25). Applying these findings to our results, we 41

can assume that at least partial vector silencing may have occurred during our transplantation experiments. This is underscored not only by the mean transduction efficiency of 72.5 %

(see 3.9), but also by the change in Mapre2 expression in the BFP- population before and after transplantation. While 48 hours after transduction, Mapre2 expression in this compartment was at 58.4 %, the Mapre2 expression in the same compartment ex vivo post transplantation was at 94.2 %, supporting the near complete silencing of the shRNA during the course of our in vivo experiment (see 3.5, 3.10). A potential workaround could be either the use of a retrovirus based vector or the use of a different promoter to induce gene knockdown (25).

4.2.3 Aggressive leukemia model

The mouse model of CALM/AF10 positive leukemia employed in this study mimicks a number of properties of its human counterpart, among them the tendency for aggressive growth. In one of the earlier reports of CALM/AF10 positive leukemia, Bohlander et al. showed a remarkably poor overall survival in a group of 7 patients at 11 ± 7.2 months (5).

Furthermore, Deshpande et al. showed that upon serial transplantation of the CALM/AF10 model, its aggressiveness increases with a median overall survival of 110 days in primary transplants and 22 days in secondary transplants (13). Therefore, smaller effects − e. g. of gene knockdown experiments such as in this study – may be overlooked in this specific leukemia model. An evaluation in a different, less aggressive leukemia mouse model could potentially show a role for MAPRE2 in leukemogenesis. Additionally, findings in a specific subset of leukemia may not reflect AML as a whole. AML is a genetically and etiologically heterogeneous disease, so the role of certain genes may vary between different subsets of

AML.

42

4.2.4 Insufficiency of single gene knockdown

AML is a genetically heterogeneous disease with a wide variety of chromosomal and genetic perturbations. While there are certain types of AML that are characterized by a specific genetic alteration, e. g. PML and the PML-RARA fusion gene, the majority of cases are classified as cytogenetically normal. While the prognostic and clinical relevance of certain genes, such as CEBPA, FLT3 or IDH1/2 are more and more being recognized and included in disease classification, recent advances in high throughput genomics have led to an increasing list of mutations found in AML. For most of these newly found and recurring mutations, clinical significance has yet to be elucidated. In the context of such a genetically complex disease with multiple driving mutations as well as passively accumulated passenger mutations, single gene knockdown may not be sufficient to alter disease phenotype, especially in an aggressive subset of AML such as CALM/AF10 positive leukemia.

Examining these genes may however further the understanding of AML disease onset and may give insight to the relevance of these recurring mutations. By employing shRNA libraries and knocking down multiple target genes, relevant mutations may be found more effectively (43). Furthermore, other gene editing tools, such as CRISPR-Cas9, among others, will certainly improve our understanding of leukemogenesis and the accompanying genetic and epigenetic changes.

4.2.5 Discrepancy between human and murine leukemia

The stem cell gene signature published by Eppert et al. was derived from human AML samples and correlated with the clinical outcomes of the corresponding patients (20). The experiments of this study were conducted exclusively in murine leukemic cells. While both human and murine cells express MAPRE2, the expression in the hematopoetic system may

43

differ. In human hematopoesis, MAPRE2 is highly expressed in the HSC compartment (51), in murine hematopoesis Mapre2 is most highly expressed in pro-B-cells and Natural Killer cells with a medium expression level in HSCs (10, 15). While we could show comparatively high expression in our murine LSC compartment of CALM/AF10 positive leukemia, gene repression effects may not be as pronounced. Moreover, the biological function and relevance for leukemogenesis may naturally differ between species and our findings may not be transferable to human AML. Further studies in human AML cell lines and patient samples might bring light to whether MAPRE2 behaves differently in murine versus human hematopoesis and leukemogenesis.

44

5 Summary

The cancer stem cell model proposes that malignant disease is initiated and maintained by a subpopulation of tumor cells with stem cell like properties, i. e. maintaining its self-renewal ability as well as multi-lineage differentiation potential. Due to their biologic features such as slow cell cycling, these cells are also thought to contribute to resistance against current treatment options as well as disease relapse. Thus, better understanding of the cancer stem cell’s (CSC) biology becomes a prerequisite for improved patient care. In the case of leukemic stem cells, it has been shown that a stem cell gene signature can predict disease outcome in human acute myeloid leukemia (AML), therefore underscoring the clinical significance of leukemia stem cells (LSC). In this study, we investigated whether MAPRE2

– a gene expressed in this stem cell gene signature – played a role in LSC biology.

We conducted a gene knockdown experiment using shRNA mediated gene silencing in a mouse model of CALM/AF10 (phosphatidylinositol binding clathrin assembly protein / mixed-lineage leukemia; translocated to chromosome 10) positive AML. We assessed both in vitro properties such as proliferation and colony formation capacities. Furthermore, we transplanted lethally irradiated mice to find out whether knockdown of Mapre2 had an effect on leukemogenesis in vivo.

We could show that reduced expression of Mapre2 had no effect on proliferation or colony formation properties in the CALM/AF10 positive mouse model. Knockdown of Mapre2 also did not have an influence on overall survival in vivo. A potential explanation for these results is the silencing of the SFFV (spleen focus-forming virus) promoter used in our lentiviral constructs, as evidence for vector silencing in vivo has been presented. Further possibilities

45

include functionally redundant cellular pathways, a discrepancy between human and murine leukemia and the corresponding genes governing stemness properties as well as insufficiency of single gene knockdowns in an aggressive leukemia model.

Future studies using different vector systems and studies in human AML cells will help to define the role of MAPRE2 in leukemogenesis and LSCs.

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7 Acknowledgement

I want to acknowledge:

• Prof. Dr. med. Christian Buske for giving me the opportunity to experience scientific

medical research.

• Dr. Parisa Eshraghi for her supervision and practical guidance during my time in the

laboratory.

• All the staff of the Institute for Experimental Cancer Research, without whom I

would never have learnt the basics of laboratory work.

• The International Graduate School in Molecular Medicine Ulm and the Experimental

Medicine programme for enabling me to work full-time on this project.

• My family and friends who have supported me through all this time.

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8 Curriculum vitae

Personal Information

Name Sascha Kenta Winkler

Date of birth 1991

Place of birth Böblingen, Germany

Nationality German

Education

1997-2001 Elementary school (Eduard-Mörike-Grundschule,

Sondelfingen)

2001-2009 High school (Friedrich-List-Gymnasium, Reutlingen)

2009-2016 Medical school (University of Ulm, Germany)

2012-2013 Doctoral student at the Institute of Experimental Cancer

Research (Prof. Dr. med. C. Buske), supported by the

International Graduate School of Molecular Medicine, Ulm

and “Promotionsprogramm Experimentelle Medizin”, Ulm

2015-2016 Practical year (University Hospital Ulm, Germany and Steve

Biko Academic Hospital, Pretoria, South Africa)

2020 Doctoral degree (Dr. med.) from the University of Ulm

Employment

From 2017 Medical Resident (Ulm University Children’s Hospital,

Department of General Pediatrics, Neonatology and Pediatric

Intensive Care and Pediatric Hematology and Oncology)

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