Role of Innate Immunity Activators in the Treatment of Acute Myeloid Leukemia Dissertation Presented in Partial Fulfillment Of

Role of Innate Immunity Activators in the Treatment of Acute Myeloid Leukemia

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Nathaniel J. Buteyn, B.S.

Graduate Program in Molecular, Cellular, and Developmental Biology

The Ohio State University

2019

Dissertation Committee:

Susheela Tridandapani, PhD; Advisor

James Blachly, MD

John Byrd, MD

Amanda Toland, PhD

Nathaniel J. Buteyn

2019

Abstract

Immune cells of myeloid origin have a unique role in the body’s response to non- self entities. The cells, including monocytes and macrophages, carry out a diverse array of functions including phagocytosis, the uptake and presentation of foreign antigens, environmental debris, and damaged cells; the release of cytokines that coordinate acute inflammatory responses; and cytotoxic effector functions that result in the destruction of targets. In acute myeloid leukemia (AML), a differentiation block in the myeloid cell lineage prevents proper maturation of monocytes and macrophages. Instead, leukemic blasts rapidly accumulate and proliferate in the bone marrow, blood, and organs preventing proper hemocytic development. Patient death is caused mainly by infection, followed by hemorrhage and organ failure. The most common form of adult leukemia,

AML has a low five year survival rate of 26.6% and a high rate of patient relapse. Taken with the high average age of diagnosis and the fact that certain elderly patients are unable to participate in the standard treatment of high-intensity chemotherapy, it is clear that there is a need for innovative, less toxic therapeutic approaches to the disease.

One such approach is the re-invigoration of the patient’s own immune system, typically suppressed in a myriad of ways due to the disease. This is explored in two novel studies presented here. The first, detailed in Chapter 2, takes advantage of the effector

function that myeloid cells naturally possess; expression of Fcγ receptors on the cell surface allow for interaction with antibody opsonized targets. By eliciting expression of the antigen for the α-CD38 antibody daratumumab on the surface of AML blasts with all-trans retinoic acid (ATRA), we demonstrated it was possible to induce antibody- dependent blast-to-blast killing amongst the cancer itself, with blasts functioning as both targets and effectors, a phenomenon we termed fratricide. This antibody-induced fratricide showed efficacy in two mouse models of AML, decreasing tumor burden and extending survival time.

The study outlined in Chapter 3 offers another approach to reactivate the innate immune system in AML. Here, the use of a synthetic bacterial peptide that induces a robust inflammatory response was examined. The peptide, muramyl tripeptide phosphatidylethanolamine (MTP-PE), together with the pro-inflammatory cytokine interferon gamma (IFN-γ), stimulates the intracellular pathogen recognition receptor

NOD2, resulting in caspase-1 dependent blast apoptosis and pro-inflammatory cytokine production. Furthermore, induction of this inflammatory response in murine models of

AML results in extended survival and the maturation of disease suppressed natural killer cells, an important cytotoxic leukocyte whose activation has been shown to correlate with improved outcomes in AML patients.

iii

The idea of harnessing the body’s natural defenses to fight malignancy has gained steam in the recent decades with the further development of innovative technologies such as CAR T cell therapy and allogeneic natural killer cell infusion. In the studies presented within, we focused not on ancillary immune cells, but rather modulating the tumor cell itself. Results gathered demonstrate potent anti-leukemic effects and suggests that these treatments may have therapeutic potential in AML.

Vita

May 2015 ...... B.S. Biotechnology, Calvin College (now Calvin University)

2015 – 2019 ...... Graduate Research Associate, MCDB program, Department of

Internal Medicine, The Ohio State University

Publications

“Anti-leukemic effects of all-trans retinoic acid in combination with Daratumumab in acute myeloid leukemia.” NJ Buteyn, K Fatehchand, R Santhanam, H Fang, GM Dettorre, S Gautam, et al. International immunology 30 (8), 375-383

“Interferon-γ promotes antibody-mediated fratricide of Acute Myeloid Leukemia cells.” K Fatehchand, EL McMichael, BF Reader, H Fang, R Santhanam, NJ Buteyn, et al. Journal of Biological Chemistry 291 (49), 25656-25666

“CD31 acts as a checkpoint molecule and is modulated by FcγR-mediated signaling in monocytes.” G Merchand-Reyes, JP Butchar, F Robledo-Avila, NJ Buteyn, et al. Journal of Immunology November 15, 2019

Fields of Study

Major Field: Molecular, Cellular, and Developmental Biology

Table of Contents

Abstract ...... ii Vita ...... v List of Tables ...... viii List of Figures ...... ix Abbreviations ...... xii CHAPTER 1: Introduction ...... 1 Overview of the Human Immune System ...... 1 Innate Immunity ...... 2 Monocytes and Macrophages ...... 5 M1 vs M2 Polarization ...... 7 Fcγ Receptors ...... 9 Acute Myeloid Leukemia ...... 17 Classification ...... 18 Standard of Care ...... 22 Emerging Therapies ...... 24 CHAPTER 2: Anti-leukemic effects of all-trans retinoic acid in combination with daratumumab in acute myeloid leukemia ...... 28 Abstract ...... 28 Introduction ...... 29 Acute promyelocytic leukemia ...... 29 All-trans retinoic acid ...... 30 Daratumumab ...... 33 Materials and Methods ...... 35 Results ...... 40 ATRA upregulates CD38 in AML cells ...... 40

ATRA triggers daratumumab-mediated immune conjugate formation and killing in vitro... 46 Single-dose ATRA elicits CD38 upregulation and confers daratumumab activity ...... 51 ATRA synergizes with daratumumab to impede AML tumor growth and extend survival in vivo ...... 53 Discussion ...... 57 CHAPTER 3: Activation of the intracellular pattern-recognition receptor, NOD2, promotes NK cell maturation and extends survival in acute myeloid leukemia ...... 63 Abstract ...... 63 Introduction ...... 65 Coley’s toxin ...... 65 Pattern recognition receptors ...... 66 NOD2 ...... 68 MTP-PE ...... 70 Materials and Methods ...... 72 Results ...... 77 IFN-γ primes NOD2 signaling pathway in AML ...... 77 MTP-PE + IFN-γ is cytotoxic to AML blasts ...... 82 MTP-PE + IFN-γ induces a pro-inflammatory response from AML cells ...... 89 MTP-PE + IFN-γ stimulates NK cell maturation and extends survival in vivo ...... 93 Endogenous IFN-γ correlates with MTP-PE responsiveness ...... 94 Discussion ...... 99 CHAPTER 4: Conclusions and Future Directions ...... 103 References: ...... 112 Appendix A: Supplementary Figures and Tables ...... 135

vii

List of Tables

APPENDIX A: SUPPLEMENTAL FIGURES

Table A.1 Characterization of AML patient samples utilized for ATRA/daratumumab study ...... 136

Table A.2 Characterization of AML patient samples utilized for MTP-PE/IFN-γ study ...... 140

viii

List of Figures

CHAPTER 2

Figure 2.1 ATRA upregulates CD38 in AML cells ...... 42

Figure 2.2 ATRA upregulates CD38 in AML cells (cont) ...... 43

Figure 2.3 ATRA upregulates CD38 via RARA ...... 44

Figure 2.4 ATRA upregulates CD38 on AML stem cells ...... 45

Figure 2.5 ATRA triggers daratumumab-mediated immune-conjugate formation in vitro ...... 48

Figure 2.6 ATRA plus daratumumab kills in vitro ...... 49

Figure 2.7 ATRA plus daratumumab kills via Fc-dependent fratricide ...... 50

Figure 2.8 Single-dose ATRA elicits CD38 upregulation and confers daratumumab activity ...... 52

Figure 2.9 ATRA synergizes with daratumumab to impede AML tumor growth ..... 54

Figure 2.10 ATRA and daratumumab extend survival in vivo ...... 55

Figure 2.11 Schematic of proposed mechanism of ATRA and daratumumab induced fratricide in AML ...... 56

CHAPTER 3

Figure 3.1 MTP-PE alone fails to elicit a response in AML ...... 79

Figure 3.2 MTP-PE internalization and NOD2 signaling pathway ...... 80

Figure 3.3 IFN-γ primes NOD2 pathway in AML ...... 81

Figure 3.4 MTP-PE + IFN-γ kills AML blasts ...... 83

Figure 3.5 MTP-PE + IFN-γ kills AML blasts (cont) ...... 84

Figure 3.6 MTP-PE cytotoxicity depends on IFN-γ ...... 85

Figure 3.7 MTP-PE + IFN-γ activates caspase-3 ...... 87

Figure 3.8 MTP-PE + IFN-γ kills via capases-1 activation ...... 88

Figure 3.9 MTP-PE + IFN-γ induces pro-inflammatory cytokine production in AML blasts ...... 91

Figure 3.10 MTP-PE + IFN-γ induced pro-inflammatory cytokines may serve to activate NK cells ...... 92

Figure 3.11 MTP-PE + IFN-γ stimulates NK cell maturation in vivo ...... 95

Figure 3.12 MTP-PE + IFN-γ stimulates NK cell maturation and decreases disease burden in vivo ...... 96

Figure 3.13 MTP-PE + IFN-γ extends survival in vivo ...... 97

Figure 3.14 Endogenous IFN-γ in HSCT AML patients ...... 98

APPENDIX A: SUPPLEMENTAL FIGURES

Figure A.1 MN1 mRNA expression versus adult AML patient cytogenetics ...... 137

Figure A.2 PRAME mRNA expression versus adult AML patient cytogenetics ...... 138

Figure A.3 CD38 mRNA expression versus adult AML patient cytogenetics ...... 139

Figure A.4 Cytokine array on AML patient samples ...... 141

Figure A.5 Neoplastic infiltration in the bone marrow of mice treated with ATRA and/or daratumumab ...... 142

Figure A.6 Exosome release induced after MTP-PE + IFN-γ treatment ...... 143

Figure A.7 ESCRT mRNA levels after MTP-PE + IFN-γ treatment ...... 144

Figure A.8 NOD2 mRNA expression versus adult AML patient cytogenetics ...... 145

Figure A.9 NOD2, NOD1, TLR2, and TLR4 mRNA expression versus adult AML patient cytogenetic risk stratification ...... 146

Abbreviations

ADCC Antibody-dependent cellular cytotoxicity AML Acute myeloid leukemia APL Acute promyelocytic leukemia ATO Arsenic trioxide ATRA All-trans retinoic acid CARD Caspase activation and recruitment domain CBF Core-binding factor; subset of AML with t(8;21), t(16;16), or inv(16) CLL Chronic lymphocytic leukemia CR Complete remission CTL Cytotoxic T lymphocyte ELISA Enzyme-linked immunosorbent assay ESCRT Endosomal sorting complexes required for transport FAB French-American-British; classification of AML based on blast morphology FcγR Fc-gamma receptor GO Gemtuzumab ozogamicin HSCT Hematopoietic stem cell transplantation IFN-γ Interferon gamma IgG Immunoglobulin G ITAM Immunoreceptor tyrosine-based activating motif ITIM Immunoreceptor tyrosine-based inhibitory motif LDH Lactate dehydrogenase assay LUBAC Linear ubiquitin chain assembly complex MDP Muramyl dipeptide MDS Myelodysplastic syndrome ML-DS Myeloid leukemia associated with Down Syndrome

xii

MTP-PE Muramyl tripeptide phosphatidylethanolamine; mifamurtide MVB Multivesicular body NCR Natural cytotoxicity receptor NK Natural killer cell NLR NOD-like receptor NOD2 Nucleotide-binding oligomerization domain-containing protein 2 NSG Non-obese diabetic severe combined immunodeficient-IL2Rγ-/- mice PAMP Pathogen-associated molecular patterns PBM peripheral blood monocytes PML Promyelocytic leukemia PRR Pattern recognition receptor qPCR Quantitative real-time polymerase chain reaction RARA Retinoic acid receptor alpha RARE Retinoic acid response element RAS Retinoic acid syndrome RCN Relative copy number R/R Relapsed or refractory RXR Retinoic X receptor TKI Tyrosine kinase inhibitor TLR Toll-like receptor TNF-α Tumor necrosis factor alpha WBC White blood cell

xiii

CHAPTER 1: Introduction

Overview of the Human Immune System

The ability of the human body to defend itself against foreign invaders and internal abnormalities like cancer is dependent on its immune system. Utilizing a diverse repertoire of cells and soluble protein systems, the immune system is able to recognize, neutralize, and destroy a vast array of non-self targets. The human immune system is split into two major arms: the adaptive and the innate. Stimulation of an immune response results in the robust, albeit delayed, activation of the adaptive arm: T and B lymphocytes that together, through both humoral and cell-mediated methods, work to eliminate their target. Furthermore, initial activation of the adaptive response against a novel pathogen leads to sustained immunological memory, the ability of specific subsets of B and T cells to react more rapidly to antigens they have encountered previously. This unique ability of adaptive immune cells is the basis for the concept of vaccination. Much work has been done investigating the role of adaptive lymphocytes in the context of disease and, specifically, cancer. In fact, one of the more exciting innovations in the past decades has been the development of artificially engineered T

cells (chimeric antigen receptor, CAR, T cells) that have shown great efficacy in certain disease scenarios.

In contrast, the innate immune system functions as the first line of defense against invading pathogens. Composed of phagocytic monocytes, macrophages, neutrophils, and dendritic cells; as well as basophils, eosinophils, and the cytotoxic natural killer (NK) cells; the cells of the innate immune system work by identifying and eliminating foreign targets in a broader, less specific sense. These cells also function to recruit additional players to the site of injury through the release of potent cytokine signals. The studies detailed here involve the modulation and/or re-invigoration of the innate immune response in the context of leukemic disease. The robust responses we see, both in in vitro cultures and murine disease models, illustrate the power of this system of defense.

Innate Immunity

The majority of cells within the innate immune system are derived from the bone marrow residing common myeloid progenitor cell; only NK cells (and one subset of dendritic cell) are derived from the common lymphoid progenitor (along with adaptive cell types, the T and B cell). From this one progenitor cell, numerous cellular fates can be realized. Cells can develop into megakaryocytes: large bone marrow cells with 2

lobated nuclei that are responsible for the production of thrombocytes, the platelet component of blood involved in clotting. Erythrocytes, or red blood cells, are hemoglobin-rich enucleated cells responsible for the transport of oxygen within the body and can also be derived from the common myeloid progenitor cell in response to low blood oxygen levels. Additionally, myeloid progenitors can undergo granulopoiesis to differentiate into polymorphonuclear neutrophils, eosinophils, and basophils that contain toxic cytoplasmic granules. Neutrophils, the most abundant white blood cell

(WBC) in the body, constitute approximately 60-70% of an adult’s WBC population.

These cells are professional phagocytes that rapidly home to sites of infection and eliminate pathogens via phagocytosis, release of granules and anti-microbial factors, and the generation of pathogen neutralizing neutrophil extracellular traps (NETs).1 The other granulocytes, typically at levels <3% of total WBCs, have more specified roles and function in targeting multi-cellular parasites (eosinophils) or in allergic reaction and histamine release (basophils). Lastly, myeloid progenitors have the ability to differentiate into monocytes and, once in tissue, further into macrophages through the process of monocytopoiesis. Cells of this lineage are proficient phagocytes and recognize pathogens through a vast array of pattern recognition receptors (PRRs) in their cytoplasm and on their cell surface that bind pathogen associated molecular patterns (PAMPs) and damage associated molecular patters (DAMPs); additional receptors recognize antibody or complement opsonized targets. In addition to their phagocytic ability, monocytes and macrophages function as antigen presenters and

potent producers of inflammatory cytokines that amplify and resolve inflammatory processes.

Innate immune cells, while diverse in their function and capabilities, are only one component of this first line of defense. Anatomical barriers, either physical, like skin and mucosal barriers; chemical, like the acidic environment of the stomach; or biological, like the hosts of commensal bacteria that reside in the gut, all in one way or another work to inhibit the entrance or establishment of pathogens within the body.

Additionally, the body produces a number of soluble proteins in the liver that together comprise the complement system. These proteins circulate in inactive forms but upon stimulation initiate a protease-dependent biological cascade resulting in target lysis, recruitment of phagocytes, and inflammation amplification.

All together, the players in the innate system function to provide a robust first reaction to invasion, providing the body with time to amplify the more specialized adaptive immune response. Escape from innate immunity and the inability to properly identify foreign or damaged material are problems that can lead to malignant cell evasion and proliferation.

Monocytes and Macrophages

Phagocytic white blood cells, first identified by Élie Metchnikoff in 1884,2 play a critical role in innate monitoring and pathogenic clearance. Unlike the abundant neutrophils, bone marrow and circulating monocytes retain the ability to proliferate and differentiate into macrophages upon tissue entry. The functional heterogeneity of these tissue-specific macrophages is reflected in their diverse names: Kupfer cells in the liver, sinus histiocytes in the lymph nodes, alveolar macrophages in the lung, microglia in the brain, and more. However, the role of the monocyte is more than just an intermediate between bone marrow progenitor and the resident macrophage; in fact, recent work has suggested that most tissue macrophages do not rely on circulating or bone marrow- derived monocytes to maintain their populations.3,4 Comprising approximately 5-10% of leukocytes in humans, monocytes represent a highly plastic effector population of cells capable of scavenging and antigen presenting5,6 and eliciting anti-microbial killing and cytokine release.7-9

Monocytes are categorized into three subsets: classical (CD14++, CD16-), intermediate (CD14++, CD16+), and non-classical (CD14low, CD16+). Classical and intermediate monocytes tend to be more potent phagocytes that elicit production of reactive oxygen species (ROS) and inflammatory cytokines in response to a broad array of microbial infection. In contrast, it has been reported that non-classical monocytes appear to be largely weak phagocytes and respond poorly to cell surface toll-like receptor (TLR) stimulation; rather, selective inflammatory cytokine production was 5

elicited to a greater extent under specific circumstances involving nucleic acid immune complexes triggering endosomal TLRs, -7 and -8.10 Moreover, evidence suggests that

CD16+ monocytes (intermediate and non-classical) function more as mobilizers during inflammatory events compared to the more scavenger focused classical subset.11,12

Predictably then, infiltrations of CD16+ monocytes often accompanies inflammatory diseases and conditions such as sepsis,13 tuberculosis,14 leishmaniasis,15 rheumatoid arthritis,16 and gastrointestinal cancer.17

Macrophages, while transcriptionally diverse amongst various tissue types,18 carry out a number of similar functions regardless of where they reside. These include phagocytosis and antigen presentation, but also tissue repair and remodeling and generation and resolution of inflammation. As mentioned above, in addition to the classical belief that circulating monocytes differentiate into macrophages during inflammatory incidence, recent discoveries have demonstrated that most adult resident macrophages are derived from embryonic progenitors deposited in tissues before birth19 and that mature tissue macrophages are capable of self-renewal without loss of functionality.20 Together, monocytes and macrophages provide a strong first defense against foreign invasion.

M1 vs M2 Polarization

Macrophages, in their response to pathogens and the subsequent resolution of inflammation, are capable of carrying out diverse and often opposing functions.

Classically activated, or M1, macrophages are induced by bacterial motifs such as LPS and/or inflammatory cytokines like IFN-γ and TNFα. These stimuli result in an inflammatory cell that responds to pathogens through expanded secretion of inflammatory cytokines, upregulation of ROS and nitrogen radicals, and increased phagocytic/cytotoxic abilities. Bacterial motifs are recognized through a series of PRRs, expressed on both the cell surface and intracellularly, that result in inflammatory signaling through factors such as NF-κB. The cytokines shown to trigger an M1 response can come from a number of sources. IFN-γ, perhaps the main cytokine associated with the M1 shift, is produced by the T cell subset, Th1, as well as activated NK cells and macrophages themselves. Moreover, IFN-γ and bacterial motifs like LPS synergize and elicit a unique gene expression compared to activation by the single agents alone.21,22

TNFα, another M1 polarizing cytokine, is produced in large amounts by activated macrophages and other antigen presenting cells. In response to stimuli, M1 macrophages produce a host of inflammatory cytokines including IL-1β, IL-12, IL-15, IL-

23, and TNFα. The inflammatory shift that IFN-γ and bacterial motifs elicit in myeloid cells, as well as a more detailed review of PRRs such as toll-like receptors and NOD-like receptors is covered in Chapter 3. While M1 responses are vital to proper clearance of pathogens, prolonged activation disrupts tissue homeostasis and repair; it is crucial then

that there exists an M2, or alternatively activated, macrophage that deals with inflammation resolution, tissue remodeling, and angiogenesis promotion.

Compared to M1 macrophages, M2 polarization is more complex; cells are further classified into three subgroups: M2a, M2b, and M2c. M2a macrophages develop after exposure to the anti-inflammatory cytokines IL-4 and IL-13, after which they temper down the inflammatory response through the release of IL-10 and TGF-β, downregulation of caspase 1, and increased expression of arginase 1.23,24 Furthermore,

M2a polarization increases expression of a variety of factors including fibronectin, beta

IG-H3, coagulation factor XIII, and IGF-1, all of which are involved in tissue repair and proliferation.25,26

M2b macrophages, also known as “type II”, are phenotypically unique compared to M1 and M2a macrophages. Induced by LPS and immune complexes, M2b macrophages produce more IL-10 compared to M2a, but still release certain inflammatory cytokine such as TNFα, IL-1β, and IL-6, suggesting an interesting role in balancing the inflammatory response. It has been demonstrated through a number of studies that, despite their pro-inflammatory functions, polarization towards an M2b macrophage promotes tumor development in cancer and dampens immune responses to pathogenic invasion.27-32

The last main subset of M2 macrophages, M2c, is induced after stimulation with

IL-10, TGF-β, and glucocorticoids. Typically thought of as ‘deactivated’ macrophages,

M2c cells are characterized by the release of IL-10 and TGF-β, that successfully downregulate pro-inflammatory cytokine production, and increased scavenging of debris and apoptotic cells through the upregulation of receptors such as CD163.

However the regulatory macrophages categorized as M2c are rather heterogeneous, and exemplify an important point when considering macrophage polarization: the classification of cells into distinct subtypes is often an over-simplification; many argue instead for a spectrum on which these cells lay along, blending phenotypes in response to environmental cues.33,34

Fcγ Receptors

Amongst the numerous receptors expressed on the surface of monocytes and macrophages, those belonging to the Fc gamma receptor (FcγR) family have a unique role in innate effector function. As the name suggests, FcγR bind to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibodies. Opposite the antigen specific Fab’2 end of the antibody, the Fc region is consistent between all antibodies within an isotype and allows for effective interaction between an antibody-opsonized target and cells expressing Fc receptors on their surface. In human monocytes and macrophages, activating Fcγ receptors, including FcγRI (CD64), FcγRIIa (CD32a), and

FcγRIIIa (CD16), signal through a cytoplasmic immunoreceptor tyrosine-based activating

motif (ITAM) upon receptor crosslinking; FcγRIIb (CD32b), the sole inhibitory receptor, signals through an immunoreceptor tyrosine-based inhibitory motif (ITIM).

These receptors have varied affinity for the four subclasses of IgG. FcγRI, the only receptor that binds to IgG with high affinity, binds IgG1, IgG3, and IgG4; the other receptors, each with their own unique specificities, are classified as low-affinity.35

Moreover, while all Fcγ receptors bind IgG-immune complexes with high avidity, only the high-affinity receptor FcγRI has the ability to bind monomeric IgG, suggesting that low-affinity receptors are the ones that remain open in circumstances of high circulation of free IgG.35,36 Yet it has been demonstrated that Fcγ receptors, even FcγRI in its constant state of saturation, only signal upon receptor crosslinking by an immune complex.37,38

FcγRI and FcγRIIIa are comprised of an α-chain that binds IgG and a common γ- chain dimer containing an ITAM motif responsible for the initiation of signaling. In contrast, FcγRIIa and FcγRIIb are each single receptors that possess both an extracellular ligand sensing domain as well as a cytoplasmic tail with an ITAM or ITIM motif. Upon aggregation of FcγR by immune complexes, src-family kinases are recruited to the ITAM on activating receptors, resulting in the phosphorylation of tyrosine residues and downstream signaling, including activation of phosphoinositide 3-kinase (PI3K) and the subsequent triggering of Bruton’s tyrosine kinase (BTK) and phospholipase Cγ (PLCγ) signaling.39-41 In contrast, activation of the inhibitory receptor FcγRIIb recruits phosphatases via its ITIM that suppress signaling. The ratio of activating versus

inhibitory receptors on the surface of a cell dictates its response to opsonized targets; expectedly, FcγRIIb knockout mice show enhanced macrophage activity.42,43 While this increased phagocyte activity may be desirable in the setting of infectious disease or cancer, suppression of inhibitory signals is detrimental in the context of autoimmune conditions. In addition to being expressed on innate immune effectors, FcγRIIb is also found on B cells. Activation of the intracellular ITIM alongside the B cell receptor (BCR) also recruits phosphatases that shut down downstream signaling.44 Moreover, FcγRIIb has been shown to function as a late checkpoint molecule during B cell development; it plays a role in the exclusion of auto-reactive B cells and its absence is associated with

IgG autoantibodies and an increased susceptibility to autoimmune diseases.45-48

FcγRIIIa, a low affinity activating receptor also known as CD16, is lowly expressed on monocytes but is the main Fcγ receptor found on NK cells. Unlike in monocytes, where FcγRIIIa associates only with the common γ-chain, in NK cells it can function through the CD3 ζ-chain (CD247) as well.49,50 Through FcγRIIIa, NK cells can be activated in a priming-independent manner resulting in direct lysis of target cells and recruitment of additional immune players through the release of inflammatory cytokines. This killing of target cells, known as antibody-dependent cellular cytotoxicity (ADCC), occurs through the release of cytoplasmic granule toxins, including the membrane pore forming perforin and the serine protease granzymes. Additionally, engagement of death receptors on the target cell such as Fas with the corresponding receptor on NK cells

(FasL) can induce apoptosis in the target via extrinsic caspase pathway activation.51

Because of these potent cytolytic abilities, NK cells have been utilized in adoptive transfer cancer immunotherapies.52,53 However, because monocytes also express Fcγ receptors, and because certain malignancies see a disruption in NK cell levels and/or maturation, monocytes can also serve as mediators of ADCC. Even in myeloid leukemias such as AML, where a maturation block results in immature myeloid cells, the cancer cells express Fcγ receptors and can function as both effector and target cells. Potential therapeutic strategies utilizing this fratricide are explored in Chapter 2.

Cancer Immunotherapy

The ability to evade immunological destruction has been classified as a hallmark of cancer.54 Indeed, individuals with suppressed immune surveillance function, such as patients after organ transplant, are more susceptible to malignant disease.55 It stands to reason then that reactivation or reinvigoration of the body’s natural defense system may serve as a deterrent to cancer progression or even as a comparatively low-toxic alternative for front-line treatment. This is evidenced in a number of malignancies where infiltrating cytotoxic T lymphocytes (CTLs) and NK cells are prognostic of improved outcome.56,57

Modern immunotherapy traces its roots back to Dr. William Coley, an American physician who developed a concoction known as Coley’s toxin at the beginning of the

20th century. Comprised of heat-killed bacteria, the treatment reinvigorated immune responses in sarcoma patients with some clinical success. Further background on Dr.

Coley and the history of immunostimulation through microbe motif receptors is detailed in Chapter 3. However, despite Coley’s observations, the focus largely turned to refining cytotoxic techniques such as radiology and the emerging field of chemotherapy through the mid-1900s.

In recent years, developments in technology have allowed for the more detailed elucidation of the molecular immunological mechanisms behind the phenomenon that

Coley described nearly 100 years ago. Alongside the burgeoning genomic revolution, these advances allowed for precise targeted therapy of genetic aberrations in lieu of the non-specific toxicity of chemotherapy. The ability to clone IgG molecules, described in the 1990s,58 was the first step in engineering and mass producing antibodies that could target cancer in such a specific way.

The utilization of antibody therapy in the treatment of malignant disease has taken many forms over the past decades with varying degrees of success. One standout story is the development of monoclonal antibody-based immune checkpoint blockade therapy. One mechanism by which cancer evades destruction is through the upregulation of co-inhibitory receptors on the tumor cell or immune cells like T lymphocytes that actively suppress effector function. Examples of these receptors include CTLA-4, a receptor on T cells that binds to CD80/CD86, blocking activation and inducing anergy, and PD-1, a receptor that binds to PD-L1 on the tumor cell and results

in T cell exhaustion. Antibodies that block the transmission of these inhibitory signals allow for robust T cell response against disease; the FDA has approved a number of these antibodies for the treatment of malignancies including melanoma (Ipilimumab,

CTLA-4; Nivolumab, PD-1), non-small cell lung cancer (Nivolumab; Pembrolizumab, PD-1;

Atezolizumab, PD-L1), renal cell carcinoma (Nivolumab), Hodgkin lymphoma

(Nivolumab, Pembrolizumab), head and neck squamous cell carcinoma (Nivolumab), urothelial carcinoma (Pembrolizumab; Atezolizumab; Avelumab, PD-L1; Durvalumab,

PD-L1), and more.59 The clinical efficacy of checkpoint inhibition, and the subsequent rapid FDA-approval of drugs targeting these molecules, demonstrates the power of immunotherapy in the context of cancer. Additional checkpoint pathways and their inhibitors are being elucidated in hopes of replicating the success of targeting PD-1/PD-

L1 and CTLA4. LAG-3,60 TIM-3,61 TIGIT,62,63 VISTA,64 and B7/H365 have been shown to be involved in pathways that negatively regulate T cell and/or natural killer cell activation and therefore serve as potential targets for this type of immunotherapy.

While the large share of the focus in developing checkpoint inhibitor treatments has been in relation to T cells, other varieties of immunotherapy have seen success targeting other cell types, including the cancer cell itself. In leukemic disease, efforts to identify cell surface antigens that could serve as targets for monoclonal antibody therapy has resulted in several therapeutic antibodies such as the α-CD33 antibody gemtuzumab66 and α-CD123 antibodies67 in acute myeloid leukemia (AML) and α-CD20

antibodies rituximab,68 obinutuzumab,69 and ofatumumab70 in chronic lymphocytic leukemia (CLL).

In AML, gemtuzumab is conjugated to a toxin, calicheamicin, which binds DNA, resulting in strand scission and cell death. The conjugate, known as gemtuzumab ozogamicin (GO), was recently approved by the FDA for newly-diagnosed CD33+ adult

AML after a previous voluntary withdrawal in 2010 due to an early stopping of a phase

III clinical trial in light of association of GO with increased early deaths (SWOG S0106).71

In contrast to previous studies that had demonstrated the benefit of GO, S0106 used a lower dose of daunorubicin in the experimental vs control arm; lower doses have since been shown to be inferior to higher doses72,73 and may explain the incongruous results obtained from the study. GO is currently approved for use as both a monotherapy and in combination with daunorubicin and cytarabine. In a randomized phase III trial (ALFA-

0701), GO in combination with chemotherapy extended event-free survival by 54.5% compared to chemotherapy alone (13.6 vs. 8.8 months).74 In a randomized phase II study (AML-19), GO proved effective as a monotherapy in newly diagnosed acute myeloid leukemia in older patients not considered candidates for intensive chemotherapy when compared to best supportive care (event-free survival of 4.9 vs. 3.6 months).75

The success of classical antibody therapy depends on the availability of the antigen. The antigen recognized by GO, CD33, is widely expressed on circulating monocytes and, at a lower level, on macrophages. Additionally, and perhaps most

importantly, the antigen is found on multipotent myeloid precursors suggesting that in addition to clearing leukemic blasts, antibodies against CD33 may target the leukemic stem cell population in AML.76 Moreover, expression of CD33 has been shown to be highest in AML compared to other myeloid neoplasms,77 further supporting the use of

CD33 targeted therapy in the disease. Upon binding to CD33, the antigen-drug complex is quickly internalized.78 Because the α-CD33 antibody is conjugated to a toxin, the internalization upon binding to the antigen is a desired effect; yet it’s been suggested that the ability of cells to renew expression of empty CD33 on the surface of the cell may correlate with sensitivity to GO.79 The availability of the antigen thus proves to be one of the most critical aspects in designing antibody therapy.

Acute Myeloid Leukemia

The studies presented here focus on myeloid function in the context of acute myeloid leukemia. AML is characterized by disruption in cellular differentiation and enhanced proliferation of early hematopoietic stem cells with subsequent accumulation of immature myeloid blasts in the bone marrow. Over 20,000 people are estimated to be diagnosed with AML and 10,000 patient die from the disease in The United States each year;80 this number is expected to grow as the population ages.81 Aggressive chemotherapy followed by allogeneic stem cell transplant is the standard approach for most patients with AML, but is ultimately often ineffective due to the high incidence of relapse. In addition, targeted therapy has only recently shown success, in part to the recognition that AML is likely multiple diseases with driving gene translocations or gene mutations involved in differentiation and proliferation.82,83 While AML largely occurs in older populations, 10-20% of childhood leukemias fall under its classification. Contrary to adult AML, pediatric disease has seen significant improvement in outcomes over the last decades, with long term survival rates up to 70%.84-86 The comparative success in pediatric versus adult disease can be attributed in part to a number of factors including superior intensive chemotherapy tolerance and increased rates of favorable-risk cytogenetic abnormalities like core-binding factor translocations. In contrast, adult AML patients under the age of 60 have a five-year survival rate of 40%;81,87 the number drops to 26.6% when all adult AML cases are considered.88

Classification

The World Health Organization, in an update to their 2008 classification of tumors of the hematopoietic and lymphoid tissues, released a classification of myeloid neoplasms and acute leukemia in 2016.89 There, they classify AML into six major subgroups: 1) AML with recurrent genetic abnormalities, 2) AML with myelodysplasia- related changes, 3) Therapy-related myeloid neoplasms, 4) AML, not otherwise specified, 5) Myeloid sarcoma, and 6) Myeloid proliferations related to Down syndrome.

Under “AML with recurrent genetic abnormalities”, the disease is classified further based on cytogenetic and molecular genetic subgroups. Those outlined here are not the only cytogenetic abnormalities that occur in AML, but represent the most common; any not listed are considered rare.90

 RUNX1-RUNX1T1: t(8;21)(q22;q22.1). Translocations between

chromosomes 8 and 21 are found in approximately 5-10%91,92 of AML

cases with higher incident among pediatric patients.93,94 The abnormality

is associated with more favorable prognosis as well as a higher frequency

of secondary genetic alterations.95 RUNX1 (AML1, CBFA2) is a DNA

binding transcription factor subunit that binds together in a

heterodimeric complex with core-binding factor β (CBFB) to regulate

differentiation of hematopoietic stem cells. RUNX1-RUNX1T1 fusion

proteins act as a constitutive transcriptional repressor, preventing

myeloid differentiation and resulting in blast accumulation.96

 CBFB-MYH11: inv(16)(p13.1q22) or t(16;16)(p13.1;q22). Another gene

associated with core-binding factor, CBFB is the non-DNA binding subunit

and functions by enhancing binding of the alpha subunit to target

sequences. In AML, the fusion product sequesters the alpha subunit of

the core-binding factor dimer, RUNX1, in the cytoplasm preventing the

activation of typical myeloid differentiation programming.97 CBFB-

MYH11 is found is approximately 6-8% of adult de novo AML and, similar

to the other core-binding factor fusion RUNX1-RUNX1T1, carries a

favorable prognosis.98,99

 PML-RARA: Typically presents as t(15;17) resulting in a fusion protein

between the retinoic acid receptor alpha (RARA) and promyelocytic

leukemia (PML). The hallmark of acute promyelocytic leukemia (APL),

PML-RARA fusions interfere with the transcriptional activities of RARA.

Curable after treatment with all-trans retinoic acid (ATRA) or arsenic

trioxide. Further details concerning PML:RARA and APL are presented in

Chapter 2.

 MLLT3-KMT2A: t(9;11)(p21.3;q23.3). Fusion proteins involving KMT2A

(mixed-lineage leukemia 1, MLL1), also known more generally as MLL

rearranged leukemias, account for around 10% of older children and

adult AML with a higher incident in infant AML (35-50%).100,101 The

t(9;11) specifically is found in approximately 2-5% of all AML and up to

25% in pediatric AML and is generally associated with poor prognosis.102

MLL encodes a methyltransferase that positively regulates gene

transcription; fusion products result in sustained HOX expression and

stalled differentiation.103,104

 DEK-NUP214: t(6;9)(p23;q34.1). Translocations resulting in the fusion

between DEK and NUP214 occur in less than 5% of AML patients and

generally correlate with poor prognosis.105,106

 GATA2, MECOM: inv(3)(q21.3q26.2) or t(3;3)(q26.2). 3q abnormalities are

found in less than 5% of AML and carry an unfavorable prognosis.107

 RBM15-MKL1: t(1;22)(p13.3;q13.3). Megakaryoblastic leukemia that

presents mainly in infants accounting for 4-20% of pediatric AML.108

 Additional categories include AML with mutated NPM1 (approximately

27% of adult AML cases109) and AML with biallelic mutations of CEBPA (8-

19% of cytogenetically normal AML;110 improved prognosis associated

with biallelic but not single mutations111). Additional provisional entities

include BCR-ABL1 fusions that, while rare, are classified as a distinct

entity due to the potential benefit from tyrosine kinase inhibitor

therapy112,113 and RUNX1 mutations not associated with cytogenetic

abnormalities that seem to carry a worse prognosis.114

The next subgroup of AML is “AML with myelodysplasia-related features”. To fit under this category, patients much meet the criteria for a diagnosis of AML (>20%

myeloblast count) and have a disease that either developed from a previous documented myelodysplastic syndrome (MDS), has cytogenetic abnormalities related to

MDS (listed in Table 18 here89), or present with morphologically identified multilineage dysplasia, defined as >50% percent of cells in two or more hematopoietic lineages. AML with myelodysplasia-related features has been shown to correlate with a decrease in overall survival and progression-free survival.115

Therapy-related neoplasms occur as a result of previous cytotoxic therapy that induces MDS or AML. Treatment and prognosis depends on cytogenetic abnormalities.

Germ line mutations may accompany in cancer susceptible genes.116

AML, not otherwise specified defines a subset of the disease that does not fit into the previously described categories. Here, the patient is classified based on morphological characteristics of their blasts that reflect the French-American-British

(FAB) classification system that was utilized before the clinical significance of cytogenetic profiling was realized. Within this category, patients are sorted into the following: AML with minimal differentiation (FAB M0), AML without maturation (FAB

M1), AML with maturation (FAB M2), acute myelomonocytic leukemia (FAB M4), acute monoblastic/monocytic leukemia (FAB M5), pure erythroid leukemia (FAB M6), acute megakaryoblastic leukemia (FAB M7), acute basophilic leukemia, and acute panmyelosis with myelofibrosis.

Myeloid sarcoma refers to an extramedullary mass consisting of myeloid blasts that isn’t as much a subtype, but rather a distinct clinical presentation of AML. Myeloid

sarcomas predate leukemic disease in a quarter of cases by months or even years, and is most commonly reported in the skin, bone, and lymph nodes. The disease is rare and large studies have not been completed, but there does not appear to be a correlation between myeloid sarcoma and outcome.117

Myeloid proliferations related to Down syndrome are diseases that develop in the setting of trisomy 21 and can be further classified into two subcategories. The first, transient abnormal myelopoiesis (TAM), develops after fetal acquisition of GATA1 mutations. The condition typically spontaneously resolves in months but can lead to the second type of DS-related myeloid proliferation, myeloid leukemia associated with

Down Syndrome (ML-DS) in 20-30% of patients.118 ML-DS is derived from the same

GATA1 mutation with additional mutations in the JAK/STAT pathway and presents as a megakaryoblastic leukemia.119

Standard of Care

Intensive induction chemotherapy is still the standard therapeutic approach for

AML patients 18-60 years of age. Some older patients, based on their cytogenetic risk stratification and co-existing conditions, may not respond well to induction therapy and may be candidates for investigational therapies. Standard “7+3” induction therapy is composed of seven days of continuous infusion cytarabine and three days intravenous anthracycline (daunorubicin, doxorubicin, idarubicin, or mitoxantrone). Cytarabine, also

known as cytosine arabinoside or ara-C, is a cytosine base attached to an arabinose sugar that is similar enough to deoxycytidine to be incorporated into DNA. Upon conversion to its active form, arabinofuranosylcytosine triphosphate, the drug inhibits

DNA polymerase activity and induces DNA damage specifically in S phase of the cell cycle. Anthracyclines are DNA intercalating agents that function by inducing cytotoxicity through the inhibition of topoisomerase II. A complete response is achieved in 60-85% of younger adults (ages 18-60) after induction therapy, but drops in older patients (>60) at 40-60%.120 As mentioned in the previous section, the addition of the α-CD33 antibody-toxin conjugate GO has shown to reduce the risk of relapse and improve survival time when combined with standard induction therapy.121

Upon remission after induction therapy, patients typically undergo consolidation therapy consisting of intermediate-dose cytarabine and, if eligible, allogeneic hematopoietic stem cell transplantation (HSCT). It has been demonstrated that cytogenetic variations in patients pre-treatment are indicative of induction response, probability of relapse, and average overall survival.122 Moreover, the cytogenetic risk stratification has shown to correlate with post-remission response to HSCT123 and is an important factor, along with age and comorbidities, in assessing the suitability of HSCT for patients.124 Patients that are classified with intermediate- or poor-risk AML typically respond quite poorly to cyatrabine consolidation, with cure rates below 15%.120 Thus, in these patients where conventional therapies are unlikely to result in long-term remission, HSCT may provide a curative option. In fact, when comparing allogeneic

HSCT to autologous transplant or consolidation chemotherapy, there was an advantage in relapse-free and overall survival in intermediate- and poor-risk but not in favorable- risk AML.125

Emerging Therapies

In spite of conventional treatment, adult AML patients under the age of 60 still have a five-year survival rate of only 40%126 demonstrating the high risk of relapse and the need for novel therapeutic strategies. As the characteristics and variance in outcome of different cytogenetic subgroups of AML is further elucidated, efforts to target specific genetic aberrations have increased.

Mutations in fms like tyrosine kinase 3 (FLT3) are found in roughly one third of adult AML patients.109,127 Mutations fall into two main categories: internal tandem duplications (ITD; 25% of cases) and tyrosine kinase domain mutations (TKD; 7-10%).

These mutations lead to aberrant FLT3 signaling and constitutive activation of downstream pathways including STAT3/5 and MAPK and result in cell survival and proliferation.128,129 FLT3 mutations generally correlate with a poor prognosis for the patient. Sorafenib is a first-generation tyrosine kinase inhibitor (TKI) that targets a number of kinases including FLT3-ITD. In a number of clinical trials, Sorafenib demonstrated ability to reduce blast counts and induce CR in relapsed/refractory (R/R)

AML, but the majority of patients relapsed within 72 days of remission.130-132

Midostaurin, another first generation TKI, saw similar success to Sorafenib but was again limited by the rapid emergence of resistance.133,134 Second generation TKIs (Quizartinib,

Crenolanib, Gilteritinib) specifically target FLT3 mutants and reduce off-target toxicities compared to first generation TKIs, but still result in only transient responses and eventual relapse.135-137 Investigating and overcoming the mechanisms of resistance, potentially with the addition of other agents, is an ongoing area of research.

STAT3 activation (i.e. tyrosine phosphorylation) has been shown to be upregulated in AML and correlates with shortened disease-free survival time.138,139

Activation of the STAT pathway can occur via the FLT3 ligand and may be one mechanism through which TKI resistance occurs.140 Small molecule STAT inhibitors

(OPB-31121) have demonstrated the ability to overcome certain FLT3 inhibitor resistance.141

IDH1 and IDH2 mutations occur in approximately 5-10% and 10-20% of AML patients, respectively109,142,143 and also are targets for small molecule inhibitors. In their unmutated form, the enzymes convert isocitrate to α-ketogluterate (α-KG) in the cytoplasm (IDH1) and mitochondria (IDH2). The mutated enzymes in AML function by converting the product of the WT IDH, α-KG, into an oncometabolite, 2- hydroxygluterate (2-HG). 2-HG has been shown to competitively inhibit α-KG-dependent dioxygenases and alter DNA and histone methylation signatures resulting in a block in cellular differentiation.144-146 Enasidenib, an IDH2 inhibitor, has shown to induce a hematological response in a phase 1/2 trial of R/R AML147 and Ivosidenib, an IDH1

inhibitor, induced durable remission and improved patient outcomes in a phase I clinical trial in R/R AML.148

DNA methyltransferase 3A (DNMT3A) mutations are common in AML, occurring in around 20-25% of adult AML and confer a poor prognosis.109,149 The mutation results in abnormal DNA methylation and gene expression. In cases where there is a loss-of- function of DNMT3A, there appears to be a correlation with increased expression of the histone 3 lysine 79 (H3K79) methyltransferase, Dot1l. After inhibition of Dot1l, terminal differentiation, cell-cycle arrest, and apoptosis induction was observed in AML cells with a DNMT3A mutation.150 In addition to DNMT3A AML, Dot1l has been implicated as being important in the H3K79 methylation that helps drive MLL-rearranged AML, another subset of AML with poor prognosis.151,152 Small molecule inhibition of the target has shown to inhibit H3K79 methylation and confer survival advantages in preclinical murine models of MLL-rearranged AML.153,154

In addition to small molecule therapies that target specific genetic aberrations, antibody-based and cell-based therapies are being explored. As mentioned earlier, the

α-CD33 antibody-toxin conjugate, GO, is currently the only antibody therapy to receive

FDA approval for the treatment of AML, but clinical and pre-clinical investigations have identified α-CD-123,155,156 α-CD45-radionuclide conjugates,157,158 and α-CD40159,160 as potential therapeutic options. In Chapter 2, a novel immunotherapy in AML is proposed using the retinol derivative ATRA to upregulate an antigen on AML blasts, the

glycoprotein CD38. When combined with an α-CD38 antibody daratumumab, it’s shown that the AML blasts can mediate fratricidal ADCC.

Cell-based therapies, while sometimes lacking the specificity of antibodies or small-molecule drugs, have also shown promise in AML. The best example of success,

HSCT, is already a mainstay in post-induction treatment for eligible patients, but other therapies are also emerging that may play a role in initial treatment or in the setting of

R/R AML. These include chimeric antigen receptor (CAR) T cells and NK cell adoptive transfer. Of note, NK cell therapies utilizing haploindentical donors have shown efficacy in the disease.52,161-163 In Chapter 3 a combination of immune stimulation peptide, muramyl tripeptide-phosphatidylethanolamine (MTP-PE), and interferon-γ (IFN-γ) is investigated; the two agents together not only induce apoptosis in AML blasts, but facilitate the maturation of diseased-suppressed NK cells. The implication of this discovery is further discussed in that chapter.

CHAPTER 2: Anti-leukemic effects of all-trans retinoic acid in combination with daratumumab in acute myeloid leukemia

Abstract

AML remains a significant health problem, with poor outcomes despite chemotherapy and HSCT. Although one form of AML, acute promyelocytic leukemia

(APL), is successfully treated with all-trans retinoic acid (ATRA), this agent is seemingly ineffective against all other forms of AML. Here, we show that ATRA upregulates CD38 expression on AML blasts to sufficient levels that promote antibody-mediated fratricide following the addition of anti-CD38 daratumumab. The combination of ATRA plus daratumumab induced Fc-dependent conjugate formation and cytotoxicity among AML blasts in vitro. Combination treatment also led to reduction in tumor volume and resulted in increased overall survival in murine engraftment models of AML. These results suggest that, although ATRA does not induce differentiation of non-APL, it may be effective as a therapy in conjunction with daratumumab.

Introduction

Acute promyelocytic leukemia

APL is a subset of AML characterized by a t(15;17) chromosomal translocation resulting in a fusion protein of retinoic acid receptor alpha (RARA) and promyelocytic leukemia gene (PML). First described as a separate entity in the 1950s, APL initially was one of the most fatal forms of cancer with a median survival in untreated cases of less than a week.164-168 Some success was observed after the administration of daunorubicin; remission rates increased from 13 to 58%.169 Despite the progress, average remission times ranged for only 11 to 25 months and fewer than half the patients were cured based on five-year-survival rates.170,171 The most striking advancement in APL treatment occurred in the early 1980s when retinoic acid first demonstrated the capability of differentiating promyelocyte blasts.172,173 A few years later, data were published on the first clinical trials utilizing ATRA in APL with all patients achieving complete remission.174,175 Subsequent studies established that concurrent treatment of ATRA with chemotherapy (daunorubicin and cytarabine) decreased incidents of relapse176-178 cementing the regiment as the standard for induction therapy in newly diagnosed APL.179

In addition to the use of ATRA in APL, studies have shown the efficacy of arsenic trioxide (ATO) in the clearance of leukemic disease as a monotherapy.180,181 The treatment was initially recommended for consolidation/maintenance therapy or for

relapsed patients,182,183 but in the last decade combinations of ATRA and ATO with and without chemotherapy have also demonstrated robust anti-leukemic effects as induction therapy in newly diagnosed APL patients.184-187 ATO induces differentiation at low doses and apoptosis at higher doses. The agent targets PML and successfully degrades the fusion protein PML:RARA allowing for proper myeloid differentiation.188,189

However, while ATRA combined with chemotherapy or arsenic trioxide results in cure of the great majority of patients with APL, it has failed to elicit the same differentiating effect in other subtypes of AML.190

All-trans retinoic acid

Retinoids, a class of compounds encompassing vitamin A and its analogs, have been extensively studied since their classification in the early 1900s. The compounds have proved essential in a variety of physiological processes including development, vision, and regulation of immune function.191-193 Classified as a vitamin, retinoids are taken up through the diet; when animal tissue is consumed this occurs in the form of retinyl esters.194 These storage retinoids are deposited largely in the liver and are biologically inactive. Retinyl esters can be hydrolyzed to retinol (vitamin A1) by retinol ester hydrolases; further oxidization of retinol in the cell by retinol dehydrogenases results in the molecule all-trans retinaldehyde. Subsequent conversion by retinaldehyde dehydrogenase produces the bioactive molecule all-trans retinoic acid (ATRA).195

Among the various functions of retinoids, ATRA in particular has been implicated in genomic regulation. The molecule binds with high affinity to retinoic acid receptors

(RARα,-β,-γ), a class of steroid/thyroid hormone nuclear receptors; the resultant ligand- receptor complex functions as a transcription factor.196-198 Specifically, in the absence of a ligand, RAR complexes in a heterodimer with RXR (retinoic X receptor) on AGGTCA repeats separated by 5 nucleotides on DNA termed retinoic acid response elements

(RARE).199-201 Unlike RAR, RXR does not bind ATRA but rather 9-cis-retinoic acid;202,203 in addition to composing a heterodimer with RAR, RXR can form a homodimer that binds to the same DNA tandem repeat separated by one nucleotide.200,204,205 Upon binding of

ATRA, RAR undergo conformational changes that modulate affinity for co-activators and co-repressors that results in gene transcription or repression. Of the three RAR, RARα is the most ubiquitously expressed; RARβ and RARγ show more restricted expression.206

Hundreds of genes are regulated in this manner by ATRA (reviewed here207); the ectoenzyme CD38 has been shown to be one that is consistently upregulated.208,209

In APL, the translocation between chromosomes 15 and 17 creates a fusion protein of PML:RARA that successfully repress the differentiating ability of retinoic acid through a variety of ways. The fusion protein, binding to RARE, sequesters RXR and recruits co-repressors including histone-deacetylase complexes and methyltransferases

DNMT1 and DNMT3A that result in the repression of typical transcriptional programming.210-212 In addition to the transcriptional regulation, PML:RARA disrupts the localization and tumor suppressor functions of PML, all of which contributes to a hyper-

proliferative expansion of immature blasts. It has been demonstrated that pharmacological doses of ATRA induces a conformational change in the PML:RARA fusion that allows for repressor disassociation and recruitment of co-activators resulting in transcription of target genes. Moreover, it appears that ATRA itself is capable of inducing degradation of the fusion protein directly by stimulating caspase cleavage.213,214

Regulation of ATRA is controlled in part by a group of monooxygenase enzymes known as cytochromes P450. Three members of this family in particular, CYP26A1,

CYP26B1, and CYP26C1, have been extensively studied in relation to the metabolism and isomerization of retinoic acid; these enzymes convert ATRA into more polar metabolites including 4-hydroxy-, 4-oxo-, and 18-hydroxy-retinoic acid.215,216 The three CYP26 genes are differentially expressed throughout tissues in the body as well as during different developmental stages. CYP26C1 seems to be primarily expressed during embryonic development,217,218 CYP26B1 is found mainly in the adult cerebellum,215 and

CYP26A1 is expressed largely in the adult liver.219,220 The metabolites produced by these enzymes are less biologically active and more easily secreted. The differential expression of the CYP26 enzymes, together with the regulated localized concentration of ATRA synthesizing enzymes (retinol- and retinaldehyde dehydrogenase), creates a dynamic system of controlled retinoic acid expression throughout the body. Moreover, it has been demonstrated that ATRA itself induces expression of the CYP26 proteins, initiating a feedback inhibition loop to control retinoic acid signaling.221-223

ATRA, under the name Tretinoin, has been approved for additional uses besides that in APL. Topical tretinoin has been approved for the treatment of acne vulgaris and has demonstrated potential in a host of additional dermatological conditions with little side effects.224 For APL, ATRA is given orally; standard dosing is 45 mg/m2 per day.225,226

Here there is possibility of patients developing a condition known as differentiation syndrome. Also called retinoic acid syndrome, differentiation syndrome occurs when

ATRA, a differentiation agent, is used to target the large population of immature leukemic blast found in diseases such as APL. The mass blast differentiation results in cellular migration and release of cytokines and vascular factors associated with tissue damage. Unchecked, differentiation syndrome can lead to respiratory distress with pulmonary infiltrates, capillary leak syndrome, and acute renal failure.227-229 However, proper monitoring and immediate or even preemptive intervention with intravenous corticoids (dexamethasone) greatly mitigates risk in patient.230-232

Daratumumab

Daratumumab is a monoclonal IgG1κ antibody against CD38, a transmembrane glycoprotein with both receptor and enzymatic functions expressed on hematopoietic cells. The intracellular enzymatic function of CD38 is capable of metabolizing NAD and

NADP resulting in the production of dynamic secondary messengers cyclic ADP ribose and NAADP.233-235 Independent of its enzymatic function, and of particular interest in the context of cancer, signaling through the CD38 receptor results in the activation,

proliferation, and survival of myeloid236,237 and lymphoid cells.238-241 Thus, targeting the receptor has become an attractive option in leukemic disease. Of note, daratumumab was approved by the FDA in 2015 for the treatment of Multiple Myeloma.242

Given the survival benefit of antibody therapy in other hematologic malignancies including ALL, CLL, NHL, and multiple myeloma, significant interest in developing antibody therapeutics for AML exists. As the effectiveness of antibody therapy is in part related to density of antigen on tumor cells, we hypothesized that efforts to up-regulate targetable antigens on these leukemia cells would offer therapeutic advantage. While the CD38 antigen is only expressed on a subset of AML patients (and therefore represents a less optimal target), it has been found to be upregulated by ATRA on a variety of oncogenic cells including APL243 and multiple myeloma.208 Here, we assessed the effects of ATRA on non-APL AML cells and found that it led to significant upregulation of CD38 and enhanced daratumumab-mediated AML-cell fratricide in vitro.

Additionally, using a murine engraftment model of AML we found that ATRA plus daratumumab significantly increased overall survival time compared to either agent alone. These results support future trials combining ATRA with daratumumab in AML.

Materials and Methods

Reagents

All-trans retinoic acid (ATRA) was purchased from Sigma-Aldrich (St. Louis, MO); daratumumab was supplied from commercial sources (The Ohio State University,

Columbus, OH, USA). AM580 was purchased from abcam (Cambridge, MA).

Cell Culture

The AML cell lines used for this study were MV4-11, OCI-AML3, MOLM-13, and U937.

Cells were purchased from American Type Culture Collection and cultured in RPMI

Medium 1640 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Grand Island, NY), 2mM L-glutamine

(Invitrogen, Grand Island, NY), and 56U/mL/56μg/mL penicillin/streptomycin

(Invitrogen) at 37°C in 5% CO2. Aphaeretic white blood cells from AML patients were obtained under written informed consent in accordance to protocol approved by the institutional review board of The Ohio State University. Samples were stored in liquid nitrogen in 20% FBS and 10% DMSO prior to use. Upon thawing, cells were cultured in

RPMI Medium 1640 (Gibco) supplemented with 20% FBS, 2mM L-glutamine (Invitrogen), and 56U/mL/56μg/mL penicillin/streptomycin (Invitrogen) at 37°C in 5% CO2.

Quantitative Real-time Polymerase Chain Reaction (qPCR)

RNA was extracted using the Total RNA Purification Plus Kit (Norgen Biotek Corp.,

Ontario, Canada). RNA was reverse-transcribed before being quantified by qPCR using

Power SYBR® Green Master Mix (Applied Biosystems, Grand Island, NY). GAPDH was used as a reference gene for normalizing target genes. Primers used were: CD38

(forward 5′-GCT CAA TGG ATC CCG CAG T-3′ and reverse 5′-TCC TGG CAR AAG TCT CTG

G-3′) and GAPDH (forward 5′-ATT CCC TGG ATT GTG AAA TAG TC-3′ and reverse 5′-ATT

AAA GTC ACC GCC TTC TGT AG-3′). Data are presented as relative copy number (RCN),

–ΔCt calculated as 2 x 100, where ΔCt is the Ct(target) – Ct(GAPDH).

Flow Cytometry

Cells (1x106/mL) were incubated with 1μg anti-human CD38 antibody conjugated to FITC

(BioLegend, San Diego, CA: Cat #303503) or to FITC-conjugated isotype (BioLegend: Cat

#400108) for 30 minutes at 4°C preceding two washes with FACs buffer (PBS, 0.09% sodium azide, 10% FBS). Samples were analyzed using an LSRII flow cytometer (BD

Bioscience, San Jose, CA) and FlowJo software (FLOWJO, LLC, Ashland, OR).

Solid-Tumor Murine Model

All animal experiments were carried out in full accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University.

Non-obese diabetic severe combined immunodeficient-IL2Rγ-/- (NSG) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and bred in a university vivarium

under the direction of Dr. Adrienne Dorrance. 2.5x106 MV4-11 cells in PBS were injected subcutaneously into the flanks of 25 3-month-old female mice. Tumors were allowed to grow for 10 days before the mice were randomly sorted into four groups: untreated,

ATRA (10 mg/kg mouse), daratumumab (1 μg/g mouse), and ATRA + daratumumab.

Intraperitoneal injections of ATRA were administered four times a week (Mo, Tu, Th, Fr) via corn-oil vehicle; daratumumab in PBS vehicle was administered via intraperitoneal injection twice a week (Tu, Fr). Treatment was carried out for 18 days, with tumor volume measured in a blinded fashion.

Leukemic Murine Model

For the leukemic model, 3.0x105 spleen cells from MV4-11-engrafted mice were injected intravenously into 75 NSG mice (male and female; 2-5 months old) and allowed to engraft for one week before commencing treatment. Grouping, dosing, scheduling, and mode of injection of ATRA and daratumumab were identical to the solid-tumor model.

Early-removal criteria consisted of 20% weight loss, paralysis or inability to stand, uncontrolled shivering, or unwillingness to eat or drink. Animal studies were approved by the Institutional Laboratory Animal Care and Use Committee at The Ohio State

University.

Conjugate Formation Assay

MV4-11 cells were plated at 1x106 cells/mL and treated with or without 1 μM ATRA and/or 20 μg/mL daratumumab for 24 hours at 37°C in 5% CO2. Cells were centrifuged at 200g for 10 minutes, culture media was removed, and the cells were washed with

PBS before being fixed in 4% paraformaldehyde for 10 minutes at 21°C. Rhodamine

Phalloidin F-actin stain (Cytoskeleton, Inc., Denver, CO) was diluted to 100 nM per manufacturer’s instruction; 200 μL were added to each sample before incubation for 30 minutes at 21°C in the dark. Samples were then washed 3 times with PBS and then counted in a blinded fashion via fluorescence microscopy. Conjugation index is defined as the number of cells with at least one conjugate per 100 cells.

Lactate Dehydrogenase Assay

MV4-11 cells were plated at 5x105 cells/mL and treated with 1 μM ATRA and/or 20

μg/mL daratumumab. At 24-hour intervals, supernatants were removed and used for a

CytoTox96® Non-Radio Cytotoxicity Assay (Promega, Madison, WI) according to manufacturer’s instruction. Percent cytotoxicity was defined as [Experimental LDH release OD490 / Maximum LDH release OD490] x 100, and was normalized to UT or ATRA- alone for all samples.

Trypan Blue Exclusion Assay

Trypan Blue (Sigma-Aldrich, St. Louis, MO) was used to stain cells according to manufacturer instructions. Cells were counted on a Luna Dual Fluorescence Cell Counter

(Logos Biosystems Inc., Annandale, VA).

Statistical Analysis

For all analyses: * denotes an α = 0.05; ** an α = 0.01; *** an α = 0.001. For the experiments with repeated measures, data were analyzed by mixed effect models, and the time or dose dependencies were analyzed by trend tests. A paired t-test was used for qPCR and flow cytometry analysis of CD38 expression. For the experiments involved multiple independent groups, data were analyzed by analysis of variance (ANOVA) followed by pairwise comparisons. Tumor volumes in the solid-tumor murine model were adjusted from their baseline and a mixed effect model incorporating repeated measures was used to test the difference in tumor growth trends and tumor volumes at the last two days; an interaction contrast was used to test the synergy of the two drugs.

Mantel-Cox log-rank tests were used to analyze the survival functions derived from the tail-vein murine model. The difference in survival probabilities among groups were analyzed by log-rank tests. The synergy between ATRA and daratumumab was tested by interaction contrasts. Multiplicity was adjusted by Holm’s method. Data analyses were performed by using SAS 9.4 (SAS 9.4, Inc; Cary, NC).

Results

ATRA upregulates CD38 in AML cells

Recent work by Yoshida et al. showed that ATRA upregulated CD38 expression in

KG-1 and U937 AML cell lines, and select primary AML samples.244 To further confirm this effect of ATRA, we treated AML cell lines and primary patient samples with 1 μM

ATRA for 24 and 48 hours, then measured CD38 mRNA expression via qPCR. Results showed that for both time points, ATRA significantly upregulated CD38 in all cell lines and primary samples (Figure 2.1A and C, respectively; patient cytogenetic and mutational status are included in Appendix A: Supplemental Figures, Table A.1). To verify that this increase in transcript levels correlated with an increase in surface antigen expression, samples were analyzed via flow cytometry. Results showed significant upregulation in surface expression of CD38 after 24 hours of ATRA treatment in all four

AML cell lines (Figure 2.1B) as well as in all nine patient samples (Figure 2.1D; average increase of 94.4%).

In order to explore dose-response relationships we then treated MV4-11 cells for

24 hours with concentrations of ATRA ranging from 0-5 μM and measured CD38 via qPCR. Although some increase in expression could be seen with as little as 2.5 nM, 1 μM of ATRA led to optimal induction (Figure 2.2A). Next, we treated MV4-11 cells with 1 μM

ATRA for time points between 0 and 72 hours and found that CD38 mRNA increased in as early as 3 hours with continued rise through the 72-hour time point observed (Figure

2.2B). These results suggest that ATRA can significantly increase mRNA and surface expression of CD38 antigen on AML cells.

It has been previously shown that retinoic acid receptor alpha (RARα), when bound to its ligand ATRA, associates with CD38 and induces transcription of the gene.245

Using a selective agonist for RARα, AM580, we tested whether direct activation of the transcription factor was sufficient to elicit the potent upregulation of CD38 we observed with ATRA. Figure 2.3A shows CD38 mRNA levels in MV4-11 cells treated with 10nM

ATRA or AM580 after 24 hours. Transcript levels, while significantly higher than the untreated cells, showed no difference between ATRA and AM580. Flow cytometry was utilized to assess surface protein levels in identical conditions (Figure 2.3B); again no difference between ATRA and AM580 treated samples was observed. These data suggest that direct activation of RARα is sufficient to upregulate CD38 expression in AML blasts.

Additionally, Lin-/CD34+ /CD123+/CD45Rα+ cells were isolated from AML patient bone marrow samples and treated with ATRA for 24 hours. As shown in Figure 2.4, significant increases in CD38 levels were observed. This population of cells are leukemic stem cells (LSCs), progenitors that are characterized by cell-cycle quiescence, disease- renewal, and resistance to chemotherapy. Upregulating an antigen for a therapeutic antibody like daratumumab on the surface of these cells may provide an avenue for

LSCs elimination, the ultimate goal in curative leukemic therapies.

A ) 30 *** ***

Untreated 20 ATRA ** ***

10 * ** CD38 RCN CD38 ** ***

0 24 hr 48 hr 24 hr 48 hr 24 hr 48 hr 24 hr 48 hr MV4-11 OCI-AML3 MOLM-13 U937 B 8000 ) **

Untreated 6000 * ATRA * ** 4000

CD38 MFI CD38 2000

0 24 hr 24 hr 24 hr 24 hr MV4-11 OCI-AML3 MOLM-13 U937

C D ) 8 ** ) 5000 * 4000 6

3000 4 2000

CD38 MFI CD38

CD38 RCN CD38 2 1000

0 0

ATRA ATRA Untreated Untreated Figure 2.1. ATRA upregulates CD38 in AML cells. (A) MV4-11 (n=5 separate experiments), OCI- AML3, MOLM-13 and U937 cells (n=3 separate experiments each) were treated with 1 μM ATRA for 24 and 48 hours (ATRA added every 24 hours). CD38 transcript was measured by qPCR. (B) Cell lines (n=3 separate experiments) were treated with 1 μM ATRA for 24 hours and analyzed for CD38 surface protein expression by flow cytometry. (C,D) Primary AML patient apheresis samples were treated with 1 μM ATRA for 24 hours and CD38 transcript level (C; n=10 donors) and surface protein expression (D; n=9 donors) were measured; representative histogram for the flow cytometry shown in D. * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

60 ***

Fold Change from UT ( from RCN) Change Fold M M M    5 nM 1 2 5 10 nM B) 2.5 nM 100 nM500 nM Untreated

60 **

Fold Change from UT ( from RCN) Change Fold

1 hr 3 hr 6 hr 16 hr 24 hr 48 hr 72 hr

Untreated

Figure 2.2. ATRA upregulates CD38 in AML cells (cont). (A) MV4-11 cells (n=3 separate experiments) were treated with concentrations of ATRA ranging from 0-5 μM and CD38 transcripts were measured after 24 hours by qPCR. (B) MV4-11 cells (n=3 separate experiments) were treated with 1 μM ATRA for 1-72 hrs and CD38 transcripts were measured by qPCR. ** denotes p≤0.01; *** p≤0.001.

10 *** ns 8

CD38 RCN CD38 2

ATRA AM580 Untreated

B) 6000 *** ns

4000

CD38 MFI CD38 2000

ATRA AM580 Untreated

Figure 2.3. ATRA upregulates CD38 via RARA. MV4-11 cells (n=3 separate experiments) were treated with 10nM ATRA or AM580 for 24 hours; CD38 levels were measured by qPCR (A) and flow cytometry (B; representative histogram shown). *** denotes p≤0.001.

***

8000

7000

6000

5000

2000

MFI CD38 (FITC) CD38 MFI 1000

ATRA Untreated

Figure 2.4. ATRA upregulates CD38 on AML stem cells. Lin-/CD34+ /CD123+/CD45Rα+ population from primary AML samples (n=5) treated with 1 μM ATRA for 24 hours. CD38 surface protein measured via flow cytometry. *** denotes p≤0.001.

ATRA triggers daratumumab-mediated immune conjugate formation and killing in vitro

Since ATRA upregulates surface expression of CD38, we next tested whether the combination of ATRA and anti-CD38 antibody daratumumab would enhance cytotoxicity of AML cells. One of daratumumab’s mechanisms of tumor elimination is antibody- dependent cellular cytotoxicity by immune cells,208 and we have previously found that

AML blasts were capable of targeting one another in an antibody-dependent manner.246

To test if ATRA with daratumumab could elicit this self-targeting, or fratricide, we treated cells for 24 hours with each as a single agent or in combination, then stained for actin using rhodamine phalloidin fluorescent dye. Results showed that combination treatment led to significant increases in cell-to-cell conjugate formation versus single- agent treatment (Figure 2.5).

Following this, MV4-11 cells were treated with single or dual agents and tested for antibody-mediated cellular cytotoxicity over the course of 72 hours via lactate dehydrogenase assay. Results showed that the combination treatment led to a significant increase in percent cytotoxicity compared to untreated and daratumumab alone at 48 and 72 hours, and a significant increase from ATRA alone at 72 hours (Figure

2.6A). To confirm this finding, we performed a Trypan Blue exclusion assay, which also showed a significant decrease in live cells for combination-treated samples compared to all control groups by 72 hours post-treatment (Figure 2.6B). ATRA as a single agent also led to cytotoxicity, albeit to a lesser extent. This is consistent with previous findings that

ATRA could induce cell death in AML cell lines, reportedly due to Bcl-2 down-regulation, loss of mitochondrial membrane potential (ΔΨm), and cytochrome c efflux.247

To confirm that this fratricide was mediated by Fcγ receptors, we generated

F(ab)’2 fragments of daratumumab via pepsin digestion (the ability of this F(ab)’2 fragment to bind to CD38 was confirmed via flow cytometry, data not shown). Pepsin digests the Fc portion of antibodies resulting in a F(ab)’2 molecule that maintains antigen affinity and the ability to confer direct effects, but cannot interact with FcγR.

The conjugate-formation and fratricide experiments were repeated and, as expected, there was complete ablation of conjugate formation when the F(ab)’2 was used in place of whole antibody (Figure 2.7A). This corresponded to a significant decrease in cytotoxicity at 72 hours with the F(ab)’2 fragment compared to whole-antibody (Figure

2.7B). These results suggest that combined ATRA plus daratumumab treatment induces fratricide through Fcγ receptor engagement.

A )

B )

Figure 2.5. ATRA triggers daratumumab-mediated immune conjugate formation in vitro. MV4-11 cells were incubated with or without 1μM ATRA and with or without 20 μg/mL daratumumab for 24 hours, after which samples were fixed and stained with rhodamine phalloidin actin dye. Conjugate formation between the cells was measured via fluorescence microscopy in a blinded fashion (A) and graphed in (B), where conjugation index = (# cells conjugated to at least one additional cell/ # total cells) (n = 3 separate experiments). ** denotes p≤0.01.

Figure 2.6. ATRA plus daratumumab kills in vitro. (MV4-11 cells were incubated with or without 1μM ATRA and with or without 20 μg/mL daratumumab for 24, 48, 72, (and 96) hours (drugs re-added every 24 hours). (A) Cytotoxicity was analyzed via a lactate dehydrogenase assay (LDH); (B) Cell viability was measured via Trypan blue exclusion (n=3 separate experiments). * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

A) 40 *** ***

Conjugation Index Conjugation

0 ATRA ATRA+Daratumumab ATRA+Daratumumab F(ab')2 B) 20 *** **

Compared to ATRA only to ATRA Compared

% Increase in Cytotoxicity %in Increase 0 ATRA ATRA+Daratumumab ATRA+Daratumumab F(ab')2

Figure 2.7. ATRA plus daratumumab kills via Fc-dependent fratricide. (A-B) MV4-11 cells were treated with 1μM ATRA + 20 μg/mL (133nM) daratumumab or 14.6 μg/mL (133 nM) daratumumab F(ab)’ . (A) After 24 hours samples were assessed for conjugate formation (n=3 2 separate experiments). (B) After 72 hours (drugs re-added every 24 hours), supernatants were collected and analyzed via LDH assays (n=3 separate experiments). ** denotes p≤ 0.01; *** p≤0.001.

Single-dose ATRA elicits CD38 upregulation and confers daratumumab activity

The above experiments utilized treatment regimens which provided a relatively constant source of ATRA, so we next sought to determine whether short-term exposure to the drug would elicit similar effects on CD38 expression and antibody-mediated fratricide. We treated MV4-11 cells with ATRA for 45 minutes and then washed it away in order to simulate the reported plasma half-life of ATRA in vivo.248 CD38 transcript was measured 24, 48, and 72 hours after treatment via qPCR and, while expression was markedly lower than non-washout controls, the single dose led to an approximate 12- fold increase in CD38 transcript that held steady throughout the 72 hour time course

(Figure 2.8A). Additionally, when combined with daratumumab, a single dose of ATRA retained its killing potential through 72 hours as measured by lactate dehydrogenase release (Figure 2.8B). Similarly, live-cell counts were reduced through 96 hours (Figure

2.8C). These results suggest that CD38 expression on AML blasts can be modulated by short-term exposure to ATRA.

A B 90 vs COMBO ) 60 *** ) 90 60 *** *** 24h 48h 72h vs COMBO *** *** UT ns *** *** DARA ns *** * *** ** 30 *** *** 24h 48h 72h 60 ATRA ns 40 *** *** UT ns 0 *** ***

Compared to UT Compared 24 hr 48 hr 72 hr 30 DARA ns *** ***

% Increase in Cytotoxicity %in Increase -30 ATRA ns *** ***

CD38 RCN CD38 20 Untreated Daratumumab ATRA ATRA+Daratumumab0 Compared to UT Compared 24 hr 48 hr 72 hr

Fold Change from UT from Change Fold

0 Cytotoxicity %in Increase -30 24 hr 48 hr 72 hr Untreated ATRA ATRA wash Untreated Daratumumab ATRA ATRA+Daratumumab

C 3.010 6 vs COMBO 3.010 6 ) 24h 48h 72h 96h 6 vs COMBO 2.010 UT ns * *** *** DARA ns *** *** *** 24h 48h 72h 96h 6 ATRA ns ns ** *** 6 2.010 1.010 UT ns * *** ***

Live Cells (MV411) Cells Live DARA ns *** *** *** 0 ATRA ns ns ** *** 24 hr 48 hr 72 hr 96 hr6 Live Cells Live 1.010 Untreated Daratumumab ATRA ATRA+Daratumumab

0 24 hr 48 hr 72 hr 96 hr Untreated Daratumumab ATRA ATRA+Daratumumab

Figure 2.8. Single-dose ATRA elicits CD38 upregulation and confers daratumumab activity. (A) MV4-11 cells were incubated with 1 μM ATRA for 45 minutes before the drug was washed off. Cells were collected at 24, 48, and 72 hours after which CD38 expression was measured via qPCR (n=3 separate experiments). (B-C) MV4-11 cells were treated with or without a 45- minute dose of 1 μM ATRA and with or without 20 μg/mL daratumumab every 24 hours for up to 96 hours. (B) LDH assays were run at 24, 48, and 72 hours to determine % cytotoxicity; (C) live cells were counted using Trypan blue exclusion (n=3 separate experiments). * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

ATRA synergizes with daratumumab to impede AML tumor growth and extend survival in vivo

Given the effectiveness of the combination of ATRA and daratumumab in vitro, we next wanted to determine its efficacy in vivo. To test this, we injected MV4-11 cells subcutaneously into the right flanks of NOD scid gamma (NSG) mice to generate solid tumors, and then treated mice with ATRA, daratumumab, or both agents. As shown in

Figure 2.9A, ATRA and daratumumab each individually had no effect on the rate of tumor growth or final tumor volume after 18 days treatment. However, the combination of ATRA plus daratumumab synergized to significantly reduce tumor growth as well as final tumor volume (Figure 2.9B).

Next, we tested the effect of combination treatment on survival in a disseminated AML mouse model. MV4-11 cells were injected intravenously into NSG mice and treated as above in the solid tumor experiment. As shown in Figure 2.10, survival was significantly increased in the combination-treated group compared to the single-drug groups. Hence, although seen as largely ineffective as a differentiation agent for non-APL AML, ATRA appears to be a strong enhancer of anti-CD38 antibody therapy for AML in vivo.

A ) 4000 Untreated

)

3 Daratumumab 3000 ATRA ATRA+Daratumumab * 2000

1000

Tumor Volume (mm Volume Tumor 0 0 5 10 15 20 Days Treatment

B )

Figure 2.9. ATRA synergizes with daratumumab to impede AML tumor growth. (A-B) NSG 6 mice were subcutaneously injected with 2.5 x 10 MV4-11 cells and treated with vehicle (untreated), ATRA, daratumumab, or the combination of ATRA and daratumumab (n=6 per group). (A) Tumor growth rate was compared between the combination of ATRA + daratumumab and vehicle control (untreated), ATRA alone, and daratumumab alone (p- values: <0.0001, <0.0001 and 0.0048, respectively). An interaction test for synergistic effects between ATRA and daratumumab was also done (p=0.0364). (B) Photographs of excised tumors. Note: Mouse #5 in the ATRA group died of disease prior to study conclusion; tumor size was recorded at the time of death (Day 17). * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

100

60 Untreated 40 Daratumumab ATRA

Percent Percent survival 20 ATRA+ Daratumumab 0 0 20 40 60 Days Post Engraftment

Figure 2.10. ATRA and daratumumab extend survival in vivo. NSG mice were intravenously 5 injected with 3.0x10 spleen cells from MV4-11-engrafted mice and treated with vehicle (untreated), ATRA, daratumumab, or the combination of ATRA and daratumumab (n=18 mice per group). Survival was compared between ATRA + daratumumab group versus untreated, ATRA, and daratumumab groups respectively (logrank p-values were 0.0030, 0.0004 and 0.0122, respectively).

Figure 2.11. Schematic of proposed mechanism of ATRA and daratumumab induced fratricide in AML. AML blast treated with ATRA upregulate their surface expression of the ectoenzyme CD38. The receptor, the antigen for the α-CD38 antibody daratumumab, allows for cell-to-cell conjugation and the induction of antibody-dependent cellular cytotoxicity upon addition of the antibody.

Discussion

In this study we demonstrate that the combination of ATRA and daratumumab has potent anti-leukemic effects, mediated through CD38 upregulation and antibody- induced fratricide, in vitro and in vivo, as modeled in Figure 2.11. Indeed, within the murine models, combination treatment led to reduced tumor size and growth rate and a

46.6% increase in mean survival time.

Attempts to replicate the efficacy of ATRA treatment in APL for other AML subtypes have seen limited success over the past decades. While a few studies suggest that ATRA in combination with low- to standard- dose chemotherapy improves complete remission rate and/or event-free and overall survival in non-APL AML patients,249,250 these results have not been replicated in subsequent studies.251-254 One study of note, a United Kingdom Medical Research Council trial in 1075 patients, failed to demonstrate any benefit of adding ATRA to standard dose chemotherapy in patients overall as well as in distinct genotypic subpopulations (FLT3/ITD, NPM1, CEBPA, and

MN1).190

A variety of mechanisms have been proposed for the observed resistance of non-

APL AML to ATRA. The overexpression of RAR complex cofactor and transcriptional repressor MN1 has been shown to mediate such effects; patients with higher expression of the protein show greater resistance to ATRA therapy.255 Unsurprisingly, as shown in

Supplemental Figure A.1, MN1 expression amongst AML cytogenetic subgroups is lowest in APL (PML:RARA) where ATRA treatment has shown to be effective. Of note,

subgroups including patients with a inv(16)(p13q22) CBFB:MYH11 fusion, complex cytogenetics, and poor risk cytogenetic abnormalities all have elevated levels of MN1 which may limit the efficacy of ATRA. In contrast, in addition to APL, patients classified with MLL translocation, poor risk [those with a translocation that is not t(9;11) or t(11;19)] seem to have relatively low MN1 expression.109 Previous work has shown that

MLL-rearranged cells are sensitive to ATRA due to H3K4me2 in the promoter region of

RARα;256 the low expression of MN1 in the subgroup might be a complementary mechanism.

Another proposed mode of ATRA resistance in AML is the expression of the dominant co-repressor of RAR signaling PRAME.257 Preferentially expressed antigen in melanoma, or PRAME, binds to RAR and recruits polycomb-group proteins that prevent transcription of RA target genes, impeding the differentiation and cell cycle arrest effects.258 Interesting, while down-regulation has been shown to sensitize melanoma cells to ATRA treatment,259 expression of PRAME correlated with higher rates of overall and disease-free survival in pediatric AML.260 However, this observation correlated with a favorable cytogenetic profile, mainly a t(8;21) RUNX1/RUNX1T1 fusion; this matched

TCGA data in adult AML as well (Supplemental Figure A.2).109 Further investigation into the benefits and drawbacks of PRAME in the context of retinoic acid signaling are warranted.

ATRA is metabolized by CYP26 enzymes and expression, especially in the bone marrow microenvironment, can result in a quenching of the retinoid activity responsible

for cellular differentiation.261,262 t(8;21) AML patient cultures treated with the CYP26 inhibitor R115866 (Talarozole) were sensitized to ATRA treatment as evidenced by increases in cellular differentiation markers.263 Additional methods of repression likely occur; of note, a vast variety of epigenetic factors have been shown to be aberrantly expressed in ATRA resistant APL and non-APL AML.211,264,265

Because ATRA led to robust CD38 upregulation in our studies despite showing no effects on differentiation, its use, when in combination with daratumumab, may elicit a response in ATRA-resistant AML. In patient samples analyzed by qPCR, expression of

CD38, was consistently upregulated by ATRA treatment, even though the same treatment failed to elicit significant upregulation of the differentiation marker CD11b

(data not shown), which is readily observed in ATRA-treated APL blasts.266 It has been suggested that the CD38 promoter is highly sensitive to ATRA, even at doses far below those traditionally used for in vivo treatments.267 Hence, it is plausible that the threshold for CD38 upregulation by ATRA is lower than that for differentiation, such that

CD38 can still be induced in non-APL AML. Data from The Cancer Genome Atlas109 show that baseline CD38 expression is variable among the numerous listed AML subgroups.

Of note, and shown in Supplemental Figure A.3, patients with a RUNX1 t(8;21) showed significantly higher CD38 transcript levels compared to patients with APL, normal karyotype, complex cytogenetics, intermediate risk cytogenetic, and the other CBF AML variant, CBFB-MYH11 translocations. The molecular mechanisms behind this observation are not immediately clear; further investigation into whether this subset of

AML is more susceptible to daratumumab therapy would be of interest. We were unable to test all such subgroups with respect to ATRA and CD38, but in our largely- representative group we observed an ATRA-mediated increase in CD38 within each sample. This suggests that ATRA may be effective at upregulating CD38 for many, if not most or all, subgroups of AML.

Contrary to the cytotoxicity that ATRA as a single agent displayed in vitro, the drug on its own ultimately conferred no advantage in vivo. This would suggest that the resistance mechanisms observed in non-APL AML are at least partially operative in our murine models. However, the significant survival advantage resulting from the combination treatment of ATRA and daratumumab suggests that ATRA is capable of upregulating the expression of CD38. Mechanistically, a binding site for the canonical

ATRA receptor, the transcription factor retinoic acid receptor alpha (RARα), has been confirmed in the first intron of the CD38 gene since 1998; binding to this retinoic acid response element, or RARE, results in gene expression.245 Our work with the RARα specific agonists, AM580, suggests that the CD38 upregulation we observed with ATRA treatment is mediated through this canonical pathway and that ATRA can upregulate

CD38 expression directly. This indicates that a combination of ATRA and CD38 antibodies, such as daratumumab, may provide an effective therapeutic option.

It is important to note that because daratumumab is specific for human CD38 and in order to avoid rejection of engrafted human cells, all in vivo experiments were carried out in immune-compromised mice. The NSG mice used for these experiments

lack mature B cells, T cells, NK cells, and complement. Additionally, there are defects reported in dendritic cells and macrophages.268,269 This permitted us to test the interactions between the human myeloid blasts themselves, as responses from the host immune system were highly unlikely. This blast-against-blast activity, fratricide, can explain the results found, both in vivo and in vitro. Indeed, the experiments utilizing the

F(ab)’2 fragment of daratumumab suggests that the main anti-leukemic mechanism we observed was Fc-dependent fratricide. And, while fratricide alone conferred potent anti- leukemic effects in our study, addition of other arms of innate and adaptive immunity would likely drive the effects of the combination therapy to greater levels. Indeed, a previous study of ATRA and daratumumab in multiple myeloma demonstrated that

ATRA downregulated complement-inhibitory proteins CD55 and CD59, effectively increasing cellular complement dependent cytotoxicity.208 Additionally, daratumumab has been shown to modulate adaptive immune functions by reducing regulatory T cells while robustly increasing T helper and cytotoxic T cell counts.270 Further studies would be required to determine whether these mechanisms occur in AML.

Clinically, ATRA treatment in APL is reportedly well tolerated. Approximately 25% of APL patients treated with ATRA develop retinoic acid syndrome (RAS) characterized by cardiorespiratory distress; in the majority of cases treatment can continue under the addition of high-dose steroids.175,271 Yet because RAS is characterized by tissue infiltration by maturing myeloid cells,272 occurrence may be avoided when treating differentiation-resistant non-APL AML. Similarly, daratumumab, approved for the

treatment of multiple myeloma, has a favorable safety profile with manageable toxicities.273

In conclusion, to our knowledge these studies are the first to describe that although ATRA itself fails to effectively differentiate non-APL AML cells, it potently upregulates the surface expression of CD38 and, when in combination with daratumumab, may be an efficacious therapy against non-APL disease. Through the activation of Fc-mediated antibody dependent blast fratricide, this combination induces anti-leukemic effects in vitro and effectively retards tumor growth and extends overall survival time in vivo. In light of favorable safety profiles and previous FDA approval for each drug, these findings warrant the further study of ATRA and daratumumab as a potential treatment for AML.

CHAPTER 3: Activation of the intracellular pattern-recognition receptor, NOD2, promotes NK cell maturation and extends survival in acute myeloid leukemia

Abstract

Toll-like receptors (TLRs), a family of membrane-bound pattern recognition receptors (PRRs) found on innate immune cells, have been well studied in the context of cancer therapy. Activation of these receptors has been shown to induce inflammatory anti-cancer events, including differentiation and apoptosis, across a wide variety of malignancies. In contrast, intracellular pattern-recognition receptors such as Nod-like receptors (NLRs) have been minimally studied. NOD2 is a member of the NLR family that initiates inflammatory signaling in response to the bacterial motif muramyl dipeptide. Herein we examine the influence of NOD2 in AML demonstrating interferon- gamma (IFN-γ) treatment upregulates expression of NOD2 signaling pathway members such as SLC15A3 and SLC15A4, downstream signaling kinase RIPK2, and the NOD2 receptor itself. This priming allowed for effective induction of caspase 1-dependent cell death upon treatment with muramyl tripeptide phosphatidylethanolamine (MTP-PE), the synthetic ligand for NOD2. Furthermore, the combination of MTP-PE and IFN-γ with

AML blasts generated an inflammatory cytokine profile that effectively activated suppressed NK cells. In a murine model of AML, dual treatment with MTP-PE and IFN-γ led to a significant increase in mature CD27- CD11b+ NK cells accompanied by a significant reduction in disease burden and extended survival. These results suggest that

NOD2 activation, primed by IFN-γ, may provide a novel therapeutic option for AML.

Introduction

Coley’s toxin

The concept of immunostimulatory agents as cancer therapeutics is not a modern proposition; records from as far back as the 18th century note the seeming connection between febrile infection and malignant remission.274 It was William Coley, an American physician in the early 1900s, who first harnessed these observations in a therapeutic now known as Coley’s toxin. As the story goes, Coley, distressed after the loss of his first patient – a young girl with sarcoma that progressed despite amputation of the affected limb, was searching through previous case records to see what may have gone wrong. What caught his attention was the case of a man with an inoperable sarcoma of the neck. Three times surgery to remove the mass was attempted, but the tumor remained. Moreover, the surgical wound would not heal and skin graft proved unsuccessful. Unsurprisingly, and yet perhaps serendipitously, the man developed an erysipelas infection that induced a potent febrile response. As the fever raged on, the mass on the man’s neck began to shrink until it disappeared completely. Coley tracked the man down and found that, besides the large scar, he had no evidence of the disease that had almost claimed his life seven years prior. Fascinated, Coley began infecting his sarcoma patients, hoping to elicit the same sort of response and subsequent protection from the disease.275,276 Yet the unpredictability of utilizing a live infection soon became apparent; while some patients saw disease regression, in others there was trouble inducing infection and, in some, the technique proved fatal. Modifying his approach,

Coley created a vaccine comprised of heat-killed Streptococcus pyogenes and Serratia marcescens that ideally would induce the same febrile response without the risk of his previous trials. Known as Coley’s toxin, the concoction saw success when administered to patients with inoperable cancer. After induction of persistently high fever, a subset of patients was effectively cured, “permanently free of any clinical evidence of their disease.”277

Yet as the 20th century progressed, the enthusiasm behind therapies like Coley’s toxins was replaced with a confidence in the cutting edge treatments of the day: radiology followed soon after by the advent of chemotherapy. Moreover, the spontaneous regression that had been observed throughout history, and that Coley had associated with febrile infection, was becoming less prevalent as antibiotics and improved sterile techniques were becoming more commonplace.278 Today, the molecular mechanisms by which agents like Coley’s toxin activate the immune system have been more fully elucidated and the idea of engaging the host’s immune response in the fight against cancer has received renewed attention.

Pattern recognition receptors

Pattern recognition receptors (PRRs) are a group of cellular sensors that recognize pathogen-associated molecular patterns (PAMPs); their therapeutic potential against a host of diseases has been extensively studied.279-285 One of the first classes of

PRRs to be characterized were the membrane-bound toll-like receptors (TLRs). Found on the surface of the cells or endosomes, members of this family recognize a range of foreign motifs including double-stranded RNA (TLR3), bacterial lipopolysaccharides (LPS;

TLR4), single-stranded RNA (TLR7/8), and CpG DNA (TLR9).286 Structurally, the receptors possess a leucine-rich repeat (LRR) in their extracellular domain and an intracellular toll/interleukin-1 receptor (TIR) homology domain responsible for signaling upon receptor dimerization.287 Every TLR except TLR3 can signal through the recruitment of

MYD88 (myeloid differentiation primary response 88) which further recruits IRAK

(interleukin-1 receptor-associated kinase) kinases; subsequent downstream signaling results in the activation of NFκB and MAPK and the triggering of the inflammatory response.286 TLR3, and TLR4 in addition to the MYD88-dependent pathway, signal through the TRIF (TIR-domain-containing adapter-inducing interferon-β) dependent pathway and regulate type-I interferon production through IRF3 (interferon regulatory factor 3).288 Of all the PRRs, TLRs have received perhaps the most attention in the context of cancer research. Agonists for TLR3, -4, -7/8, and -9 were included in a NCI top 20 list of agents with high potential for therapeutic use.289 In fact, numerous clinical trials are currently underway testing the efficacy of these agonists against a host of malignancies (clinicaltrials.gov).

The intracellular PAMP-sensing NOD-like receptor (NLR) family contains similarities to membrane bound PRRs like TLRs. Both recognize pathogen-associated motifs and elicit potent inflammatory signaling cascades resulting in NF-κB activation.290

Additionally, these receptors have a role in activation of the inflammasome and the subsequent cleavage and release of specific inflammatory cytokines such as IL-1β.291

However, the therapeutic potential of NLRs has not been fully explored, particularly in context of leukemic disease.

NOD2

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a member of the NLR family that recognizes muramyl dipeptide (MDP), a peptidoglycan motif present on both gram-negative and gram-positive bacteria. Mechanistically, upon degradation of targeted bacteria in the endolysosome, two peptide transporters,

SLC15A3 and SLC15A4, facilitate the shuttling of MDP into the cytosol. These transporters, while shown to be essential for endosomally derived MDP signaling, have also been suggested to recruit NOD2 and downstream signaling partners to the endosomal membrane.292 Work utilizing surface plasmon resonance has demonstrated the direct high-affinity binding of MDP to NOD2,293 yet in its inactive form, NOD2 is auto-inhibited through interactions within its NACHT domain.294 Upon activation and ligand binding via the C-terminal LRR domain, NOD2 self-oligomerizes and recruits the serine/threonine kinase RIPK2 by CARD-CARD domain interactions.295-297 Additionally, activated NOD2 recruits inhibitor of apoptosis proteins (IAPs) cIAP1, cIAP2, and XIAP

which function to induce conjugation of lysine-linked ubiquitin chains on RIPK2.298-301

XIAP, through its C-terminal RING domain ubiquitin ligase (E3) activity, provides the ubiquitin scaffold needed for the subsequent recruitment of the linear ubiquitin chain assembly complex (LUBAC).301 LUBAC itself is composed of three subunits: the catalytic

HOIP and the regulatory subunits HOIL-1 and SHARPIN.302,303 As the name suggests, the recruitment of LUBAC catalyzes the conjugation of linear ubiquitin chains (linked through the N-terminal methionine, M1); it has been proposed that these M1-linked ubiquitin chains are needed for the efficient activation of the catalytic subunits of the

NF-κB-activating IκB kinase (IKK) complex.302,303 Successful recruitment to the NOD2 signaling complex results in potent downstream NF-κB activation and the induction of the inflammatory response.

In addition to its activation of NF-κB upon ligand binding, NOD2 also plays a role in the formation of the NLRP1 inflammasome. Contrary to the other NLR-family inflammasomes, NLRP1 has a C-terminal extension containing a CARD domain; NOD2, containing its own CARD domain at its N-terminal, has shown to utilize these CARD-

CARD interactions to complex with NLRP1 and activate caspase-1.291 Caspase-1, upon activation by the inflammasome, is able to process inflammatory cytokines pro- interleukin-1β (IL-1β) and pro-interleukin-18 (IL-18) into their bioactive forms.304,305

MTP-PE

Muramyl tripeptide phosphatidylethanolamine (MTP-PE) is a synthetic lipophilic derivative of MDP and has the advantage of being less pyrogenic and having longer plasma half-life compared to MDP.306 Upon internalization, MTP-PE is degraded to MDP in the cytosol and is free to bind to NOD2 and initiate inflammatory signaling.307 The agent has been used to stimulate macrophage activity in pediatric osteosarcoma with clinical success.308 Osteosarcoma, the most common primary malignancy of osteoid tissue in bone, occurs mainly in children and adolescents. Standard care of neoadjuvant multiagent chemotherapy followed by surgical resection results in 60-70% five-year event-free survival for localized non-metastatic cases, yet poor prognosis remains for those with metastatic disease.309 Interestingly however, unlike the vast majority of malignant disease where tumor-associated macrophages (TAMs) are associated with tumorigenesis,310-313 in osteosarcoma these infiltrating macrophages have shown to confer anti-osteosarcoma activity and correlate with a decrease risk of metastasis.314,315

The differential response to TAMs in varying cancers has been attributed to the polarization of the macrophage itself. While alternatively activated M2 macrophages promote angiogenesis and anti-inflammatory cytokines that support tumor proliferation, classically activated M1 macrophages exhibit anti-tumor effects both through the production of pro-inflammatory cytokines and reactive oxygen species and the induction of potent effector cells such as natural killer cells and T cells. 316,317 MTP-

PE administration, and the resultant activation of the NOD2 signaling pathway, shifts

macrophages towards an M1 phenotype and thus may have significant clinical benefit as a macrophage activating agent in these diseases.

AML differs from a solid tumor disease like osteosarcoma in many ways, but the idea of activating monocyte/macrophage, here not the TAM, but rather the leukemic blast itself, to promote anti-cancer effects is intriguing. We have shown previously that these blasts can act as effector cells and execute antibody-dependent killing between one another;246,318 we next asked if MTP-PE could polarize them into effective M1-like cells that would elicit anti-leukemic effects in the absence of an antibody. Herein, we sought to determine the influence MTP-PE would have on AML viability. The disease is very effective at circumventing the body’s natural immune functions,319-322 yet we hypothesized that MTP-PE, a known immune-stimulating peptide, could reinvigorate the suppressed immune cells in AML. However, we found that MTP-PE alone did not induce antitumor effects, but its use in conjunction with IFN-γ led to significant and super additive AML cell apoptosis.

In addition, combined MTP-PE and IFN-γ induced pro-inflammatory cytokines

TNFα and IL-1β, and stimulated maturation of NK cells both in vitro and in a MllPTD/WT

Flt3ITD/WT murine adaptive transfer model of AML. Mice treated with this combination also showed a significantly lower disease burden and increased survival compared to single agent treatment. Taken together, these data indicate that NOD2 agonists may have therapeutic potential for AML.

Materials and Methods

Reagents

Mifamurtide (MTP-PE) and Caspase 1 inhibitor Ac-YZAD-CMK were purchased from

Millipore Sigma (St. Louis, MO). Recombinant human and mouse interferon gamma and caspase inhibitor Z-VAD-FMK were purchased from R&D Systems (Minneapolis, MN).

CellEventTM Caspase-3/7 Green Flow Cytometry Assay Kit was purchased from Thermo

Fisher Scientific (Waltham, MA). LEGENDplexTM Multi-Analyte Flow Assay Kit was purchased from Biolegend (San Diego, CA).

Cell Culture

Cell lines were purchased from American Type Culture Collection and cultured in RPMI

Medium 1640 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Grand Island, NY), 2mM L-glutamine

(Invitrogen, Grand Island, NY), and 56U/mL/56μg/mL penicillin/streptomycin

RPMI Medium 1640 (Gibco) supplemented with 20% FBS, 2mM L-glutamine (Invitrogen), and 56U/mL/56μg/mL penicillin/streptomycin (Invitrogen) at 37°C in 5% CO2.

Enzyme-linked Immunosorbent Assay (ELISA)

Supernatants from cell cultures treated with/without 10 μg MTP-PE and/or 10 ng IFN-γ were analyzed at 24 or 48 hours using Human TNF-alpha DuoSet ELISA (R&D Systems) according to manufacturer’s instruction.

Quantitative Real-time Polymerase Chain Reaction (qPCR)

RNA was extracted using the Total RNA Purification Plus Kit (Norgen Biotek Corp.,

Ontario, Canada). RNA was reverse-transcribed, then quantified by qPCR using Power

SYBR® Green Master Mix (Applied Biosystems, Grand Island, NY). GAPDH was used as a reference gene for normalizing target genes. Data are presented as relative copy number (RCN), calculated as 2–ΔCt x 100, where ΔCt is the Ct(target) – Ct(GAPDH).

Lactate Dehydrogenase Assay (LDH)

MOLM-13 cells were plated at 5x105 cells/mL and treated with/without 10 μg MTP-PE and/or 10 ng IFN-γ. After 48 hours, supernatants were removed and used for a

Colony Formation Assay

MOLM-13 cells were treated with/without MTP-PE (10 ug/mL) and/or IFN-γ (10 ng/mL) and plated in MethocultTM H4100 (STEMCELL Technologies, Cambridge, MA) at a concentration of 1x103 cells/mL. Colonies were grown for 7 days at 37°C before quantification.

Western Blots

Briefly, cells were lysed with TN1 buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM

Na4P2O7, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na3VO4, and 10 μg/ml each aprotinin and leupeptin) and protein lysates were boiled in Laemmli sample buffer.

These were separated by SDS-PAGE, transferred to nitrocellulose membrane using

Trans-Blot® Turbo™ Transfer System (Bio-Rad Laboratories, Hercules, CA), probed with antibody of interest overnight and next with secondary HRP-conjugated antibody.

Membranes were developed using Pierce ECL 2 Western Blotting Substrate (Thermo

Fisher Scientific).

Microarray Analysis

Microarray data were acquired and analyzed as part of a previous publication.323 Briefly, peripheral blood monocytes (PBM) were isolated using ficoll separation followed by negative selection with the MACS Monocyte Isolation Kit (Miltenyi Biotec). PBM were incubated at 5x106/mL in RPMI media plus 10% FBS for 24 hours, with or without 25 ng/ml recombinant human IFN-γ. RNA was extracted using Trizol, column-purified using

RNeasy columns (Qiagen, Valencia, CA), and then hybridized to Affymetrix hgu133plus2 chips at the campus microarray core facility. Expression values were calculated using the

“gcrma” package in BioConductor (www.bioconductor.org) and the resulting data were analyzed for differential expression using the “limma” package.

Murine Model

All animal experiments were carried out in full accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University.

MllPTD/WT Flt3ITD/WT murine models have been previously described.324,325 Wild type syngeneic mice were irradiated and transplanted with 1x106 MllPTD/WT Flt3ITD/WT cells and disease was allowed to engraft for 2 weeks. MTP-PE (1mg/kg326,327) and recombinant murine IFN-γ (2000 U/mouse246) were administered via intraperitoneal injection 3 times per week for 3 weeks, after which mice were euthanized and tissue extracted for

analysis via flow cytometry. Survival experiments were carried out in the same model with identical drug dosing and scheduling for 65 days post engraftment.

Statistical Analysis

For all analyses: * denotes an α = 0.05; ** an α = 0.01; *** an α = 0.001. A paired t-test was used for analysis in Figure 1A,E. For the experiments with repeated measures, data were analyzed by mixed effect models. For the experiments involved multiple independent groups, data were analyzed by analysis of variance (ANOVA) followed by pairwise comparisons. Interaction contrast was used to test the synergy of the two drugs in the in vivo experiments. Log rank tests were used to analyze survival curves.

Results

IFN-γ primes NOD2 signaling pathway in AML

Stimulation of NOD2 has been well documented to induce an antibacterial environment through the activation of NF-κB and the subsequent release of inflammatory cytokines.290 Much of this work was performed in the context of intestinal immunity and inflammatory bowel disease, where mutations in NOD2 accompany increased susceptibility to Crohn’s disease.328-330 However, since NOD2 is expressed in myeloid cells, we tested here whether NOD2 stimulation could elicit the release of pro- inflammatory cytokines in AML blasts and what effects this might have on these cells.

Unlike the robust activation seen in healthy human monocytes (Figure 3.1A), the AML cell lines MOLM-13 (Figure 3.1B) and MV-4-11 (data not shown) did not respond to

MTP-PE on its own as measured by TNFα production. It has been shown previously that

IFN-γ primes healthy-donor macrophages for stronger inflammatory responses, in part through the upregulation of NOD2.331 Therefore, we tested the effect of dual treatment with MTP-PE + IFN-γ on the cell lines. Results showed that this dual treatment could indeed elicit a significant increase in the production of TNFα (Figure 3.1B).

Based on the above findings, we next examined the mechanism behind the priming effect conferred by IFN-γ. MTP-PE enters the cell via clathrin- and dynamin- dependent endocytosis.332 Upon cell entry, endolysosomal peptide transporters

SLC15A3 and SLC15A4 facilitate transfer of the ligand into the cytosol.292 The NOD2 receptor itself contains three domains: a C-terminal leucine-rich repeat (LRR) domain

responsible for auto-inhibition in the absence of ligand, a central NACHT domain, and an

N-terminal effector domain comprised of two caspase activation and recruitment domains (CARD).333 As depicted in Figure 3.2, downstream signaling occurs via CARD-

CARD domain interactions with the serine-threonine kinase RIPK2, leading to NF-κB and

MAPK activation.296,334 To examine whether IFN-γ might influence this pathway, we first searched a previously performed gene microarray from healthy donor peripheral blood monocytes treated with and without IFN-γ323 (Figure 3.3A). Interestingly, we found a significant increase in the expression of the transport proteins SLC15A3 and SLC15A4, as well as the downstream signaling kinase RIPK2 in IFN-γ treated cells; NOD2 expression was not significantly changed, but trended upward (p-value = 0.055). To test whether the same changes occurred in AML cells, we evaluated transcript levels in patient apheresis samples (patient characteristics detailed in Supplementary Table A.2). As shown in Figure 3.3B, SLC15A3, SLC15A4, NOD2, and RIPK2 all showed significant upregulation after IFN-γ treatment. Western blot analyses further confirmed that IFN-γ potently upregulated members of the NOD2 signaling pathway, including the receptor itself, in the AML cell line MOLM-13 (Figure 3.3C)

A B ) ) ** 250 * 60 ** ** 200 40 150

(pg/mL)

  100 20

TNF TNF 50

0 0  

IFN- MTP-PE MTP-PE Untreated Untreated MTP-PE+IFN-

Figure 3.1. MTP-PE alone fails to elicit a response in AML. A) Peripheral blood monocytes from healthy donors were stimulated with 10 μg/mL MTP-PE for 24 hours; TNFα levels were measured by ELISA (n=3). B) MOLM-13 cells were stimulated with 10 μg/mL MTP-PE and/or 10 ng/mL IFN-γ for 48 hours; subsequently, TNFα levels measured by ELISA (n= 3). * denotes p≤0.05; ** p≤ 0.01.

Figure 3.2. MTP-PE internalization and NOD2 signaling pathway.

Figure 3.3. IFN-γ primes NOD2 pathway in AML. A) Expression of NOD2 pathway members were extracted from gene arrays from healthy-donor peripheral blood monocytes treated with/without 25 ng/mL IFN-γ. B) Primary blasts from AML patients (n=7) were treated with/without 10 ng/mL IFN-γ for 24 hours; transcript levels were measured by qPCR. C) MOLM-13 cells were treated with/without 10 ng/mL IFN-γ for 24 hours. Whole cell lysates were separated by western blots and probed for the molecules indicated; membranes were reprobed with calreticulin antibody as a loading control (n=3). * denotes p≤0.05; ** p≤0.01.

MTP-PE + IFN-γ is cytotoxic to AML blasts

Morphologically, we observed that both patient AML blasts as well as AML cell lines treated with the combination of MTP-PE and IFN-γ demonstrated a distinct clustering/clumping pattern (Figure 3.4). While this aggregation is often detected when free DNA and cell debris exist in culture, indicative of cell lysis, it has been previously reported that MDP upregulates cellular adhesion proteins Intercellular Adhesion

Molecule 1 (ICAM1) and Integrin Beta 2 (ITGB2; CD18).335 To test whether MTP-PE + IFN-

γ was eliciting killing of the blasts or simply stimulating cellular adhesion, cellular damage was assessed via a lactate dehydrogenase release assay. Results shown in

Figure 3.5A demonstrate that the dual treatment did indeed induce significant cell killing compared to untreated and single treatment after 48 hours. Additionally, colony formation assays, in which cells are plated in single-cell suspension in semi-solid methylcellulose based media, showed significant reduction in live, proliferative colonies after treatment with MTP-PE + IFN-γ after seven days of culture (Figure 3.5B). Lactate dehydrogenase release assays and colony formation assays were repeated in MV-4-11 cells; results obtained matched those observed in the MOLM-13 cells (data not shown.

LDH: n = 3, p-value: 0.0008; Colony assay: n = 2). LDH assays were repeated in MOLM-

13 cells with varying doses of IFN-γ. Shown in Figure 3.6, the dosing of IFN-γ proved to be essential in eliciting cellular death in the AML blasts.

Figure 3.4. MTP-PE + IFN-γ kills AML blasts. Bright-field microscopy images of MOLM-13 cells (top) and AML apheresis samples (bottom) treated with/without 10 μg/mL MTP-PE and/or 10ng/mL IFN-γ after 48 hours.

Figure 3.5. MTP-PE + IFN-γ in AML kills AML blasts (cont). A) Cellular cytotoxicity assessed via lactate dehydrogenase assay in MOLM-13 cells treated with/without 10 μg/mL MTP-PE and/or 10ng/mL IFN-γ for 48 hours. B) MOLM-13 cells treated with/without 10 μg/mL MTP- PE and/or 10ng/mL IFN-γ plated in methylcellulose-based media for 7 days after which colonies were quantified (n=3). *** denotes p≤0.001.

Figure 3.6. MTP-PE cytotoxicity depends on IFN-γ. Cellular cytotoxicity assessed via lactate dehydrogenase assay in MOLM-13 cells treated with/without 10 μg/mL MTP- PE and escalating doses of IFN-γ

To explore the mechanism behind the observed cell death, we tested whether

MTP-PE + IFN-γ led to activation of executioner caspases. Cells were treated with MTP-

PE and IFN-γ and probed with a fluorogenic substrate to assay for enzymatically active caspase-3. As shown in Figure 3.7, dual treatment significantly upregulated activity of caspase-3 compared to untreated and single treatments. Cell viability was recovered when MTP-PE + IFN-γ treated cells were incubated with a caspase inhibitor (ZVAD).

Caspase-1, an inflammatory caspase and inflammasome member, has the ability to convert pro-caspase-3 into its active form.336 Because NOD2 signaling activates

NLRP1, NLRP3 and, subsequently, caspase-1,291,337 we asked if the observed caspase-3 mediated apoptotic death was being initiated through caspase-1. Consistent with this notion, inhibition of caspase-1 with the caspase-1-specific inhibitor YVAD also rescued viability in MTP-PE + IFN-γ treated cells (Figure 3.8A). Additionally, caspase-1 inhibition led to significant reduction of caspase-3 activity, on par with the inhibition by ZVAD

(Figure 3.8B). Enzymatic studies previously published have confirmed specificity of YVAD for caspase-1 and not caspase-3.338,339 Collectively, these results suggest that caspase-1 is activated following MTP-PE + IFN-γ treatment, and that this activation drives caspase-

3 activation and leads to blast apoptosis.

*** 5000 ***

4000

3000 MTP-PE 2000 IFN- 1000 MTP-PE + IFN-

(Baseline Subtracted) (Baseline Caspase 3 Activity (MFI) 3 Activity Caspase 0

Figure 3.7. MTP-PE + IFN-γ activates caspase-3. MOLM-13 cells treated with MTP-PE and/or IFN-γ for 48 hours were assessed for active caspase-3 function by flow cytometry (n=3 experiments); representative histogram shown. *** denotes p≤0.001.

A) *** 100 ***

% Viable Cells % Viable 20

0 DMSO ZVAD YVAD Untreated MTP-PE + IFN-

B) 3000 ** **

2000

1000

Caspase 3 MFI (FITC) 3 MFI Caspase 0 DMSO ZVAD YVAD Untreated MTP-PE + IFN-

Figure 3.8. MTP-PE + IFN-γ kills via caspase-1 activation. MOLM-13 cells were treated with MTP-PE and IFN-γ, in the presence of pan-caspase inhibitor ZVAD or caspase-1 specific inhibitor YVAD for 72 hours. Cell viability was assessed via trypan blue exclusion (A, n=3) and active caspase-3 function assessed by flow cytometry (B, n=3). ** denotes p≤0.01; *** p≤0.001.

MTP-PE + IFN-γ induces a pro-inflammatory response from AML cells

The interaction between NOD2 and the inflammasome, and the resulting production and activation of the pro-inflammatory cytokine IL-1β, has been well characterized in human mononuclear cells.291,304,340,341 To determine whether the dual treatment would lead to IL-1b production and/or other cytokines, we co-treated AML patient apheresis samples with MTP-PE and IFN-γ and measured cytokine production using a multiplex bead-based assay. Results shown in Figure 3.9A demonstrate that dual-treated cells produced significantly higher levels of TNFα and IL-1β after 24 hours compared to cells treated with MTP-PE or IFN-γ alone. Consistent with this, qPCR analysis of 7 patient samples showed a significant induction of both TNFa and IL-1b transcript (Figure 3.9B). Among the other cytokines tested, many showed increases after

MTP-PE + IFN-γ treatment that, while statistically significant, were relatively modest

(IFNα, IL-18, CCL2, IL-6, IL-8, and IL-12p70; Supplemental Figure A.4). Overall these trends indicate a shift towards a pro-inflammatory state. Of note, the detection of IL-1β in the supernatant is consistent with the activation of caspase-1 as mentioned above.

Since TNFα and IL-1β have been shown to stimulate NK cell cytolytic activity and

IFN-γ release,342-344 we next asked whether AML cells treated with MTP-PE and IFN-g would enhance NK cell activity, as measured by the production of IFN-g. NK cell activity has been shown to be suppressed in AML; efforts to push these cells to a more active state may have significant therapeutic benefit in the disease.325,345,346 For this, we first performed an in vitro assay with isolated NK cells from healthy-donors and then in an in

vivo study in a murine model of AML. First, NK cells were co-cultured for 24 hours with primary AML cells in the presence of single or dual treatments. IFN-γ transcript was measure by qPCR. Results showed that whereas NK cells alone produced an insignificant amount of IFN-γ transcript, those levels increased significantly when co-cultured with

AML cells and treated with the combination of MTP-PE and IFN-γ (Figure 3.10A). IFN-γ was not transcribed in the AML apheresis samples alone. This suggests that dual treatment induces cytokine production from the AML blasts which then may activate the co-cultured NK cells. Based on our in vitro data, we propose a model outlined in

Figure 3.10B where MTP-PE and IFN-γ synergize and elicit production of NK-activating cytokines in AML blasts. These activate NK cells and induce production of IFN-γ, which can feed back onto the leukemic blasts.

A ) TNF IL-1 150 ** 80 * * * *** ** 60 100

pg/mL 50 20

0 0

B ) TNF IL-1

80 * 100 ** ** * ** ** 80 60 60 40

RCN 40

20 20

(Fold Change from UT) from Change (Fold 0 0 Untreated MTP-PE IFN- MTP-PE + IFN-

Figure 3.9. MTP-PE + IFN-γ induces pro-inflammatory cytokine productionIL-1 in AML blasts. A) AML patient cells from apheresed samples (n=6)6 were incubated*** in complete* RPMI with/without MTP-PE and/or IFN-γ for 24 hours. Supernatants were collected and tested on a multi-analyte cytokine panel. Shown here are IL-1b and TNFa. B) Apheresis samples from AML patients (n=7) treated with/without MTP-PE and/or4 IFN-γ for 24 hours. Transcript levels measured by qPCR. * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

pg/mL (log2) pg/mL

0 91 24 hr 48 hr

A ** ** ) 300 *** **

200 Untreated MTP-PE

RCN  IFN-

IFN- 100 MTP-PE + IFN-

0 AML AML+NK NK B )

Figure 3.10. MTP-PE + IFN-γ induced pro-inflammatory cytokines may serve to activate NK cells. A) NK cells isolated from blood and AML apheresis samples were cultured singly or together with/without MTP-PE and/or IFN-γ for 24 hours after which IFN-γ transcript levels were evaluated by qPCR (n=3 experiments). B) Model: AML cell – NK cell cytokine loop. ** denotes p≤0.01; *** p≤0.001.

MTP-PE + IFN-γ stimulates NK cell maturation and extends survival in vivo

One mechanism by which AML evades the innate immune system is through the suppression of mature cytolytic NK cells; numerous studies have linked a decrease in NK activity with increased risk of relapse.320,321,347-349 Here, based on our above model, we tested whether MTP-PE + IFN-γ could also elicit maturation of NK cells in a murine model of AML previously described.325 For this, irradiated mice were transplanted with

MllPTD/WT Flt3ITD/WT blasts and leukemia was allowed to develop. MTP-PE and murine IFN-

γ were administered for three weeks, as described in the methods, after which blood, bone marrow, and spleen samples were collected. Murine NK cells (NK1.1+, CD3-) were isolated from each tissue compartment and stained for surface markers that distinguish distinct functional subsets. From least to most mature these are CD27-/CD11b-,

CD27+/CD11b-, CD27+/CD11b+ and CD27-/CD11b+. Flow cytometry was used to measure these subsets and, as shown in Figure 3.11, there was a significant increase in percent mature CD27-/CD11b+ cells in the blood, bone marrow, and spleens of dual-treated mice compared to untreated or single-treated mice (representative scatterplots shown in

Figure 3.12A). Furthermore, decreased disease burden was evidenced by a significant reduction in white blood cells (Figure 3.12B) as well as spleen weight (Figure 3.12C)

Based on our above findings that the combination of MTP-PE and IFNg promotes blast apoptosis and leads to NK cell maturation, and the fact that the disease burden was significantly lowered in the blood and spleen, we asked whether this dual treatment would have an effect on overall survival in the AML mouse model. As outlined in Figure

3.13, mice treated with the combination of MTP-PE and IFN-γ showed a significant increase in survival compared to single agent and untreated mice [Logrank test p-values

= 0.0021 (vehicle control), 0.0018 (MTP-PE), and 0.0018 (IFN-γ)].

Endogenous IFN-γ correlates with MTP-PE responsiveness

IFN-γ appears to be critical for the effectiveness of MTP-PE, yet recombinant IFN-γ is known to elicit adverse events when administered clinically.350,351 It has been proposed however, that certain interventions, such as allogeneic HSCT, prompt homeostatic increases of inflammatory cytokines in AML patients.352 This may provide a therapeutic window where NOD2 stimulation by MTP-PE could be elicited in cooperation with endogenous IFN-γ. To test this we measured IFN-γ levels in AML patient plasma samples pre- versus post-HSCT and found that there was a significant increase at day 30 post-

HSCT (Figure 3.14A). Next, to test whether the levels of IFN-γ correlated with the antitumor effects of MTP-PE, we incubated MOLM-13 cells with plasma from pre- or post-

HSCT patients and measured cell viability. Results showed that post-HSCT plasma led to significant reductions in cell viability when combined with MTP-PE (Figure 3.14B), suggesting that administering MTP-PE, under conditions where endogenous IFNg levels are elevated, may provide an attractive therapeutic option.

Blood 100 *** *** *** 80 CD27- CD11b- 60 CD27+ CD11b- CD27+ CD11b+ 40 CD27- CD11b+ 20

% NK Population Total of 0 Untreated MTP-PE IFN- MTP-PE +IFN- Bone Marrow 100 *** ** ** 80 CD27- CD11b- 60 CD27+ CD11b- CD27+ CD11b+ 40 CD27- CD11b+ 20

% NK Population Total of 0 Untreated MTP-PE IFN- MTP-PE +IFN- Spleen 100 *** ** *** 80 CD27- CD11b- 60 CD27+ CD11b- CD27+ CD11b+ 40 CD27- CD11b+ 20

% NK Population Total of 0 Untreated MTP-PE IFN- MTP-PE +IFN-

Figure 3.11. MTP-PE + IFN-γ stimulates NK cell maturation in vivo. Blood, bone marrow, and PTD/WT ITD/WT spleen samples from Mll Flt3 transplant mice treated with/without 1mg/kg MTP-PE and/or 2,000 U recombinant murine IFN-γ for 21 days (3 treatments/week; M,W,F) were + - analyzed by flow cytometry for the maturation state of NK1.1 CD3 NK cells. NK-cell maturation was characterized by CD27 and CD11b expression (blood= 3 mice per group; bone marrow and spleen= 4 mice per group).

A) Untreated MTP-PE

CD27 (FITC) IFN-γ MTP-PE + IFN-γ

CD11b (PerCP-Cy5.5)

B) C)

5.010 5 *** 500 *** *** *** * 4.010 5 400

3.010 5 300

2.010 5 200

WBC/uL

1.010 5 100

Spleen Weight (mg) Spleen

0 0    

IFN- IFN- MTP-PE MTP-PE Untreated Untreated

MTP-PE+IFN- MTP-PE+IFN-

Figure 3.12. MTP-PE + IFN-γ stimulates NK cell maturation and decreases disease burden in vivo. A) Representative contour plots of NK cells isolated from blood from one mouse per group; CD27 and CD11b gating determined by isotype controls. B) White-blood-cell count were taken from blood samples and (C) spleen weights were determined. * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

100

Percent Percent survival 20 **

0 0 25 30 35 40 45 50 55 60 65 Time (days) Vehicle MTP-PE IFN- MTP-PE + IFN-

PTD/WT ITD/WT Figure 3.13. MTP-PE + IFN-γ extends survival in vivo. Mll Flt3 transplant mice were treated with/without 1mg/kg MTP-PE and/or 2,000 U recombinant murine IFN-γ three times a week for 65 days. The MTP-PE + IFN-γ showed a significant increase in survival compared to the vehicle control, MTP-PE alone, and IFN-γ alone (p-value = 0.0021, 0.0018, 0.0018, respectively).

A )

Day 90

Day 30

***

pre-HSCT

0 20 40 60 80 100 pg/mL IFN-

B 100 ns *** ) 80

60 Untreated MTP-PE 40

% Viable Cells % Viable 20

0 Pre- 30 Days HSCT Post-HSCT

Figure 3.14. Endogenous IFN-γ in HSCT AML patients . A) Plasma samples collected from AML patients (n=56) pre-, 30 days, and 90 days post-HSCT were assayed for IFN-γ levels by ELISA. B) MOLM-13 cells plated in 75% patient plasma (pre- or 30 days post-HSCT) and 25% complete RPMI, and with/without 10 μg/mL MTP-PE were assayed for cell viability via trypan blue exclusion after 24 hours (n=5). *** denotes p≤0.001

Discussion

Chronic inflammation has long been associated with an increased risk of malignancy,353-355 yet controlled activation of the innate immune system, through the administration of immune-activating agents, has been shown to mediate anti-cancer effects. For example, favorable results have been observed utilizing the Bacillus

Calmette–Guérin vaccine (TLR2,4,9) against bladder356 and colon cancer,357 and in combination with chemotherapy for breast cancer358 and AML.359 Imiquimod (TLR7) is used for superficial basal cell carcinoma.360 CpG oligodeoxynucleotides (TLR9) are effective as monotherapy against metastatic melanoma,361 glioblastoma362 and other malignancies.285,363,364 However, the cytosolic NLR family to which NOD2 belongs, while in many ways similar to membrane-bound TLRs, has gone relatively unstudied in the context of leukemic disease. In this study we demonstrate that the NOD2 ligand MTP-

PE, together with IFN-γ, stimulates the innate immune response, measured here through the release of inflammatory cytokines and the activation of NK cells, as well as inducing direct apoptotic effects on leukemic blasts themselves.

MTP-PE (mifamurtide, MEPACT®) has gone through extensive testing and is currently approved in Europe for the treatment of high-grade resectable nonmetastatic osteosarcoma after macroscopically complete surgical resection in children, adolescents and young adults. A manageable safety profile has been reported with the most frequent adverse effects being headache, chills, tachycardia, nausea, and pyrexia.308,365-

367 In 2007, the FDA declined approval for mifamurtide in the treatment of

osteosarcoma, requesting data from additional trials to demonstrate benefit.368

Utilization of the drug in different disease contexts, such as AML, may elicit renewed interest in its future.

CD27 and CD11b have been shown to be markers that effectively categorize the maturation state of NK cells in mice,369 with the CD27-/CD11b+ subset being the most mature and the most cytolytically functional.370 In our murine MllPTD/WT Flt3ITD/WT transplant model of AML, significant reduction in disease burden was accompanied by increased maturation of NK cells as measured by these markers. AML has shown to exhibit high sensitivity to NK cell-mediated cytotoxicity compared to solid tumors.371 In fact, adoptive immunotherapy with haploidentical NK cells has demonstrated safety and efficacy in the disease.52,161-163 However, endogenous NK cell maturation and activity has been shown to be suppressed in AML populations.325,345,346 Patients with an immature NK cell profile have been shown to have reduced overall survival as well as a dramatically reduced three year relapse-free survival [0%, compared to intermediate maturation (52.6%) and hypermaturation (73.3%)].349 Thus there appears to be great therapeutic potential for agents that could successfully re-stimulate these cells.

It has been proposed that reactivation of cytomegalovirus, and the subsequent expansion of active NK cells, after allogeneic hematopoietic cell transplantation may confer this type of protection against leukemic relapse.372,373 This claim is disputed,374 but there appears to be agreement that, whatever benefit may exist, the risk of nonrelapse-related mortality may outweigh it.372,374 Here, if one could elicit a similar

100

maturation of disease-suppressed NK cells in a potent, yet controlled, manner, there is potential for the same type of protection with fewer adverse results.

While our study examined the NK cell population as a whole, further efforts to characterize the mature CD27-/CD11b+ subset are warranted. For example, down- regulation of natural cytotoxicity receptors (NCRs) on NK cells have been shown to correlate with poor survival in AML.346 Our preliminary in vitro data suggest that MTP-PE

+ IFN-γ may increase the levels of these receptors; we found a significant increase in

NCR2 transcript with dual treatment of NK cells versus untreated (data not shown).

Furthermore, leukemic blasts may also evade immune detection through the shedding of NK-activating ligands;319 characterizing how MTP-PE + IFN-γ treatment may influence ligands on the blasts themselves will better elucidate the mechanisms of leukemic clearance we observe in vivo.

In the same transplant murine model of AML, the dual treatment of MTP-PE and

IFN-γ significantly increased survival compared to the single agent- and vehicle- treated mice (median survival increased by 28.9%). This observation, together with the NK cell maturation data, suggests that the combination of MTP-PE and IFN-γ may have a therapeutic role as a consolidation or maintenance therapy. In terms of clinical translation, while recombinant IFN-γ is generally well tolerated, some adverse events may yet occur.350,351 However, certain interventions, such as allogeneic HSCT, prompt homeostatic increases of inflammatory cytokines in AML patients.352 Indeed, when we tested AML patient plasma samples pre- and post-HSCT transplant for IFN-γ levels, we

101

found significantly elevated levels 30 days post-transplant. Additionally, the patient plasma post-transplant were able to synergize with MTP-PE to induce cell death in the

AML cell line MOLM-13; the same cell death was not seen in the pre-HSCT patient plasma cultured with MTP-PE. Further studies are needed to elucidate whether there exists a therapeutic window where endogenous IFN-γ could cooperate with NOD2 stimulation to promote anti-leukemic effects.

In summary, we demonstrate here for the first time, to our knowledge, the anti- leukemic effects of NOD2 stimulation in the context of AML. While not sufficient on its own to induce an inflammatory response, MTP-PE led to robust activation when paired with IFN-γ. In addition to inducing direct apoptosis in AML blasts in a caspase-1- dependent manner, the combination of MTP-PE and IFN-γ provoked a pro-inflammatory response and successfully drove NK cells to their most mature state. In a murine transplant model of AML, MTP-PE and IFN-γ significantly increased the ratio of mature

CD27-CD11b+ NK cells in the blood, bone marrow, and spleen; a significant increase in survival was also observed. These data suggest that MTP-PE, together with IFN-γ, may provide a therapeutic benefit in the treatment of AML.

102

CHAPTER 4: Conclusions and Future Directions

The emerging subfield of cell-based therapeutic immunology has shown great promise in the context of malignant disease. The idea of harnessing the power of the body’s own immune system, or immune cells from a donor, in lieu of systemic toxic options like chemotherapy, are particularly attractive in cancers like AML where there is a large elderly population that cannot always tolerate high intensity chemotherapy. The types of cell-based therapies are diverse: from CAR T cells and HSCT, to fratricidal blast ADCC and PRR NK cell maturation described here; each may have a distinct place in the treatment regimen – as a frontline treatment in de novo AML, together with chemotherapeutics or as a monotherapy, or perhaps as consolidation or maintenance post-remission. The pre-clinical work here lays the groundwork for possible further investigation into the efficacy of the proposed treatments and what role, if any, they might play in the treatment of leukemic disease. In this chapter, conclusions and implications of the projects detailed in the previous chapters will be discussed as well as future directions of study.

103

Chapter 2 outlined a novel therapeutic approach in AML: the combination of

ATRA, a retinol derivative capable of inducing expression of the glycoprotein CD38, and the α-CD38 antibody daratumumab. After the induction of Fc-dependent conjugation, the combination led to the elimination of the leukemic cells in in vitro cultures by blast- to-blast fratricide. Furthermore, this combination treatment resulted in both a reduction in tumor load and an extension in survival in murine models of AML.

One of the questions that often emerges when investigating new treatments in the context of cancer, specifically leukemic disease, is the efficiency by which the agents can eliminate or mature leukemic stem cells (LSCs). Derived from normal hematopoietic stem cells, LSCs are characterized by their ability for self-renewal and disease initiation in serial transplantation models. The idea that a small population of clonogenic progenitors could be responsible for the bulk leukemic disease was first elucidated in

AML in the mid-1980s.375,376 Additional research in the last decades has identified the largely-quiescent LSCs as resistant to chemotherapy and correlative with poor prognosis.377-379 Thus, targeting LSCs is imperative in the development of AML therapeutic strategies.

As shown in Figure 2.4, ATRA was able to upregulate the expression of CD38 on a population of Lin-/CD34+ /CD123+/CD45Rα+ primary AML bone marrow samples (n =

5 patients). While there is some debate as to what receptors are present on AML LSCs, the markers used here are supported in the literature as those that can isolate LSC poplations.380,381 These results suggest a few possibilities. The first is that the

104

upregulation of CD38, a marker that arises upon the transition of hematopoietic stem cells from primitive CD34+ cells to more mature unipotent myeloid or lymphoid progenitors, successfully matures LSCs and limits their proliferative ability. Given

ATRA’s potent ability to upregulate CD38 in vitro, but not to extend survival or significantly reduce disease burden in vivo, it appears that this maturation push, if occurring, is not sufficient enough on its own to confer an advantage. However, the other possibility is that, much like the blast fratricide shown in the rest of Chapter 2, the upregulation of CD38 on LSCs could sensitize them to daratumumab treatment.

Histopathology reports on mice treated with ATRA and/or daratumumab showed clearance of AML blasts in the bone marrow only in the daratumumab and ATRA + daratumumab treated mice suggesting that the antibody was able to elicit elimination within the compartment where AML LSCs mainly reside (Supplementary Figure A.5). Yet penetrance of the α-CD38 antibody isn’t theoretically sufficient, as ATRA is needed to upregulate the antigen on LSCs, traditionally characterized as CD38 negative.382 The resistance to ATRA therapy, outlined earlier in Chapter 2, occurs through a variety of mechanisms including the heightened expression of retinoid metabolizers within the bone marrow. Investigations into how these limit ATRA functionality, possible mechanisms to inhibit them, and further characterization of any elimination or shifting of phenotype in the LSC population would strengthen the argument for the use of this combination in the treatment of AML.

105

Murine engraftment models often better recapitulate human disease and offer an attractive option for LSC studies. As shown in Chapter 2, ATRA and daratumumab confer anti-leukemic effects in murine models of AML; to assess their effect on LSCs, serial transplantations, where bone marrow from treated mice is re-transplanted in WT mice and engraftment efficiency is observed, would be utilized. Additionally, phenotypic changes in the LSC population could be monitored from bone marrow samples of treated mice by flow cytometry.

Our study of ATRA and daratumumab in the context of AML focused largely on the phenomenon of fratricide, inducing the AML blasts themselves to kill their neighboring blasts. The shift in these blasts from solely immunological targets to now both effector and target, resulted in a reduction in disease burden and extended survival. However, the models that we utilized to study this phenomenon were consequently deficient of any additional immune effector cells. Moving forward, studies into the effects on these other immune players are warranted. As discussed in Chapter

3, NK cells can play a vital role in the resolution of AML. The effects of retinoic acid on

NK cells have been studied in this context, but there doesn’t appear to be a clear consensus as to their benefit. It has been demonstrated that retinoic acid, known to influence gene transcription through its RAR transcriptional factor associations, has the ability to upregulate antigens (such as NKG2D ligands) on the surface of diseased cells that makes them more susceptible to targeting and elimination by NK cells (hepatic stellate cells383 and AML384,385). However, direct effects of ATRA on NK cells may lead to

106

impaired cytolytic function386 and decreased sensitivity to interferon signaling.387 CD38, in addition to being upregulated on monocytic blasts after ATRA treatment, has been shown to be expressed on immunosuppressive subsets of T cells. Indeed, daratumumab treatment in CLL has demonstrated the ability to decrease levels of T regulatory cells and enhanced cytotoxic T lymphocyte proliferation and cytolytic ability.388 Further investigation into how these agents influence other players in the immune system would elucidate the potential of this therapy.

Taken together with the clinical success that daratumumab has demonstrated in multiple myeloma, the data presented in our study suggests that ATRA and daratumumab may have clinical relevance as a treatment for AML. On that note, Syros

Pharmaceutical has recently begun conducting a clinical trial with SY-1425, a selective

RARA agonist, in combination with daratumumab in a pilot cohort of 9 AML patients.

Released data shows that eight out of the nine patients had upregulation of CD38 surface expression on myeloid blast cells (average of 1.57 fold increase).389

In Chapter 3, a combination of the synthetic bacterial peptide MTP-PE and IFN-γ and the resultant inflammatory response was studied. IFN-γ was essential for proper

MTP-PE stimulation; apparently through its potent upregulation of NOD2, the receptor for the bacterial ligand, and other pathway proteins including SLC15A3, SLC15A4, and

RIPK2. Upon addition of MTP-PE and IFNγ, AML cells underwent caspase-1 mediated apoptosis and release of pro-inflammatory cytokines. In NK cell/AML primary blast co- culture experiments, NK cells in the combination treated samples saw significant

107

increases in IFN-γ transcript production, signifying activation. These results were further supported by experiments in an Mll/FLT3-ITD murine model of AML where NK cells were pushed to their most mature phenotype by the combination of MTP-PE and IFN-γ; survival time in these mice was significantly extended.

The implication of maturing NK cells in the context of AML is potentially quite impactful. A statistic mentioned earlier, that three-year disease free survival drops to

0% in patients with immature NK cells,349 demonstrates the power the cells play in disease control. In our study detailed in Chapter 3, while we showed the maturation profile shift in these cells; further characterization of their functionality may be beneficial. While this idea was briefly explored at the end of Chapter 3, with relation to examining NK cell markers and ligands on AML cells, further investigation into how these agents stimulate NK cells is currently underway.

Activation of the inflammasome has shown to increase extracellular vesicle release and modulation of their content.390 As the literature and our recent work in

AML suggests, NOD2 activation activates inflammasomes, including NLRP1 and NLRP3, resulting in cleavage of pro-IL-1β to its active form. We examined levels of these extracellular vesicles, or exosomes, and found that they are released at higher levels in

AML cell lines upon treatment with MTP-PE and IFN-γ (Supplementary Figure A.6).

Exosomes themselves are derived from an endosomal multivesicular body (MVB) that contains within it numerous intraluminal vesicles (ILVs) that bud into the endosomal lumen. Upon fusing of the MVB with the plasma membrane, ILVs, now classified as

108

exosomes, are released. The endosomal sorting complexes required for transport

(ESCRT), is a system of protein complexes responsible for MVB biogenesis and release of exosomes. Under the treatment of MTP-PE and IFN-γ, a number of the ESCRT proteins are upregulated at the transcript level (Supplementary Figure A.7). These include HGS, an early ESCRT-0 complex protein that functions to cluster ubiquitin selected cargo for inclusion in the newly formed MVB. The role of ubiquitination in the exosome packing process is not fully understood; while ubiquitinated proteins are found in exosomal

ILVs,391 there are also enzymes that can remove the ubiquitin on endosomal cargo before they are packaged in the mature ILV.392 Elucidating the mechanisms by which

MTP-PE and IFN-γ upregulate this ESCRT machinery, and identifying any cargo that is carried in these exosomes, may further explain how neighboring cells, such as the maturing NK cells, are being signaled.

In addition to studies on the mechanisms of action that MTP-PE and IFN-γ utilize in AML, further investigation into a timeframe and patient population where a treatment like this would be most effective would be informative. Briefly mentioned at the end of Chapter 3, endogenous IFN-γ levels were assayed pre- and post-HSCT transplant, with elevated IFN-γ levels observed 30 days post-HSCT. Investigating whether these patients have heightened expression of PRR pathways such as the NOD2 signaling pathway, and whether that expression correlates with susceptibility to treatment as suggested in our study, would be of interest. Furthermore, haploidentical

NK cell therapy has shown promise in bridging refractory AML or MDS patients to

109

HSCT;393 if MTP-PE and IFN-γ could elicit NK cytotoxic effects, now from the patient’s own endogenous pool, the cost, time, and any side effects of donor infusions could be potentially avoided.

If the sensitivity to MTP-PE and IFN-γ is indeed correlative with expression levels of the NOD2 receptor, as suggested in Chapter 3, certain subsets of AML patients may be better candidates for this type of therapy. According to data from the Cancer

Genome Atlas,109 patients with a RUNX1-RUNX1T1 t(8;21), BCR-ABL1, and poor risk cytogenetic abnormalities may have lower levels of NOD2 (Supplemental Figure A.8).

Determining whether or not these patient subtypes are able to upregulate the NOD2 pathway and subsequently induce an inflammatory response to MTP-PE and IFN-γ would be an important step moving forward. Interestingly, when examining the expression of additional PRRs, such as NOD1, TLR2, and TLR4, it seems like patients classified according to their cytogenetics as poor risk express lower levels of these receptors compared to intermediate or good risk groups (Supplemental Figure A.9).

Elucidating the molecular mechanisms behind this observation may help to explain any discrepancies in response between patient subgroups.

In conclusion, the studies presented here offer a variation on the traditional therapeutic approach to leukemic disease. In contrast to the cytotoxic therapies that seek to destroy diseased cells, albeit alongside collateral damage to healthy cells, these agents offer relatively nontoxic approaches to modulating the leukemic blast itself.

Ultimately still resulting in the apoptosis of the cell, along the way the combination of

110

ATRA and daratumumab transformed the target leukemic blast into a fratricidal effector cell capable of inducing ADCC amongst the neighboring cancer cells. Likewise, the synthetic bacterial motif MTP-PE together with IFN-γ also induced blast apoptosis, this time through controlled stimulation of acute inflammation and subsequent activation of inflammasome-related caspase pathways, all the while re-invigorating and maturing the typically suppressed NK cell population. Both combination treatments, while varying in their mechanism of action, resulted in reduced disease burden and extended survival in murine models of AML. Our investigation into these agents suggests that these types of immunotherapeutic strategies may have significant clinical benefit in the treatment of leukemic disease.

111

References:

1. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532-1535. 2. Metchnikoff E. Ueber die Beziehung der Phagocyten zu Milzbrandbacillen. Virchow’s Archiv für pathologische Anatomie und Physiologie und für klinische Medicin. 1884;97:502-526. 3. Hashimoto D, Chow A, Noizat C, et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity. 2013;38(4):792-804. 4. Yona S, Kim KW, Wolf Y, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 2013;38(1):79-91. 5. Auffray C, Fogg D, Garfa M, et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science. 2007;317(5838):666-670. 6. Jakubzick C, Gautier EL, Gibbings SL, et al. Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes. Immunity. 2013;39(3):599-610. 7. Geissmann F, Auffray C, Palframan R, et al. Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses. Immunol Cell Biol. 2008;86(5):398-408. 8. Serbina NV, Jia T, Hohl TM, Pamer EG. Monocyte-mediated defense against microbial pathogens. Annu Rev Immunol. 2008;26:421-452. 9. Strauss-Ayali D, Conrad SM, Mosser DM. Monocyte subpopulations and their differentiation patterns during infection. J Leukoc Biol. 2007;82(2):244-252. 10. Cros J, Cagnard N, Woollard K, et al. Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity. 2010;33(3):375-386. 11. Wong KL, Yeap WH, Tai JJ, Ong SM, Dang TM, Wong SC. The three human monocyte subsets: implications for health and disease. Immunol Res. 2012;53(1-3):41-57. 12. Zawada AM, Rogacev KS, Schirmer SH, et al. Monocyte heterogeneity in human cardiovascular disease. Immunobiology. 2012;217(12):1273-1284. 13. Fingerle G, Pforte A, Passlick B, Blumenstein M, Strobel M, Ziegler-Heitbrock HW. The novel subset of CD14+/CD16+ blood monocytes is expanded in sepsis patients. Blood. 1993;82(10):3170-3176. 14. Castano D, Garcia LF, Rojas M. Increased frequency and cell death of CD16+ monocytes with Mycobacterium tuberculosis infection. Tuberculosis (Edinb). 2011;91(5):348-360. 15. Soares G, Barral A, Costa JM, Barral-Netto M, Van Weyenbergh J. CD16+ monocytes in human cutaneous leishmaniasis: increased ex vivo levels and correlation with clinical data. J Leukoc Biol. 2006;79(1):36-39. 16. Kawanaka N, Yamamura M, Aita T, et al. CD14+,CD16+ blood monocytes and joint inflammation in rheumatoid arthritis. Arthritis Rheum. 2002;46(10):2578-2586. 17. Saleh MN, Khazaeli MB, Wheeler RH, et al. Phase II trial of murine monoclonal antibody D612 combined with recombinant human monocyte colony-stimulating factor (rhM-CSF) in patients with metastatic gastrointestinal cancer. Cancer Res. 1995;55(19):4339-4346. 112

18. Gautier EL, Shay T, Miller J, et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat Immunol. 2012;13(11):1118-1128. 19. Epelman S, Lavine KJ, Randolph GJ. Origin and functions of tissue macrophages. Immunity. 2014;41(1):21-35. 20. Sieweke MH, Allen JE. Beyond stem cells: self-renewal of differentiated macrophages. Science. 2013;342(6161):1242974. 21. Martinez FO, Gordon S, Locati M, Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. J Immunol. 2006;177(10):7303-7311. 22. Nau GJ, Richmond JF, Schlesinger A, Jennings EG, Lander ES, Young RA. Human macrophage activation programs induced by bacterial pathogens. Proc Natl Acad Sci U S A. 2002;99(3):1503-1508. 23. Cerretti DP, Kozlosky CJ, Mosley B, et al. Molecular cloning of the interleukin-1 beta converting enzyme. Science. 1992;256(5053):97-100. 24. Thornberry NA, Bull HG, Calaycay JR, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992;356(6372):768-774. 25. Gratchev A, Guillot P, Hakiy N, et al. Alternatively activated macrophages differentially express fibronectin and its splice variants and the extracellular matrix protein betaIG-H3. Scand J Immunol. 2001;53(4):386-392. 26. Torocsik D, Bardos H, Nagy L, Adany R. Identification of factor XIII-A as a marker of alternative macrophage activation. Cell Mol Life Sci. 2005;62(18):2132-2139. 27. Asai A, Tsuchimoto Y, Ohama H, et al. Host antitumor resistance improved by the macrophage polarization in a chimera model of patients with HCC. Oncoimmunology. 2017;6(4):e1299301. 28. Ito I, Bhopale KK, Nishiguchi T, et al. The Polarization of M2b Monocytes in Cultures of Burn Patient Peripheral CD14(+) Cells Treated with a Selected Human CCL1 Antisense Oligodeoxynucleotide. Nucleic Acid Ther. 2016;26(5):269-276. 29. Lefevre L, Gales A, Olagnier D, et al. PPARgamma ligands switched high fat diet-induced macrophage M2b polarization toward M2a thereby improving intestinal Candida elimination. PLoS One. 2010;5(9):e12828. 30. Liu RX, Wei Y, Zeng QH, et al. Chemokine (C-X-C motif) receptor 3-positive B cells link interleukin-17 inflammation to protumorigenic macrophage polarization in human hepatocellular carcinoma. Hepatology. 2015;62(6):1779-1790. 31. Nishiguchi T, Ito I, Lee JO, Suzuki S, Suzuki F, Kobayashi M. Macrophage polarization and MRSA infection in burned mice. Immunol Cell Biol. 2017;95(2):198-206. 32. Tsuchimoto Y, Asai A, Tsuda Y, et al. M2b Monocytes Provoke Bacterial Pneumonia and Gut Bacteria-Associated Sepsis in Alcoholics. J Immunol. 2015;195(11):5169-5177. 33. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8(12):958-969. 34. Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 2014;41(1):14-20. 35. Bruhns P, Iannascoli B, England P, et al. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood. 2009;113(16):3716- 3725.

113

36. van der Poel CE, Spaapen RM, van de Winkel JG, Leusen JH. Functional characteristics of the high affinity IgG receptor, FcgammaRI. J Immunol. 2011;186(5):2699-2704. 37. Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CM, Tsai M. Mast cells as "tunable" effector and immunoregulatory cells: recent advances. Annu Rev Immunol. 2005;23:749-786. 38. Gould HJ, Sutton BJ, Beavil AJ, et al. The biology of IGE and the basis of allergic disease. Annu Rev Immunol. 2003;21:579-628. 39. Wang AV, Scholl PR, Geha RS. Physical and functional association of the high affinity immunoglobulin G receptor (Fc gamma RI) with the kinases Hck and Lyn. J Exp Med. 1994;180(3):1165-1170. 40. Ghazizadeh S, Bolen JB, Fleit HB. Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells. J Biol Chem. 1994;269(12):8878-8884. 41. Kiener PA, Rankin BM, Burkhardt AL, et al. Cross-linking of Fc gamma receptor I (Fc gamma RI) and receptor II (Fc gamma RII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase. J Biol Chem. 1993;268(32):24442-24448. 42. Clynes R, Maizes JS, Guinamard R, Ono M, Takai T, Ravetch JV. Modulation of immune complex-induced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors. J Exp Med. 1999;189(1):179-185. 43. Yuasa T, Kubo S, Yoshino T, et al. Deletion of fcgamma receptor IIB renders H-2(b) mice susceptible to collagen-induced arthritis. J Exp Med. 1999;189(1):187-194. 44. Bolland S, Ravetch JV. Inhibitory pathways triggered by ITIM-containing receptors. Adv Immunol. 1999;72:149-177. 45. Jiang Y, Hirose S, Abe M, et al. Polymorphisms in IgG Fc receptor IIB regulatory regions associated with autoimmune susceptibility. Immunogenetics. 2000;51(6):429-435. 46. Jiang Y, Hirose S, Sanokawa-Akakura R, et al. Genetically determined aberrant down- regulation of FcgammaRIIB1 in germinal center B cells associated with hyper-IgG and IgG autoantibodies in murine systemic lupus erythematosus. Int Immunol. 1999;11(10):1685-1691. 47. Paul E, Nelde A, Verschoor A, Carroll MC. Follicular exclusion of autoreactive B cells requires FcgammaRIIb. Int Immunol. 2007;19(4):365-373. 48. Pritchard NR, Cutler AJ, Uribe S, Chadban SJ, Morley BJ, Smith KG. Autoimmune-prone mice share a promoter haplotype associated with reduced expression and function of the Fc receptor FcgammaRII. Curr Biol. 2000;10(4):227-230. 49. Anderson P, Caligiuri M, O'Brien C, Manley T, Ritz J, Schlossman SF. Fc gamma receptor type III (CD16) is included in the zeta NK receptor complex expressed by human natural killer cells. Proc Natl Acad Sci U S A. 1990;87(6):2274-2278. 50. Lanier LL. Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol. 2008;9(5):495-502. 51. Smyth MJ, Cretney E, Kelly JM, et al. Activation of NK cell cytotoxicity. Mol Immunol. 2005;42(4):501-510. 52. Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood. 2005;105(8):3051-3057. 53. Fujisaki H, Kakuda H, Shimasaki N, et al. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res. 2009;69(9):4010-4017.

114

54. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674. 55. Vajdic CM, van Leeuwen MT. Cancer incidence and risk factors after solid organ transplantation. Int J Cancer. 2009;125(8):1747-1754. 56. Pages F, Galon J, Dieu-Nosjean MC, Tartour E, Sautes-Fridman C, Fridman WH. Immune infiltration in human tumors: a prognostic factor that should not be ignored. Oncogene. 2010;29(8):1093-1102. 57. Nelson BH. The impact of T-cell immunity on ovarian cancer outcomes. Immunol Rev. 2008;222:101-116. 58. Winter G, Milstein C. Man-made antibodies. Nature. 1991;349(6307):293-299. 59. Hargadon KM, Johnson CE, Williams CJ. Immune checkpoint blockade therapy for cancer: An overview of FDA-approved immune checkpoint inhibitors. Int Immunopharmacol. 2018;62:29-39. 60. Goldberg MV, Drake CG. LAG-3 in Cancer Immunotherapy. Curr Top Microbiol Immunol. 2011;344:269-278. 61. He Y, Cao J, Zhao C, Li X, Zhou C, Hirsch FR. TIM-3, a promising target for cancer immunotherapy. Onco Targets Ther. 2018;11:7005-7009. 62. Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity. Nat Immunol. 2018;19(7):723-732. 63. Johnston RJ, Yu X, Grogan JL. The checkpoint inhibitor TIGIT limits antitumor and antiviral CD8(+) T cell responses. Oncoimmunology. 2015;4(9):e1036214. 64. Lines JL, Pantazi E, Mak J, et al. VISTA is an immune checkpoint molecule for human T cells. Cancer Res. 2014;74(7):1924-1932. 65. Lee YH, Martin-Orozco N, Zheng P, et al. Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function. Cell Res. 2017;27(8):1034- 1045. 66. Jen EY, Ko CW, Lee JE, et al. FDA Approval: Gemtuzumab Ozogamicin for the Treatment of Adults with Newly Diagnosed CD33-Positive Acute Myeloid Leukemia. Clin Cancer Res. 2018;24(14):3242-3246. 67. Abdollahpour-Alitappeh M, Razavi-Vakhshourpour S, Abolhassani M. Development of a new anti-CD123 monoclonal antibody to target the human CD123 antigen as an acute myeloid leukemia cancer stem cell biomarker. Biotechnol Appl Biochem. 2018;65(6):841-847. 68. Hallek M, Fischer K, Fingerle-Rowson G, et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 2010;376(9747):1164-1174. 69. Goede V, Fischer K, Engelke A, et al. Obinutuzumab as frontline treatment of chronic lymphocytic leukemia: updated results of the CLL11 study. Leukemia. 2015;29(7):1602-1604. 70. Wierda WG, Kipps TJ, Mayer J, et al. Ofatumumab as single-agent CD20 immunotherapy in fludarabine-refractory chronic lymphocytic leukemia. J Clin Oncol. 2010;28(10):1749-1755. 71. Petersdorf SH, Kopecky KJ, Slovak M, et al. A phase 3 study of gemtuzumab ozogamicin during induction and postconsolidation therapy in younger patients with acute myeloid leukemia. Blood. 2013;121(24):4854-4860. 72. Lowenberg B, Ossenkoppele GJ, van Putten W, et al. High-dose daunorubicin in older patients with acute myeloid leukemia. N Engl J Med. 2009;361(13):1235-1248. 73. Fernandez HF, Sun Z, Yao X, et al. Anthracycline dose intensification in acute myeloid leukemia. N Engl J Med. 2009;361(13):1249-1259.

115

74. Castaigne S, Pautas C, Terre C, et al. Effect of gemtuzumab ozogamicin on survival of adult patients with de-novo acute myeloid leukaemia (ALFA-0701): a randomised, open-label, phase 3 study. Lancet. 2012;379(9825):1508-1516. 75. Amadori S, Suciu S, Selleslag D, et al. Randomized trial of two schedules of low-dose gemtuzumab ozogamicin as induction monotherapy for newly diagnosed acute myeloid leukaemia in older patients not considered candidates for intensive chemotherapy. A phase II study of the EORTC and GIMEMA leukaemia groups (AML-19). Br J Haematol. 2010;149(3):376- 382. 76. Laszlo GS, Estey EH, Walter RB. The past and future of CD33 as therapeutic target in acute myeloid leukemia. Blood Rev. 2014;28(4):143-153. 77. Jilani I, Estey E, Huh Y, et al. Differences in CD33 intensity between various myeloid neoplasms. Am J Clin Pathol. 2002;118(4):560-566. 78. Linenberger ML. CD33-directed therapy with gemtuzumab ozogamicin in acute myeloid leukemia: progress in understanding cytotoxicity and potential mechanisms of drug resistance. Leukemia. 2005;19(2):176-182. 79. Jedema I, Barge RM, van der Velden VH, et al. Internalization and cell cycle-dependent killing of leukemic cells by Gemtuzumab Ozogamicin: rationale for efficacy in CD33-negative malignancies with endocytic capacity. Leukemia. 2004;18(2):316-325. 80. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65(1):5-29. 81. Stein EM, Tallman MS. Emerging therapeutic drugs for AML. Blood. 2016;127(1):71-78. 82. Rowe JM. AML in 2016: Where we are now? Best Pract Res Clin Haematol. 2016;29(4):315-319. 83. Estey E, Levine RL, Lowenberg B. Current challenges in clinical development of "targeted therapies": the case of acute myeloid leukemia. Blood. 2015;125(16):2461-2466. 84. Abrahamsson J, Forestier E, Heldrup J, et al. Response-guided induction therapy in pediatric acute myeloid leukemia with excellent remission rate. J Clin Oncol. 2011;29(3):310- 315. 85. Entz-Werle N, Suciu S, van der Werff ten Bosch J, et al. Results of 58872 and 58921 trials in acute myeloblastic leukemia and relative value of chemotherapy vs allogeneic bone marrow transplantation in first complete remission: the EORTC Children Leukemia Group report. Leukemia. 2005;19(12):2072-2081. 86. Pession A, Rondelli R, Basso G, et al. Treatment and long-term results in children with acute myeloid leukaemia treated according to the AIEOP AML protocols. Leukemia. 2005;19(12):2043-2053. 87. Dombret H, Gardin C. An update of current treatments for adult acute myeloid leukemia. Blood. 2016;127(1):53-61. 88. Howlader N NA, Krapcho M, Miller D, Bishop K, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA SEER Cancer Statistics Review, 1975-2013. In: Institute NC ed. Bethesda, MD; 2016. 89. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391- 2405. 90. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood. 2010;116(3):354-365.

116

91. Cheng Y, Wang Y, Wang H, et al. Cytogenetic profile of de novo acute myeloid leukemia: a study based on 1432 patients in a single institution of China. Leukemia. 2009;23(10):1801- 1806. 92. Sanderson RN, Johnson PR, Moorman AV, et al. Population-based demographic study of karyotypes in 1709 patients with adult acute myeloid leukemia. Leukemia. 2006;20(3):444-450. 93. Raimondi SC, Chang MN, Ravindranath Y, et al. Chromosomal abnormalities in 478 children with acute myeloid leukemia: clinical characteristics and treatment outcome in a cooperative pediatric oncology group study-POG 8821. Blood. 1999;94(11):3707-3716. 94. Rubnitz JE, Raimondi SC, Halbert AR, et al. Characteristics and outcome of t(8;21)- positive childhood acute myeloid leukemia: a single institution's experience. Leukemia. 2002;16(10):2072-2077. 95. Krauth MT, Eder C, Alpermann T, et al. High number of additional genetic lesions in acute myeloid leukemia with t(8;21)/RUNX1-RUNX1T1: frequency and impact on clinical outcome. Leukemia. 2014;28(7):1449-1458. 96. Wildonger J, Mann RS. The t(8;21) translocation converts AML1 into a constitutive transcriptional repressor. Development. 2005;132(10):2263-2272. 97. Adya N, Stacy T, Speck NA, Liu PP. The leukemic protein core binding factor beta (CBFbeta)-smooth-muscle myosin heavy chain sequesters CBFalpha2 into cytoskeletal filaments and aggregates. Mol Cell Biol. 1998;18(12):7432-7443. 98. von Neuhoff C, Reinhardt D, Sander A, et al. Prognostic impact of specific chromosomal aberrations in a large group of pediatric patients with acute myeloid leukemia treated uniformly according to trial AML-BFM 98. J Clin Oncol. 2010;28(16):2682-2689. 99. Appelbaum FR, Kopecky KJ, Tallman MS, et al. The clinical spectrum of adult acute myeloid leukaemia associated with core binding factor translocations. Br J Haematol. 2006;135(2):165-173. 100. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev Cancer. 2007;7(11):823-833. 101. Muntean AG, Hess JL. The pathogenesis of mixed-lineage leukemia. Annu Rev Pathol. 2012;7:283-301. 102. Huret JL, Dessen P, Le Minor S, Bernheim A. The "Atlas of genetics and cytogenetics in oncology and haematology" on the internet and a review on infant leukemias. Cancer Genet Cytogenet. 2000;120(2):155-159. 103. Martin ME, Milne TA, Bloyer S, et al. Dimerization of MLL fusion proteins immortalizes hematopoietic cells. Cancer Cell. 2003;4(3):197-207. 104. Milne TA, Briggs SD, Brock HW, et al. MLL targets SET domain methyltransferase activity to Hox gene promoters. Mol Cell. 2002;10(5):1107-1117. 105. Song Y, Bixby D, Roulston D, Magenau J, Choi SW. The Challenge of t (6;9) and FLT3- Positive Acute Myelogenous Leukemia in a Young Adult. J Leuk (Los Angel). 2014;2. 106. Tarlock K, Alonzo TA, Moraleda PP, et al. Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is associated with poor outcome in childhood AML regardless of FLT3-ITD status: a report from the Children's Oncology Group. Br J Haematol. 2014;166(2):254-259. 107. Lugthart S, Groschel S, Beverloo HB, et al. Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and various other 3q abnormalities in acute myeloid leukemia. J Clin Oncol. 2010;28(24):3890-3898. 108. Gokce M, Aytac S, Unal S, Altan I, Gumruk F, Cetin M. Acute Megakaryoblastic Leukemia with t(1;22) Mimicking Neuroblastoma in an Infant. Turk J Haematol. 2015;32(1):64-67.

117

109. Cancer Genome Atlas Research N, Ley TJ, Miller C, et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368(22):2059-2074. 110. Dufour A, Schneider F, Metzeler KH, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol. 2010;28(4):570-577. 111. Wouters BJ, Lowenberg B, Erpelinck-Verschueren CA, van Putten WL, Valk PJ, Delwel R. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood. 2009;113(13):3088-3091. 112. Konoplev S, Yin CC, Kornblau SM, et al. Molecular characterization of de novo Philadelphia chromosome-positive acute myeloid leukemia. Leuk Lymphoma. 2013;54(1):138- 144. 113. Soupir CP, Vergilio JA, Dal Cin P, et al. Philadelphia chromosome-positive acute myeloid leukemia: a rare aggressive leukemia with clinicopathologic features distinct from chronic myeloid leukemia in myeloid blast crisis. Am J Clin Pathol. 2007;127(4):642-650. 114. Schnittger S, Dicker F, Kern W, et al. RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis. Blood. 2011;117(8):2348-2357. 115. Weinberg OK, Seetharam M, Ren L, et al. Clinical characterization of acute myeloid leukemia with myelodysplasia-related changes as defined by the 2008 WHO classification system. Blood. 2009;113(9):1906-1908. 116. Churpek JE, Marquez R, Neistadt B, et al. Inherited mutations in cancer susceptibility genes are common among survivors of breast cancer who develop therapy-related leukemia. Cancer. 2016;122(2):304-311. 117. Avni B, Koren-Michowitz M. Myeloid sarcoma: current approach and therapeutic options. Ther Adv Hematol. 2011;2(5):309-316. 118. Roy A, Roberts I, Vyas P. Biology and management of transient abnormal myelopoiesis (TAM) in children with Down syndrome. Semin Fetal Neonatal Med. 2012;17(4):196-201. 119. Lange BJ, Kobrinsky N, Barnard DR, et al. Distinctive demography, biology, and outcome of acute myeloid leukemia and myelodysplastic syndrome in children with Down syndrome: Children's Cancer Group Studies 2861 and 2891. Blood. 1998;91(2):608-615. 120. Dohner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. N Engl J Med. 2015;373(12):1136-1152. 121. Hills RK, Castaigne S, Appelbaum FR, et al. Addition of gemtuzumab ozogamicin to induction chemotherapy in adult patients with acute myeloid leukaemia: a meta-analysis of individual patient data from randomised controlled trials. Lancet Oncol. 2014;15(9):986-996. 122. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002;100(13):4325-4336. 123. Slovak ML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study. Blood. 2000;96(13):4075-4083. 124. Sorror ML, Storb RF, Sandmaier BM, et al. Comorbidity-age index: a clinical measure of biologic age before allogeneic hematopoietic cell transplantation. J Clin Oncol. 2014;32(29):3249-3256.

118

125. Koreth J, Schlenk R, Kopecky KJ, et al. Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: systematic review and meta-analysis of prospective clinical trials. JAMA. 2009;301(22):2349-2361. 126. Burnett A, Wetzler M, Lowenberg B. Therapeutic advances in acute myeloid leukemia. J Clin Oncol. 2011;29(5):487-494. 127. Small D. FLT3 mutations: biology and treatment. Hematology Am Soc Hematol Educ Program. 2006:178-184. 128. Hayakawa F, Towatari M, Kiyoi H, et al. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene. 2000;19(5):624-631. 129. Mizuki M, Fenski R, Halfter H, et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood. 2000;96(12):3907-3914. 130. Borthakur G, Kantarjian H, Ravandi F, et al. Phase I study of sorafenib in patients with refractory or relapsed acute leukemias. Haematologica. 2011;96(1):62-68. 131. Crump M, Hedley D, Kamel-Reid S, et al. A randomized phase I clinical and biologic study of two schedules of sorafenib in patients with myelodysplastic syndrome or acute myeloid leukemia: a NCIC (National Cancer Institute of Canada) Clinical Trials Group Study. Leuk Lymphoma. 2010;51(2):252-260. 132. Zhang W, Konopleva M, Shi YX, et al. Mutant FLT3: a direct target of sorafenib in acute myelogenous leukemia. J Natl Cancer Inst. 2008;100(3):184-198. 133. Fischer T, Stone RM, Deangelo DJ, et al. Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3. J Clin Oncol. 2010;28(28):4339-4345. 134. Stone RM, DeAngelo DJ, Klimek V, et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood. 2005;105(1):54-60. 135. Cortes JE, Kantarjian H, Foran JM, et al. Phase I study of quizartinib administered daily to patients with relapsed or refractory acute myeloid leukemia irrespective of FMS-like tyrosine kinase 3-internal tandem duplication status. J Clin Oncol. 2013;31(29):3681-3687. 136. Zhang H, Savage S, Schultz AR, et al. Clinical resistance to crenolanib in acute myeloid leukemia due to diverse molecular mechanisms. Nat Commun. 2019;10(1):244. 137. Perl AE, Altman JK, Cortes J, et al. Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1-2 study. Lancet Oncol. 2017;18(8):1061-1075. 138. Benekli M, Xia Z, Donohue KA, et al. Constitutive activity of signal transducer and activator of transcription 3 protein in acute myeloid leukemia blasts is associated with short disease-free survival. Blood. 2002;99(1):252-257. 139. Steensma DP, McClure RF, Karp JE, et al. JAK2 V617F is a rare finding in de novo acute myeloid leukemia, but STAT3 activation is common and remains unexplained. Leukemia. 2006;20(6):971-978. 140. Zhou J, Bi C, Janakakumara JV, et al. Enhanced activation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML. Blood. 2009;113(17):4052-4062.

119

141. Hayakawa F, Sugimoto K, Harada Y, et al. A novel STAT inhibitor, OPB-31121, has a significant antitumor effect on leukemia with STAT-addictive oncokinases. Blood Cancer J. 2013;3:e166. 142. Green CL, Evans CM, Zhao L, et al. The prognostic significance of IDH2 mutations in AML depends on the location of the mutation. Blood. 2011;118(2):409-412. 143. Marcucci G, Maharry K, Wu YZ, et al. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol. 2010;28(14):2348-2355. 144. Dang L, White DW, Gross S, et al. Cancer-associated IDH1 mutations produce 2- hydroxyglutarate. Nature. 2009;462(7274):739-744. 145. Wang JH, Chen WL, Li JM, et al. Prognostic significance of 2-hydroxyglutarate levels in acute myeloid leukemia in China. Proc Natl Acad Sci U S A. 2013;110(42):17017-17022. 146. Xu W, Yang H, Liu Y, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell. 2011;19(1):17-30. 147. Stein EM, DiNardo CD, Pollyea DA, et al. Enasidenib in mutant IDH2 relapsed or refractory acute myeloid leukemia. Blood. 2017;130(6):722-731. 148. DiNardo CD, Stein EM, de Botton S, et al. Durable Remissions with Ivosidenib in IDH1- Mutated Relapsed or Refractory AML. N Engl J Med. 2018;378(25):2386-2398. 149. Ribeiro AF, Pratcorona M, Erpelinck-Verschueren C, et al. Mutant DNMT3A: a marker of poor prognosis in acute myeloid leukemia. Blood. 2012;119(24):5824-5831. 150. Rau RE, Rodriguez BA, Luo M, et al. DOT1L as a therapeutic target for the treatment of DNMT3A-mutant acute myeloid leukemia. Blood. 2016;128(7):971-981. 151. Bernt KM, Zhu N, Sinha AU, et al. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell. 2011;20(1):66-78. 152. Kuhn MW, Hadler MJ, Daigle SR, et al. MLL partial tandem duplication leukemia cells are sensitive to small molecule DOT1L inhibition. Haematologica. 2015;100(5):e190-193. 153. Daigle SR, Olhava EJ, Therkelsen CA, et al. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood. 2013;122(6):1017-1025. 154. Daigle SR, Olhava EJ, Therkelsen CA, et al. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell. 2011;20(1):53-65. 155. Lee EM, Yee D, Busfield SJ, et al. Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice. Haematologica. 2015;100(7):914-926. 156. Li F, Sutherland MK, Yu C, et al. Characterization of SGN-CD123A, A Potent CD123- Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia. Mol Cancer Ther. 2018;17(2):554-564. 157. Matthews DC, Appelbaum FR, Eary JF, et al. Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood. 1999;94(4):1237-1247. 158. Orozco JJ, Zeller J, Pagel JM. Radiolabeled antibodies directed at CD45 for conditioning prior to allogeneic transplantation in acute myeloid leukemia and myelodysplastic syndrome. Ther Adv Hematol. 2012;3(1):5-16. 159. Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med. 2006;12(10):1167-1174.

120

160. Vey N, Delaunay J, Martinelli G, et al. Phase I clinical study of RG7356, an anti-CD44 humanized antibody, in patients with acute myeloid leukemia. Oncotarget. 2016;7(22):32532- 32542. 161. Curti A, Ruggeri L, D'Addio A, et al. Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients. Blood. 2011;118(12):3273-3279. 162. Curti A, Ruggeri L, Parisi S, et al. Larger Size of Donor Alloreactive NK Cell Repertoire Correlates with Better Response to NK Cell Immunotherapy in Elderly Acute Myeloid Leukemia Patients. Clin Cancer Res. 2016;22(8):1914-1921. 163. Rubnitz JE, Inaba H, Ribeiro RC, et al. NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia. J Clin Oncol. 2010;28(6):955-959. 164. Pisciotta AV, Schulz EJ. Fibrinolytic purpura in acute leukemia. Am J Med. 1955;19(5):824-828. 165. Cooperberg AA, Neiman GM. Fibrinogenopenia and fibrinolysis in acute myelogenous leukemia. Ann Intern Med. 1955;42(3):706-711. 166. Fisher S, Ramot B, Kreisler B. Fibrinolysis in acute leukemia; a case report. Isr Med J. 1960;19:195-198. 167. Ghitis J. Acute promyelocytic leukemia? Blood. 1963;21:237-240. 168. Larson RA, Kondo K, Vardiman JW, Butler AE, Golomb HM, Rowley JD. Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia. Am J Med. 1984;76(5):827-841. 169. Bernard J, Weil M, Boiron M, Jacquillat C, Flandrin G, Gemon MF. Acute promyelocytic leukemia: results of treatment by daunorubicin. Blood. 1973;41(4):489-496. 170. Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly fatal to highly curable. Blood. 2008;111(5):2505-2515. 171. Fenaux P, Wang ZZ, Degos L. Treatment of acute promyelocytic leukemia by retinoids. Curr Top Microbiol Immunol. 2007;313:101-128. 172. Flynn PJ, Miller WJ, Weisdorf DJ, Arthur DC, Brunning R, Branda RF. Retinoic acid treatment of acute promyelocytic leukemia: in vitro and in vivo observations. Blood. 1983;62(6):1211-1217. 173. Nilsson B. Probable in vivo induction of differentiation by retinoic acid of promyelocytes in acute promyelocytic leukaemia. Br J Haematol. 1984;57(3):365-371. 174. Huang ME, Ye YC, Chen SR, et al. All-trans retinoic acid with or without low dose cytosine arabinoside in acute promyelocytic leukemia. Report of 6 cases. Chin Med J (Engl). 1987;100(12):949-953. 175. Huang ME, Ye YC, Chen SR, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood. 1988;72(2):567-572. 176. Fenaux P, Chastang C, Chevret S, et al. A randomized comparison of all transretinoic acid (ATRA) followed by chemotherapy and ATRA plus chemotherapy and the role of maintenance therapy in newly diagnosed acute promyelocytic leukemia. The European APL Group. Blood. 1999;94(4):1192-1200. 177. Lengfelder E, Reichert A, Schoch C, et al. Double induction strategy including high dose cytarabine in combination with all-trans retinoic acid: effects in patients with newly diagnosed acute promyelocytic leukemia. German AML Cooperative Group. Leukemia. 2000;14(8):1362- 1370.

121

178. Sanz MA, Martin G, Rayon C, et al. A modified AIDA protocol with anthracycline-based consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia. PETHEMA group. Blood. 1999;94(9):3015-3021. 179. Johnson DE, Redner RL. An ATRActive future for differentiation therapy in AML. Blood Rev. 2015;29(4):263-268. 180. Mathews V, George B, Chendamarai E, et al. Single-agent arsenic trioxide in the treatment of newly diagnosed acute promyelocytic leukemia: long-term follow-up data. J Clin Oncol. 2010;28(24):3866-3871. 181. Ghavamzadeh A, Alimoghaddam K, Rostami S, et al. Phase II study of single-agent arsenic trioxide for the front-line therapy of acute promyelocytic leukemia. J Clin Oncol. 2011;29(20):2753-2757. 182. Niu C, Yan H, Yu T, et al. Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood. 1999;94(10):3315- 3324. 183. Soignet SL, Frankel SR, Douer D, et al. United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol. 2001;19(18):3852-3860. 184. Iland HJ, Bradstock K, Supple SG, et al. All-trans-retinoic acid, idarubicin, and IV arsenic trioxide as initial therapy in acute promyelocytic leukemia (APML4). Blood. 2012;120(8):1570- 1580; quiz 1752. 185. Ravandi F, Estey E, Jones D, et al. Effective treatment of acute promyelocytic leukemia with all-trans-retinoic acid, arsenic trioxide, and gemtuzumab ozogamicin. J Clin Oncol. 2009;27(4):504-510. 186. Shen ZX, Shi ZZ, Fang J, et al. All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. Proc Natl Acad Sci U S A. 2004;101(15):5328-5335. 187. Estey E, Garcia-Manero G, Ferrajoli A, et al. Use of all-trans retinoic acid plus arsenic trioxide as an alternative to chemotherapy in untreated acute promyelocytic leukemia. Blood. 2006;107(9):3469-3473. 188. Zhu J, Koken MH, Quignon F, et al. Arsenic-induced PML targeting onto nuclear bodies: implications for the treatment of acute promyelocytic leukemia. Proc Natl Acad Sci U S A. 1997;94(8):3978-3983. 189. Chen GQ, Shi XG, Tang W, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells. Blood. 1997;89(9):3345-3353. 190. Burnett AK, Hills RK, Green C, et al. The impact on outcome of the addition of all-trans retinoic acid to intensive chemotherapy in younger patients with nonacute promyelocytic acute myeloid leukemia: overall results and results in genotypic subgroups defined by mutations in NPM1, FLT3, and CEBPA. Blood. 2010;115(5):948-956. 191. McCollum EV, Davis M. Nutrition classics from The Journal of Biological Chemistry 15:167-175, 1913. The necessity of certain lipins in the diet during growth. By E. V. McCollum and Marguerite Davis. Nutr Rev. 1973;31(9):280-281. 192. Wald G. Molecular basis of visual excitation. Science. 1968;162(3850):230-239. 193. Semba RD. The role of vitamin A and related retinoids in immune function. Nutr Rev. 1998;56(1 Pt 2):S38-48.

122

194. Schreiber R, Taschler U, Preiss-Landl K, Wongsiriroj N, Zimmermann R, Lass A. Retinyl ester hydrolases and their roles in vitamin A homeostasis. Biochim Biophys Acta. 2012;1821(1):113-123. 195. Ni X, Hu G, Cai X. The success and the challenge of all-trans retinoic acid in the treatment of cancer. Crit Rev Food Sci Nutr. 2019;59(sup1):S71-S80. 196. Petkovich M, Brand NJ, Krust A, Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature. 1987;330(6147):444-450. 197. Giguere V, Ong ES, Segui P, Evans RM. Identification of a receptor for the morphogen retinoic acid. Nature. 1987;330(6149):624-629. 198. Evans RM, Mangelsdorf DJ. Nuclear Receptors, RXR, and the Big Bang. Cell. 2014;157(1):255-266. 199. de The H, Vivanco-Ruiz MM, Tiollais P, Stunnenberg H, Dejean A. Identification of a retinoic acid responsive element in the retinoic acid receptor beta gene. Nature. 1990;343(6254):177-180. 200. Mangelsdorf DJ, Umesono K, Kliewer SA, Borgmeyer U, Ong ES, Evans RM. A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR. Cell. 1991;66(3):555-561. 201. Durand B, Saunders M, Leroy P, Leid M, Chambon P. All-trans and 9-cis retinoic acid induction of CRABPII transcription is mediated by RAR-RXR heterodimers bound to DR1 and DR2 repeated motifs. Cell. 1992;71(1):73-85. 202. Levin AA, Sturzenbecker LJ, Kazmer S, et al. 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature. 1992;355(6358):359-361. 203. Heyman RA, Mangelsdorf DJ, Dyck JA, et al. 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992;68(2):397-406. 204. Umesono K, Murakami KK, Thompson CC, Evans RM. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell. 1991;65(7):1255-1266. 205. Kliewer SA, Umesono K, Heyman RA, Mangelsdorf DJ, Dyck JA, Evans RM. Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling. Proc Natl Acad Sci U S A. 1992;89(4):1448-1452. 206. Sucov HM, Evans RM. Retinoic acid and retinoic acid receptors in development. Mol Neurobiol. 1995;10(2-3):169-184. 207. Balmer JE, Blomhoff R. Gene expression regulation by retinoic acid. J Lipid Res. 2002;43(11):1773-1808. 208. Nijhof IS, Groen RW, Lokhorst HM, et al. Upregulation of CD38 expression on multiple myeloma cells by all-trans retinoic acid improves the efficacy of daratumumab. Leukemia. 2015;29(10):2039-2049. 209. Mihara K, Yoshida T, Ishida S, et al. All-trans retinoic acid and interferon-alpha increase CD38 expression on adult T-cell leukemia cells and sensitize them to T cells bearing anti-CD38 chimeric antigen receptors. Blood Cancer J. 2016;6:e421. 210. Pandolfi PP. In vivo analysis of the molecular genetics of acute promyelocytic leukemia. Oncogene. 2001;20(40):5726-5735. 211. Di Croce L, Raker VA, Corsaro M, et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science. 2002;295(5557):1079-1082.

123

212. Villa R, Morey L, Raker VA, et al. The methyl-CpG binding protein MBD1 is required for PML-RARalpha function. Proc Natl Acad Sci U S A. 2006;103(5):1400-1405. 213. Nervi C, Ferrara FF, Fanelli M, et al. Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein. Blood. 1998;92(7):2244- 2251. 214. Zhu J, Gianni M, Kopf E, et al. Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor alpha (RARalpha) and oncogenic RARalpha fusion proteins. Proc Natl Acad Sci U S A. 1999;96(26):14807-14812. 215. White JA, Ramshaw H, Taimi M, et al. Identification of the human cytochrome P450, P450RAI-2, which is predominantly expressed in the adult cerebellum and is responsible for all- trans-retinoic acid metabolism. Proc Natl Acad Sci U S A. 2000;97(12):6403-6408. 216. White JA, Guo YD, Baetz K, et al. Identification of the retinoic acid-inducible all-trans- retinoic acid 4-hydroxylase. J Biol Chem. 1996;271(47):29922-29927. 217. Tahayato A, Dolle P, Petkovich M. Cyp26C1 encodes a novel retinoic acid-metabolizing enzyme expressed in the hindbrain, inner ear, first branchial arch and tooth buds during murine development. Gene Expr Patterns. 2003;3(4):449-454. 218. Reijntjes S, Gale E, Maden M. Generating gradients of retinoic acid in the chick embryo: Cyp26C1 expression and a comparative analysis of the Cyp26 enzymes. Dev Dyn. 2004;230(3):509-517. 219. Xi J, Yang Z. Expression of RALDHs (ALDH1As) and CYP26s in human tissues and during the neural differentiation of P19 embryonal carcinoma stem cell. Gene Expr Patterns. 2008;8(6):438-442. 220. Wang Y, Zolfaghari R, Ross AC. Cloning of rat cytochrome P450RAI (CYP26) cDNA and regulation of its gene expression by all-trans-retinoic acid in vivo. Arch Biochem Biophys. 2002;401(2):235-243. 221. Ray WJ, Bain G, Yao M, Gottlieb DI. CYP26, a novel mammalian cytochrome P450, is induced by retinoic acid and defines a new family. J Biol Chem. 1997;272(30):18702-18708. 222. Ross AC, Cifelli CJ, Zolfaghari R, Li NQ. Multiple cytochrome P-450 genes are concomitantly regulated by vitamin A under steady-state conditions and by retinoic acid during hepatic first-pass metabolism. Physiol Genomics. 2011;43(1):57-67. 223. Yamamoto Y, Zolfaghari R, Ross AC. Regulation of CYP26 (cytochrome P450RAI) mRNA expression and retinoic acid metabolism by retinoids and dietary vitamin A in liver of mice and rats. FASEB J. 2000;14(13):2119-2127. 224. Baldwin HE, Nighland M, Kendall C, Mays DA, Grossman R, Newburger J. 40 years of topical tretinoin use in review. J Drugs Dermatol. 2013;12(6):638-642. 225. Warrell RP, Jr., Frankel SR, Miller WH, Jr., et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med. 1991;324(20):1385- 1393. 226. Tallman MS, Altman JK. How I treat acute promyelocytic leukemia. Blood. 2009;114(25):5126-5135. 227. Frankel SR, Eardley A, Lauwers G, Weiss M, Warrell RP, Jr. The "retinoic acid syndrome" in acute promyelocytic leukemia. Ann Intern Med. 1992;117(4):292-296. 228. Luesink M, Pennings JL, Wissink WM, et al. Chemokine induction by all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia: triggering the differentiation syndrome. Blood. 2009;114(27):5512-5521.

124

229. Nicolls MR, Terada LS, Tuder RM, Prindiville SA, Schwarz MI. Diffuse alveolar hemorrhage with underlying pulmonary capillaritis in the retinoic acid syndrome. Am J Respir Crit Care Med. 1998;158(4):1302-1305. 230. De Botton S, Dombret H, Sanz M, et al. Incidence, clinical features, and outcome of all trans-retinoic acid syndrome in 413 cases of newly diagnosed acute promyelocytic leukemia. The European APL Group. Blood. 1998;92(8):2712-2718. 231. de Botton S, Chevret S, Coiteux V, et al. Early onset of chemotherapy can reduce the incidence of ATRA syndrome in newly diagnosed acute promyelocytic leukemia (APL) with low white blood cell counts: results from APL 93 trial. Leukemia. 2003;17(2):339-342. 232. Montesinos P, Sanz MA. The differentiation syndrome in patients with acute promyelocytic leukemia: experience of the pethema group and review of the literature. Mediterr J Hematol Infect Dis. 2011;3(1):e2011059. 233. Howard M, Grimaldi JC, Bazan JF, et al. Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38. Science. 1993;262(5136):1056-1059. 234. Guse AH. Regulation of calcium signaling by the second messenger cyclic adenosine diphosphoribose (cADPR). Curr Mol Med. 2004;4(3):239-248. 235. Yamasaki M, Churchill GC, Galione A. Calcium signalling by nicotinic acid adenine dinucleotide phosphate (NAADP). FEBS J. 2005;272(18):4598-4606. 236. Frasca L, Fedele G, Deaglio S, et al. CD38 orchestrates migration, survival, and Th1 immune response of human mature dendritic cells. Blood. 2006;107(6):2392-2399. 237. Musso T, Deaglio S, Franco L, et al. CD38 expression and functional activities are upregulated by IFN-gamma on human monocytes and monocytic cell lines. J Leukoc Biol. 2001;69(4):605-612. 238. Ausiello CM, la Sala A, Ramoni C, Urbani F, Funaro A, Malavasi F. Secretion of IFN- gamma, IL-6, granulocyte-macrophage colony-stimulating factor and IL-10 cytokines after activation of human purified T lymphocytes upon CD38 ligation. Cell Immunol. 1996;173(2):192- 197. 239. Campana D, Suzuki T, Todisco E, Kitanaka A. CD38 in hematopoiesis. Chem Immunol. 2000;75:169-188. 240. Mallone R, Funaro A, Zubiaur M, et al. Signaling through CD38 induces NK cell activation. Int Immunol. 2001;13(4):397-409. 241. Zubiaur M, Izquierdo M, Terhorst C, Malavasi F, Sancho J. CD38 ligation results in activation of the Raf-1/mitogen-activated protein kinase and the CD3-zeta/zeta-associated protein-70 signaling pathways in Jurkat T lymphocytes. J Immunol. 1997;159(1):193-205. 242. Landgren O, Iskander K. Modern multiple myeloma therapy: deep, sustained treatment response and good clinical outcomes. J Intern Med. 2017;281(4):365-382. 243. Malavasi F, Funaro A, Roggero S, Horenstein A, Calosso L, Mehta K. Human CD38: a glycoprotein in search of a function. Immunol Today. 1994;15(3):95-97. 244. Yoshida T, Mihara K, Takei Y, et al. All-trans retinoic acid enhances cytotoxic effect of T cells with an anti-CD38 chimeric antigen receptor in acute myeloid leukemia. Clin Transl Immunology. 2016;5(12):e116. 245. Kishimoto H, Hoshino S, Ohori M, et al. Molecular mechanism of human CD38 gene expression by retinoic acid. Identification of retinoic acid response element in the first intron. J Biol Chem. 1998;273(25):15429-15434. 246. Fatehchand K, McMichael EL, Reader BF, et al. Interferon-gamma Promotes Antibody- mediated Fratricide of Acute Myeloid Leukemia Cells. J Biol Chem. 2016;291(49):25656-25666.

125

247. Zheng A, Mantymaa P, Saily M, Siitonen T, Savolainen ER, Koistinen P. An association between mitochondrial function and all-trans retinoic acid-induced apoptosis in acute myeloblastic leukaemia cells. Br J Haematol. 1999;105(1):215-224. 248. Adamson PC. Clinical and pharmacokinetic studies of all-trans-retinoic acid in pediatric patients with cancer. Leukemia. 1994;8(11):1813-1816. 249. Venditti A, Stasi R, Del Poeta G, et al. All-trans retinoic acid and low-dose cytosine arabinoside for the treatment of 'poor prognosis' acute myeloid leukemia. Leukemia. 1995;9(7):1121-1125. 250. Schlenk RF, Frohling S, Hartmann F, et al. Phase III study of all-trans retinoic acid in previously untreated patients 61 years or older with acute myeloid leukemia. Leukemia. 2004;18(11):1798-1803. 251. Estey EH, Thall PF, Pierce S, et al. Randomized phase II study of fludarabine + cytosine arabinoside + idarubicin +/- all-trans retinoic acid +/- granulocyte colony-stimulating factor in poor prognosis newly diagnosed acute myeloid leukemia and myelodysplastic syndrome. Blood. 1999;93(8):2478-2484. 252. Milligan DW, Wheatley K, Littlewood T, Craig JI, Burnett AK, Group NHOCS. Fludarabine and cytosine are less effective than standard ADE chemotherapy in high-risk acute myeloid leukemia, and addition of G-CSF and ATRA are not beneficial: results of the MRC AML-HR randomized trial. Blood. 2006;107(12):4614-4622. 253. Burnett AK, Milligan D, Prentice AG, et al. A comparison of low-dose cytarabine and hydroxyurea with or without all-trans retinoic acid for acute myeloid leukemia and high-risk myelodysplastic syndrome in patients not considered fit for intensive treatment. Cancer. 2007;109(6):1114-1124. 254. Belhabri A, Thomas X, Wattel E, et al. All trans retinoic acid in combination with intermediate-dose cytarabine and idarubicin in patients with relapsed or refractory non promyelocytic acute myeloid leukemia: a phase II randomized trial. Hematol J. 2002;3(1):49-55. 255. Heuser M, Argiropoulos B, Kuchenbauer F, et al. MN1 overexpression induces acute myeloid leukemia in mice and predicts ATRA resistance in patients with AML. Blood. 2007;110(5):1639-1647. 256. Sakamoto K, Imamura T, Yano M, et al. Sensitivity of MLL-rearranged AML cells to all- trans retinoic acid is associated with the level of H3K4me2 in the RARalpha promoter region. Blood Cancer J. 2014;4:e205. 257. Bullinger L, Schlenk RF, Gotz M, et al. PRAME-induced inhibition of retinoic acid receptor signaling-mediated differentiation--a possible target for ATRA response in AML without t(15;17). Clin Cancer Res. 2013;19(9):2562-2571. 258. Epping MT, Wang L, Edel MJ, Carlee L, Hernandez M, Bernards R. The human tumor antigen PRAME is a dominant repressor of retinoic acid receptor signaling. Cell. 2005;122(6):835-847. 259. Passeron T, Valencia JC, Namiki T, et al. Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid. J Clin Invest. 2009;119(4):954-963. 260. Steinbach D, Viehmann S, Zintl F, Gruhn B. PRAME gene expression in childhood acute lymphoblastic leukemia. Cancer Genet Cytogenet. 2002;138(1):89-91. 261. Van heusden J, Wouters W, Ramaekers FC, et al. The antiproliferative activity of all- trans-retinoic acid catabolites and isomers is differentially modulated by liarozole-fumarate in MCF-7 human breast cancer cells. Br J Cancer. 1998;77(8):1229-1235.

126

262. Su M, Alonso S, Jones JW, et al. All-Trans Retinoic Acid Activity in Acute Myeloid Leukemia: Role of Cytochrome P450 Enzyme Expression by the Microenvironment. PLoS One. 2015;10(6):e0127790. 263. Su M, Jones R, Ghiaur G. Bone Marrow (BM) Stromal Expression Of Cytochrome P450 (CYP) Enzymes Protects Acute Myeloid Leukemia (AML) From All-Trans Retinoic Acid (atRA). Blood. 2013 122:1449;. 264. He LZ, Tolentino T, Grayson P, et al. Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia. J Clin Invest. 2001;108(9):1321-1330. 265. Subramanyam D, Belair CD, Barry-Holson KQ, et al. PML-RAR{alpha} and Dnmt3a1 cooperate in vivo to promote acute promyelocytic leukemia. Cancer Res. 2010;70(21):8792- 8801. 266. De Marchis ML, Ballarino M, Salvatori B, Puzzolo MC, Bozzoni I, Fatica A. A new molecular network comprising PU.1, interferon regulatory factor proteins and miR-342 stimulates ATRA-mediated granulocytic differentiation of acute promyelocytic leukemia cells. Leukemia. 2009;23(5):856-862. 267. Chillemi A, Zaccarello G, Quarona V, et al. Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule. Mol Med. 2013;19:99-108. 268. Ishikawa F, Yasukawa M, Lyons B, et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood. 2005;106(5):1565- 1573. 269. Shultz LD, Lyons BL, Burzenski LM, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174(10):6477-6489. 270. Krejcik J, Casneuf T, Nijhof IS, et al. Daratumumab depletes CD38+ immune regulatory cells, promotes T-cell expansion, and skews T-cell repertoire in multiple myeloma. Blood. 2016;128(3):384-394. 271. Patatanian E, Thompson DF. Retinoic acid syndrome: a review. J Clin Pharm Ther. 2008;33(4):331-338. 272. Tallman MS. Retinoic acid syndrome: a problem of the past? Leukemia. 2002;16(2):160- 161. 273. Lokhorst HM, Plesner T, Laubach JP, et al. Targeting CD38 with Daratumumab Monotherapy in Multiple Myeloma. N Engl J Med. 2015;373(13):1207-1219. 274. Nowotny. Antitumor effects of endotoxins. Handbook of Endotoxin. 1985;3:389-448. 275. Coley WB. The treatment of malignant tumors by repeated inoculations of erysipelas. With a report of ten original cases. 1893. Clin Orthop Relat Res. 1991(262):3-11. 276. Coley WB. II. Contribution to the Knowledge of Sarcoma. Ann Surg. 1891;14(3):199-220. 277. Wiemann B, Starnes CO. Coley's toxins, tumor necrosis factor and cancer research: a historical perspective. Pharmacol Ther. 1994;64(3):529-564. 278. Hoption Cann SA, van Netten JP, van Netten C. Dr William Coley and tumour regression: a place in history or in the future. Postgrad Med J. 2003;79(938):672-680. 279. Lin E, Freedman JE, Beaulieu LM. Innate immunity and toll-like receptor antagonists: a potential role in the treatment of cardiovascular diseases. Cardiovasc Ther. 2009;27(2):117-123.

127

280. Balak DM, van Doorn MB, Arbeit RD, et al. IMO-8400, a toll-like receptor 7, 8, and 9 antagonist, demonstrates clinical activity in a phase 2a, randomized, placebo-controlled trial in patients with moderate-to-severe plaque psoriasis. Clin Immunol. 2017;174:63-72. 281. Li M, Wang ZN, Yang LF, et al. TLR4 antagonist suppresses airway remodeling in asthma by inhibiting the T-helper 2 response. Exp Ther Med. 2017;14(4):2911-2916. 282. Beutner KR, Geisse JK, Helman D, Fox TL, Ginkel A, Owens ML. Therapeutic response of basal cell carcinoma to the immune response modifier imiquimod 5% cream. J Am Acad Dermatol. 1999;41(6):1002-1007. 283. Amos SM, Pegram HJ, Westwood JA, et al. Adoptive immunotherapy combined with intratumoral TLR agonist delivery eradicates established melanoma in mice. Cancer Immunol Immunother. 2011;60(5):671-683. 284. Kanzler H, Barrat FJ, Hessel EM, Coffman RL. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med. 2007;13(5):552-559. 285. Adams S. Toll-like receptor agonists in cancer therapy. Immunotherapy. 2009;1(6):949- 964. 286. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11(5):373-384. 287. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol. 2003;21:335-376. 288. Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol. 2014;5:461. 289. Cheever. Twelve immunotherapy drugs that could cure cancers. Immunol Rev. 2008(222):357-368. 290. Caruso R, Warner N, Inohara N, Nunez G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity. 2014;41(6):898-908. 291. Hsu LC, Ali SR, McGillivray S, et al. A NOD2-NALP1 complex mediates caspase-1- dependent IL-1beta secretion in response to Bacillus anthracis infection and muramyl dipeptide. Proc Natl Acad Sci U S A. 2008;105(22):7803-7808. 292. Nakamura N, Lill JR, Phung Q, et al. Endosomes are specialized platforms for bacterial sensing and NOD2 signalling. Nature. 2014;509(7499):240-244. 293. Grimes CL, Ariyananda Lde Z, Melnyk JE, O'Shea EK. The innate immune protein Nod2 binds directly to MDP, a bacterial cell wall fragment. J Am Chem Soc. 2012;134(33):13535- 13537. 294. Maekawa S, Ohto U, Shibata T, Miyake K, Shimizu T. Crystal structure of NOD2 and its implications in human disease. Nat Commun. 2016;7:11813. 295. Park JH, Kim YG, McDonald C, et al. RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs. J Immunol. 2007;178(4):2380-2386. 296. Kobayashi K, Inohara N, Hernandez LD, et al. RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature. 2002;416(6877):194-199. 297. Inohara N, Nunez G. The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene. 2001;20(44):6473-6481. 298. Bertrand MJ, Doiron K, Labbe K, Korneluk RG, Barker PA, Saleh M. Cellular inhibitors of apoptosis cIAP1 and cIAP2 are required for innate immunity signaling by the pattern recognition receptors NOD1 and NOD2. Immunity. 2009;30(6):789-801. 299. Hasegawa M, Fujimoto Y, Lucas PC, et al. A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation. EMBO J. 2008;27(2):373-383.

128

300. Yang Y, Yin C, Pandey A, Abbott D, Sassetti C, Kelliher MA. NOD2 pathway activation by MDP or Mycobacterium tuberculosis infection involves the stable polyubiquitination of Rip2. J Biol Chem. 2007;282(50):36223-36229. 301. Damgaard RB, Nachbur U, Yabal M, et al. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol Cell. 2012;46(6):746-758. 302. Kirisako T, Kamei K, Murata S, et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 2006;25(20):4877-4887. 303. Tokunaga F, Nakagawa T, Nakahara M, et al. SHARPIN is a component of the NF-kappaB- activating linear ubiquitin chain assembly complex. Nature. 2011;471(7340):633-636. 304. Ferwerda G, Kramer M, de Jong D, et al. Engagement of NOD2 has a dual effect on proIL- 1beta mRNA transcription and secretion of bioactive IL-1beta. Eur J Immunol. 2008;38(1):184- 191. 305. Chavarria-Smith J, Vance RE. The NLRP1 inflammasomes. Immunol Rev. 2015;265(1):22- 34. 306. Meyers PA. Muramyl tripeptide (mifamurtide) for the treatment of osteosarcoma. Expert Rev Anticancer Ther. 2009;9(8):1035-1049. 307. Ando K, Mori K, Corradini N, Redini F, Heymann D. Mifamurtide for the treatment of nonmetastatic osteosarcoma. Expert Opin Pharmacother. 2011;12(2):285-292. 308. Nardin A, Lefebvre ML, Labroquere K, Faure O, Abastado JP. Liposomal muramyl tripeptide phosphatidylethanolamine: Targeting and activating macrophages for adjuvant treatment of osteosarcoma. Curr Cancer Drug Targets. 2006;6(2):123-133. 309. Luetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment - where do we stand? A state of the art review. Cancer Treat Rev. 2014;40(4):523-532. 310. Hagemann T, Wilson J, Burke F, et al. Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 2006;176(8):5023-5032. 311. Lee CH, Espinosa I, Vrijaldenhoven S, et al. Prognostic significance of macrophage infiltration in leiomyosarcomas. Clin Cancer Res. 2008;14(5):1423-1430. 312. Lissbrant IF, Stattin P, Wikstrom P, Damber JE, Egevad L, Bergh A. Tumor associated macrophages in human prostate cancer: relation to clinicopathological variables and survival. Int J Oncol. 2000;17(3):445-451. 313. van Dongen M, Savage ND, Jordanova ES, et al. Anti-inflammatory M2 type macrophages characterize metastasized and tyrosine kinase inhibitor-treated gastrointestinal stromal tumors. Int J Cancer. 2010;127(4):899-909. 314. Buddingh EP, Kuijjer ML, Duim RA, et al. Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clin Cancer Res. 2011;17(8):2110-2119. 315. Kleinerman ES, Raymond AK, Bucana CD, et al. Unique histological changes in lung metastases of osteosarcoma patients following therapy with liposomal muramyl tripeptide (CGP 19835A lipid). Cancer Immunol Immunother. 1992;34(4):211-220. 316. Sica A, Larghi P, Mancino A, et al. Macrophage polarization in tumour progression. Semin Cancer Biol. 2008;18(5):349-355. 317. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell. 2010;141(1):39-51. 318. Buteyn NJ, Fatehchand K, Santhanam R, et al. Anti-leukemic effects of all-trans retinoic acid in combination with Daratumumab in acute myeloid leukemia. Int Immunol. 2018;30(8):375-383.

129

319. Salih HR, Antropius H, Gieseke F, et al. Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood. 2003;102(4):1389-1396. 320. Tajima F, Kawatani T, Endo A, Kawasaki H. Natural killer cell activity and cytokine production as prognostic factors in adult acute leukemia. Leukemia. 1996;10(3):478-482. 321. Tratkiewicz JA, Szer J. Loss of natural killer activity as an indicator of relapse in acute leukaemia. Clin Exp Immunol. 1990;80(2):241-246. 322. Le Dieu R, Taussig DC, Ramsay AG, et al. Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts. Blood. 2009;114(18):3909-3916. 323. Butchar JP, Parsa KV, Marsh CB, Tridandapani S. IFNgamma enhances IL-23 production during Francisella infection of human monocytes. FEBS Lett. 2008;582(7):1044-1048. 324. Zorko NA, Bernot KM, Whitman SP, et al. Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias. Blood. 2012;120(5):1130-1136. 325. Mundy-Bosse BL, Scoville SD, Chen L, et al. MicroRNA-29b mediates altered innate immune development in acute leukemia. J Clin Invest. 2016;126(12):4404-4416. 326. Biteau K, Guiho R, Chatelais M, et al. L-MTP-PE and zoledronic acid combination in osteosarcoma: preclinical evidence of positive therapeutic combination for clinical transfer. Am J Cancer Res. 2016;6(3):677-689. 327. Redini F, Biteau K, Taurelle J, et al. Preclinical evidence of positive effect of l-MTP-PE alone or combined with zoledronic acid in osteosarcoma. Journal of Clinical Oncology. 2014;32(15_suppl):10048-10048. 328. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature. 2001;411(6837):603-606. 329. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature. 2001;411(6837):599-603. 330. Kobayashi KS, Chamaillard M, Ogura Y, et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science. 2005;307(5710):731-734. 331. Totemeyer S, Sheppard M, Lloyd A, et al. IFN-gamma enhances production of nitric oxide from macrophages via a mechanism that depends on nucleotide oligomerization domain- 2. J Immunol. 2006;176(8):4804-4810. 332. Marina-Garcia N, Franchi L, Kim YG, et al. Clathrin- and dynamin-dependent endocytic pathway regulates muramyl dipeptide internalization and NOD2 activation. J Immunol. 2009;182(7):4321-4327. 333. Tanabe T, Chamaillard M, Ogura Y, et al. Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition. EMBO J. 2004;23(7):1587-1597. 334. Ogura Y, Inohara N, Benito A, Chen FF, Yamaoka S, Nunez G. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J Biol Chem. 2001;276(7):4812-4818. 335. Heinzelmann M, Mercer-Jones MA, Gardner SA, Wilson MA, Polk HC. Bacterial cell wall products increase monocyte HLA-DR and ICAM-1 without affecting lymphocyte CD18 expression. Cell Immunol. 1997;176(2):127-134. 336. Tewari M, Quan LT, O'Rourke K, et al. Yama/CPP32 beta, a mammalian homolog of CED- 3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995;81(5):801-809.

130

337. Lee SI, Kang SK, Jung HJ, Chun YH, Kwon YD, Kim EC. Muramyl dipeptide activates human beta defensin 2 and pro-inflammatory mediators through Toll-like receptors and NLRP3 inflammasomes in human dental pulp cells. Clin Oral Investig. 2015;19(6):1419-1428. 338. Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, Thornberry NA. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem. 1998;273(49):32608-32613. 339. Rabuffetti M, Sciorati C, Tarozzo G, Clementi E, Manfredi AA, Beltramo M. Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. J Neurosci. 2000;20(12):4398-4404. 340. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013;13(6):397-411. 341. Wagner RN, Proell M, Kufer TA, Schwarzenbacher R. Evaluation of Nod-like receptor (NLR) effector domain interactions. PLoS One. 2009;4(4):e4931. 342. Cooper MA, Fehniger TA, Ponnappan A, Mehta V, Wewers MD, Caligiuri MA. Interleukin- 1beta costimulates interferon-gamma production by human natural killer cells. Eur J Immunol. 2001;31(3):792-801. 343. Tripp CS, Wolf SF, Unanue ER. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc Natl Acad Sci U S A. 1993;90(8):3725-3729. 344. Ostensen ME, Thiele DL, Lipsky PE. Tumor necrosis factor-alpha enhances cytolytic activity of human natural killer cells. J Immunol. 1987;138(12):4185-4191. 345. Khaznadar Z, Boissel N, Agaugue S, et al. Defective NK Cells in Acute Myeloid Leukemia Patients at Diagnosis Are Associated with Blast Transcriptional Signatures of Immune Evasion. J Immunol. 2015;195(6):2580-2590. 346. Fauriat C, Just-Landi S, Mallet F, et al. Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction. Blood. 2007;109(1):323-330. 347. Pizzolo G, Trentin L, Vinante F, et al. Natural killer cell function and lymphoid subpopulations in acute non-lymphoblastic leukaemia in complete remission. Br J Cancer. 1988;58(3):368-372. 348. Lowdell MW, Craston R, Samuel D, et al. Evidence that continued remission in patients treated for acute leukaemia is dependent upon autologous natural killer cells. Br J Haematol. 2002;117(4):821-827. 349. Chretien AS, Fauriat C, Orlanducci F, et al. Natural Killer Defective Maturation Is Associated with Adverse Clinical Outcome in Patients with Acute Myeloid Leukemia. Front Immunol. 2017;8:573. 350. Sriskandan K, Garner P, Watkinson J, et al. A toxicity study of recombinant interferon- gamma given by intravenous infusion to patients with advanced cancer. Cancer Chemother Pharmacol. 1986;18(1):63-68. 351. Thompson JA, Cox WW, Lindgren CG, et al. Subcutaneous recombinant gamma interferon in cancer patients: toxicity, pharmacokinetics, and immunomodulatory effects. Cancer Immunol Immunother. 1987;25(1):47-53.

131

352. Melenhorst JJ, Tian X, Xu D, et al. Cytopenia and leukocyte recovery shape cytokine fluctuations after myeloablative allogeneic hematopoietic stem cell transplantation. Haematologica. 2012;97(6):867-873. 353. Hudson JD, Shoaibi MA, Maestro R, Carnero A, Hannon GJ, Beach DH. A proinflammatory cytokine inhibits p53 tumor suppressor activity. J Exp Med. 1999;190(10):1375- 1382. 354. Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420(6917):860-867. 355. Kuper H, Adami H, Trichopoulos D. Infections as a major preventable cause of human cancer. J Intern Med. 2002;Sep;248(3):171-183. 356. Lamm DL, Blumenstein BA, Crawford ED, et al. A randomized trial of intravesical doxorubicin and immunotherapy with bacille Calmette-Guerin for transitional-cell carcinoma of the bladder. N Engl J Med. 1991;325(17):1205-1209. 357. Mosolits S, Nilsson B, Mellstedt H. Towards therapeutic vaccines for colorectal carcinoma: a review of clinical trials. Expert Rev Vaccines. 2005;4(3):329-350. 358. Gutterman JU, Cardenas JO, Blumenschein GR, et al. Chemoimmunotherapy of advanced breast cancer: prolongation of remission and survival with BCG. Br Med J. 1976;2(6046):1222-1225. 359. Powles RL, Lister TA, Crowther D, McElwain T, Alexander P, Fairley GH. Immunotherapy for acute myelogenous leukemia. Bibl Haematol. 1975(40):737-749. 360. Geisse J, Caro I, Lindholm J, Golitz L, Stampone P, Owens M. Imiquimod 5% cream for the treatment of superficial basal cell carcinoma: results from two phase III, randomized, vehicle-controlled studies. J Am Acad Dermatol. 2004;50(5):722-733. 361. Pashenkov M, Goess G, Wagner C, et al. Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma. J Clin Oncol. 2006;24(36):5716-5724. 362. Carpentier A, Laigle-Donadey F, Zohar S, et al. Phase 1 trial of a CpG oligodeoxynucleotide for patients with recurrent glioblastoma. Neuro Oncol. 2006;8(1):60-66. 363. Liu Y, Zeng G. Cancer and innate immune system interactions: translational potentials for cancer immunotherapy. J Immunother. 2012;35(4):299-308. 364. Braunstein MJ, Kucharczyk J, Adams S. Targeting Toll-Like Receptors for Cancer Therapy. Target Oncol. 2018;13(5):583-598. 365. Anderson PM, Meyers P, Kleinerman E, et al. Mifamurtide in metastatic and recurrent osteosarcoma: a patient access study with pharmacokinetic, pharmacodynamic, and safety assessments. Pediatr Blood Cancer. 2014;61(2):238-244. 366. Bravo SP, Cascajares SC, Cebas AL, Goyache-Goñi MP, Piquero JMF. Mifamurtide for the treatment of non-metastatic osteosarcoma: Evaluation of the effectiveness and safety. European Journal of Clinical Pharmacy. 2007;19:253-257. 367. Venkatakrishnan K, Kramer WG, Synold TW, Goodman DB, Sides E, 3rd, Oliva C. A pharmacokinetic, pharmacodynamic, and electrocardiographic study of liposomal mifamurtide (L-MTP-PE) in healthy adult volunteers. Eur J Clin Pharmacol. 2012;68(10):1347-1355. 368. Carlson B. Approvals, FDA actions, clinical trials. Biotechnol Healthc. 2007;4(5):14-15. 369. Chiossone L, Chaix J, Fuseri N, Roth C, Vivier E, Walzer T. Maturation of mouse NK cells is a 4-stage developmental program. Blood. 2009;113(22):5488-5496. 370. Fu B, Wang F, Sun R, Ling B, Tian Z, Wei H. CD11b and CD27 reflect distinct population and functional specialization in human natural killer cells. Immunology. 2011;133(3):350-359. 371. Guillerey C, Huntington ND, Smyth MJ. Targeting natural killer cells in cancer immunotherapy. Nat Immunol. 2016;17(9):1025-1036.

132

372. Green ML, Leisenring WM, Xie H, et al. CMV reactivation after allogeneic HCT and relapse risk: evidence for early protection in acute myeloid leukemia. Blood. 2013;122(7):1316- 1324. 373. Litjens NHR, van der Wagen L, Kuball J, Kwekkeboom J. Potential Beneficial Effects of Cytomegalovirus Infection after Transplantation. Front Immunol. 2018;9:389. 374. Teira P, Battiwalla M, Ramanathan M, et al. Early cytomegalovirus reactivation remains associated with increased transplant-related mortality in the current era: a CIBMTR analysis. Blood. 2016;127(20):2427-2438. 375. Griffin JD, Lowenberg B. Clonogenic cells in acute myeloblastic leukemia. Blood. 1986;68(6):1185-1195. 376. McCulloch EA. Stem cells in normal and leukemic hemopoiesis (Henry Stratton Lecture, 1982). Blood. 1983;62(1):1-13. 377. Ishikawa F, Yoshida S, Saito Y, et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol. 2007;25(11):1315-1321. 378. Saito Y, Kitamura H, Hijikata A, et al. Identification of therapeutic targets for quiescent, chemotherapy-resistant human leukemia stem cells. Sci Transl Med. 2010;2(17):17ra19. 379. van Rhenen A, Feller N, Kelder A, et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res. 2005;11(18):6520-6527. 380. Jordan CT, Upchurch D, Szilvassy SJ, et al. The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia. 2000;14(10):1777- 1784. 381. Zeijlemaker W, Kelder A, Oussoren-Brockhoff YJ, et al. A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia. Leukemia. 2016;30(2):439-446. 382. Laverdiere I, Boileau M, Neumann AL, et al. Leukemic stem cell signatures identify novel therapeutics targeting acute myeloid leukemia. Blood Cancer J. 2018;8(6):52. 383. Radaeva S, Wang L, Radaev S, Jeong WI, Park O, Gao B. Retinoic acid signaling sensitizes hepatic stellate cells to NK cell killing via upregulation of NK cell activating ligand RAE1. Am J Physiol Gastrointest Liver Physiol. 2007;293(4):G809-816. 384. Poggi A, Catellani S, Garuti A, Pierri I, Gobbi M, Zocchi MR. Effective in vivo induction of NKG2D ligands in acute myeloid leukaemias by all-trans-retinoic acid or sodium valproate. Leukemia. 2009;23(4):641-648. 385. Schlegel P, Ditthard K, Lang P, et al. NKG2D Signaling Leads to NK Cell Mediated Lysis of Childhood AML. J Immunol Res. 2015;2015:473175. 386. Sanchez-Martinez D, Krzywinska E, Rathore MG, et al. All-trans retinoic acid (ATRA) induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity. Int J Biochem Cell Biol. 2014;49:42-52. 387. Abb J, Abb H, Deinhardt F. Effect of retinoic acid on the spontaneous and interferon- induced activity of human natural killer cells. Int J Cancer. 1982;30(3):307-310. 388. Manna A, Akhtar S, Yi P, et al. Daratumumab Decreases Treg-Mediated Immunosuppression and Potentiates CD8+ T-Cell-Induced Killing of Chronic Lymphocytic Leukemia (CLL) Cells Ex Vivo. Blood. 2017;130:1736.

133

389. Syros_Pharmaceuticals. A Biomarker-Directed Phase 2 Trial of SY-1425 in Patients With Acute Myeloid Leukemia or Myelodysplastic Syndrome. Vol. NCT02807558. clinicaltrials.gov; 2019. 390. Cypryk W, Nyman TA, Matikainen S. From Inflammasome to Exosome-Does Extracellular Vesicle Secretion Constitute an Inflammasome-Dependent Immune Response? Front Immunol. 2018;9:2188. 391. Buschow SI, Liefhebber JM, Wubbolts R, Stoorvogel W. Exosomes contain ubiquitinated proteins. Blood Cells Mol Dis. 2005;35(3):398-403. 392. Raiborg C, Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature. 2009;458(7237):445-452. 393. Bjorklund AT, Carlsten M, Sohlberg E, et al. Complete Remission with Reduction of High- Risk Clones following Haploidentical NK-Cell Therapy against MDS and AML. Clin Cancer Res. 2018;24(8):1834-1844.

134

Appendix A: Supplementary Figures and Tables

135

Table A.1. AML patient sample characteristics. Detailed characterization of primary patient samples used in Figure 1C,D.

136

*** * * MN1 *** * *** *** *** 15 **

(log2) 5

expression mRNA

(RNA Seq V2 RSEM) V2 Seq (RNA 0

N.D.:

PML-RARA: BCR-ABL1: CBFB-MYH11:

RUNX1-RUNX1T1: Normal Karyotype:

Complex Cytogenetics:

MLL translocation poor risk: Intermediate Risk Cytogenetic Poor Risk Cytogenetic Abnormal

Figure A.1. MN1 mRNA expression versus adult AML patient cytogenetics. Data obtained from TCGA database. * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

137

*** *** PRAME *** *** *** *** 6 * ***

(log2)

mRNA expression mRNA

RSEM) V2 Seq (RNA -2

BCR-ABL1PML-RARA CBFB-MYH11 RUNX1-RUNX1T1 Normal Karyotype

Complex Cytogenetics

MLL translocation poor risk Intermediate Risk Cytogenetic Poor Risk Cytogenetic Abnormal

Figure A.2. PRAME mRNA expression versus adult AML patient cytogenetics. Data obtained from TCGA database. * denotes p≤0.05; *** p≤0.001.

138

* * ** CD38 13 * * 12

(log2) 9

expression mRNA

(RNA Seq V2 RSEM) V2 Seq (RNA 7 6

N.D.

BCR-ABL1 PML-RARA CBFB-MYH11 RUNX1-RUNX1T1 Normal Karyotype

Complex Cytogenetics MLL translocation t(9;11) MLL translocation poor risk Intermediate Risk Cytogenetic Poor Risk Cytogenetic Abnormal

Figure A.3. CD38 mRNA expression versus adult AML patient cytogenetics. Data obtained from TCGA database. * denotes p≤0.05; ** p≤0.01.

139

Patient ID Sex Karyotype Mutations

S01 F 46,XX [20]/nonclonal [1] NA

46,XX,t(12;22)(p13;q12)[1]/46,sl,del(20)(q11.2)[14]/47,sdl1,+8[1]/ nonclonal S02 F w/clonal abnormalities[1]/46,XX[3].nuc ish(ETV6x2)(5' ETV6 sep 3' NA ETV6x1)[163/211] S03 M 46,XY [20] NPM1

S04 M 47,XY,+13[cp2]/48,sl,+4,del(4)(p12p14),del(4)(p14p16)[cp8]/46,XY[10] FLT3 ITD/NPM1

S05 M 46,XY,t(6;11)(q27;q23)[19]/nonclonal[1] NA S06 M 46,XY[19]/nonclonal[1] NA S07 M 46,XY[19]/nonclonal[1] FLT3 ITD/NPM1 46,XY,t(9;22)(q34'q11.2)[18]/nonclonal w/clonal[2] S08 M NPM1 S0,XX,+6,+8,+10,+13[11]/46,XX,der(2)t(2;3)(p21'p25),der(3)t(3;11)(p21;p15) der(11)t(3;11)(p21;p15)t(2;3)(p21;p25)[2]/nonclonal w/clonal S09 F FLT3 TKD abnormalities[2]/46,XX[5] S10 M 47,XY,del(7)(q22q36),+8[4]/46,XY[1] CEBPA S11 F 46,XX[19]/nonclonal[1] NA S12 M 46,XY[18]/nonclonal[2] NA

Table A.2. Characterization of AML patient samples. Sex, karyotype, and select tested mutations for primary AML patient samples used in the MTP-PE/IFN-γ study.

140

Figure A.4. Cytokine array on AML patient samples. Primary AML samples (n=6) treated with/without MTP-PE and/or IFN-γ after 24 hours. * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

141

100

Bone Marrow Bone 20

Infiltration % Neoplastic 0

ATRA Untreated

Daratumumab

ATRA + Daratumumab

Figure A.5. Neoplastic infiltration in the bone marrow of mice treated with ATRA and/or daratumumab. Tissues extracted from mice treated in an identical fashion to those in the experiment outlined in Figure 2.10

142

A B ) ) 10 * 250

8 200

6 150

4 100

2 50

Particles/mL (e+10) Particles/mL

0 Size (nm) Exosome Mean 0     IFN- IFN- MTP-PE MTP-PE Untreated Untreated MTP-PE+IFN- MTP-PE+IFN-

Figure A.6. Exosome release induced after MTP-PE + IFN-γ treatment. Exosome isolated by nanosight from THP-1 cells after 48 hours treatment with/without MTP-PE and/or IFN-γ (n=2). A) Quantity and B) mean size of particles collected.

143

HGS STAM1 VPS4A 4 *** 4 *** *** *** *** 3 *** 3 3 2 2 2

RCN RCN RCN

1 1 1

0 0 0       UT UT UT IFN- IFN- IFN- MTP-PE MTP-PE MTP-PE

MTP-PE + IFN MTP-PE + IFN MTP-PE + IFN

VPS4B ALIX VTA1 ** 2.5 3 ** 3 ** 2.0 2 2 1.5

RCN RCN 1.0 RCN 1 1 0.5

0 0.0 0       UT UT UT IFN- IFN- IFN- MTP-PE MTP-PE MTP-PE

MTP-PE + IFN MTP-PE + IFN MTP-PE + IFN

Figure A.7. ESCRT mRNA levels after MTP-PE + IFN-γ treatment. Transcript levels of ESCRT proteins quantified by qPCR after treatment with/without MTP-PE and/or IFN-γ after 24 hours in THP-1 cells.

144

NOD2

(log2)

mRNA expression mRNA

RSEM) V2 Seq (RNA

-2

BCR-ABL1PML-RARA CBFB-MYH11 RUNX1-RUNX1T1 Normal Karyotype

Complex Cytogenetics Poor Risk Cytogenetic

MLL translocation poor risk Intermediate Risk Cytogenetic

Figure A.8. NOD2 mRNA expression versus adult AML patient cytogenetics. Data obtained from TCGA database.

145

NOD2 NOD1 * * 4 4

2 2

(log2)

mRNA expression mRNA 0 mRNA expression mRNA 0

(RNA Seq V2 RSEM) V2 Seq (RNA

-2 -2

Good Poor Good Poor

Intermediate Intermediate

TLR4 TLR2 6 ** *** 4 4

2 2

(log2)

mRNA expression mRNA

(RNA Seq V2 RSEM) V2 Seq (RNA 0

mRNA expression mRNA 0

(RNA Seq V2 RSEM) V2 Seq (RNA

-2 -2

Good Poor Good Poor

Intermediate Intermediate

Figure A.9. NOD2, NOD1, TLR2, and TLR4 mRNA expression versus adult AML patient cytogenetic risk stratification. Data obtained from TCGA database. * denotes p≤0.05; ** p≤0.01; *** p≤0.001.

146