Exploiting epigenetically mediated changes: Acute myeloid leukemia, leukemia stem cells and the bone marrow microenvironment

CHAPTER SIX

Exploiting epigenetically mediated changes: Acute myeloid leukemia, leukemia stem cells and the bone marrow microenvironment Aksinija A. Kogana,b, Rena G. Lapidusb,c, Maria R. Baerb,c, Feyruz V. Rassoola,b,*

a Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD, United States b University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD, United States c Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, United States *Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Bone marrow microenvironment (BME) 2.1 Normal hematopoietic stem cells (HSCs) 2.2 Immune cells in the BME and effects on HSCs 2.3 Leukemia stem cells (LSCs) 3. Epigenetic deregulation in AML 3.1 DNA modifications 3.2 Histone modifications 3.3 Other epigenetic modifiers 4. Dysregulation of the epigenetic microenvironment 4.1 Epigenetic dysregulation 4.2 Dysregulation of specific microenvironmental factors 4.3 Immune microenvironment and effects on LSCs 5. Targeted epigenetic therapy 5.1 Targeting DNA methylation 5.2 Targeting histone modification 5.3 Targeting microRNA 5.4 Targeting immune factors and the microenvironment 6. Conclusions and future directions References

Advances in Cancer Research, Volume 141 ISSN 0065-230X https://doi.org/10.1016/bs.acr.2018.12.005

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Abstract Acute myeloid leukemia (AML) derives from the clonal expansion of immature myeloid cells in the bone marrow, and results in the disruption of normal hematopoiesis and subsequent bone marrow failure. The bone marrow microenvironment (BME) and its immune and other supporting cells are regarded to facilitate the survival, differentiation and proliferation of leukemia stem cells (LSCs), which enables AML cells to persist and expand despite treatment. Recent studies have identified epigenetic modifications among AML cells and BME constituents in AML, and have shown that epigenetic therapy can potentially reprogram these alterations. In this review, we summarize the interactions between the BME and LSCs, and discuss changes in how the BME and immune cells interact with AML cells. After describing the epigenetic modifications seen across chromatin, DNA, the BME, and the immune microenvironment, we explore how demethylating agents may reprogram these pathological interactions, and potentially re-sensitize AML cells to treatment.

1. Introduction AML (Table 1) is a genetically heterogeneous disease characterized by malignant clonal proliferation of immature myeloid cells in the bone marrow, leading to disruption of normal hematopoiesis and bone marrow failure (Conway O’Brien, Prideaux, & Chevassut, 2014; Plass, Oakes, Blum, & Marcucci, 2008). Cytogenetic and molecular analysis has defined genetic alterations that contribute to the initiation and maintenance of this disease (Plass et al., 2008). AML is currently classified into distinct groups with defined prognosis based on the presence or absence of these specific recurrent cytogenetic and molecular abnormalities (Kumar, 2011) (Table 2). Specific recurrent chromosome abnormalities can be identified by cytogenetic analysis in approximately 60% of AML cases. This cytogenetic information is the single most important tool to classify AML into three prognostic categories at diagnosis (Table 2) (De Kouchkovsky & AbdulHay, 2016). Cytogenetic abnormalities that confer favorable risk include inv(16), which results in a fusion between the core binding factor beta (CBFB) and the myosin heavy chain 11 (MYH11) genes on the p and q arms of chromosome 16, respectively, and the t(8;21) translocation, which fuses the RUNX1 (formerly AML1) gene on chromosome 21 and the RUNX1T1 (formerly ETO) gene on chromosome 8 (De Kouchkovsky & Abdul-Hay, 2016). In contrast, cytogenetic abnormalities that confer a poor prognosis include monosomy 5, monosomy 7, translocations of chromosome 11q23 that involve juxtaposition of the KMT2A (formerly MLL) gene on

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Table 1 Relevant acronyms and definitions. Acronym Definition

ABL

Tyrosine-protein kinase

AML

Acute myeloid leukemia

AML1

Acute myeloid leukemia 1 protein (Runx1/CBFA)

APAF1

Apoptotic protease activating factor 1

APL

Acute promyelocytic leukemia

Ara-C

Cytarabine

BME

Bone marrow microenvironment

BMI1

B lymphoma Mo-MLV insertion region 1 homolog

CBFB

Core-binding factor subunit beta (Runx2)

CBX7

Chromobox protein homolog 7

CDX2

Caudal type homeobox 2 protein coding gene

CEBPA

CCAAT enhancer-binding protein alpha

c-KIT

Mast/stem cell growth factor

CpG

Cytosine and guanine only separated by one phosphate in stretches of DNA

CSC

Cancer stem cell

CTLA-4

Cytotoxic T-lymphocyte associated protein 4

CXCL12

C-X-C motif chemokine 12

CXCR4

Chemokine receptor type 4

DC

Dendritic cell

DNMT

DNA methyltransferase

DNMTi

DNA methyltransferase inhibitor

ECM

Extracellular matrix

ERK

Extracellular regulated kinase

ETO

Runx1 translocation partner 1

EZH2

Enhancer of Zeste 2 polycomb repressive complex 2 subunit

FLT3-ITD FMS-like tyrosine kinase 3 internal tandem duplication GATA6

GATA binding protein 6

G-CSF

Granulocyte colony stimulating factor Continued

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Table 1 Relevant acronyms and definitions.—cont’d Acronym Definition

GM-CSF

Granulocyte-macrophage colony-stimulating factor

G-MDSCs Granulocyte-myeloid derived suppressor cells H3K4me3

Histone 3 lysine 4 methylation

H3K79me2 Histone 3 lysine 79 methylation HAT

Histone acetyltransferase

HDAC

Histone deacetylase

HDACi

Histone deacetylase inhibitors

HIF-1

Hypoxia inducible factor 1

HMT

Histone methyl transferase

HOXB

Homeobox B

HSC

Hematopoietic stem cell

HSPCs

Hematopoietic stem and progenitor cells

IL-2

Interleukin-2

IL-6

Interleukin-6

IL-35

Interleukin-35

Jag1

Jagged 1—ligand for notch receptor

KDM1

Demethylase of H3K4me1cauing gene repression

LNA

Locked-nucleic-acid

LSC

Leukemic stem cell

LSD1

Lysine specific demethylase 1

LTC-IC

Long term culture-initiating cell assay

MDS

Myelodysplastic syndrome

MDSC

Myeloid derived suppressor cells

MHC1

Major histocompatibility complex 1

MLL

Mixed lineage leukemia

M-MDSCs Monocytic-myeloid derived suppressor cells MSCs

Mesenchymal stem cells

MYC

Myc—proto-oncogene

MYH11

Myosin heavy chain 11

Epigenetics, AML LSCs and microenvironment

Table 1 Relevant acronyms and definitions.—cont’d Acronym Definition

NC

Normal cytogenetics

NK

Natural killer cell

NLK

Nemo like kinase—Serine/threonine-protein kinase

NPM1

Nucleophosmin 1

NY-ESO-1 Human tumor antigen of the cancer/testis family PcG

Polycomb group protein

PD-1

Programmed death-1

PD-L1

Programmed death-ligand 1

PML

Promyelocytic leukemia protein

PRC1

Polycomb repressive complex 1

PRC2

Polycomb repressive complex 2

RAR alpha Retinoic acid receptor alpha Runx2

Runt related transcription factor 2

T-ALL

T-cell acute lymphoblastic leukemia

TCR

T-cell receptor

TIM-3

T cell immunoglobulin mucin-3

TLR

Toll-like receptor

TNF-alpha Tumor necrosis factor-alpha TNFR

Tumor necrosis factor receptor

TP53INP1 Tumor protein P53 inducible nuclear protein 1 T-regs

Regulatory T cell

Sall4

Spalt like transcription factor 4

SCF

Stem cell factor

STAT1

Signal transducer and activator of transcription 1

Suz12

Polycomb Repressive Complex 2 Subunit

VCAM-1

Vascular cell adhesion molecule-1

VEGF

Vascular endothelial growth factor

VLA-4

Very late antigen-4

ZEB1

Zinc finger E-Bix binding homeobox 1

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Table 2 AML classification based on cytogenetics and molecular markers. Abnormality Risk

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

Favorable

inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 NPM1 mutation CEBPA mutation t(9;11)(p21.3;q23.3); MLLT3-KMT2A

Intermediate

Normal karyotype Cytogenetic abnormalities not classified as favorable or adverse t(6;9)(p23;q34.1); DEK-NUP214

Adverse

t(v;11q23.3); KMT2A rearrangement t(9;22)(q34.1;q11.2); BCR-ABL1 inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2,MECOM (EVI1)
5 or del(5q);
7;
17/abn(17p) Monosomy 5 Monosomy 7 Complex karyotype FLT3-ITD RUNX1 mutation ASXL1 mutation TP53 mutation

chromosome 11 with multiple gene partners, and complex karyotypes, among others. In recent years, a variety of novel molecular markers have refined risk stratification of AML, and particularly in AML with normal karyotype. For instance, FLT3 internal tandem duplications (ITD) are associated with a poor prognosis (Chu et al., 2012), while NPM1 mutations and double CEBPA mutations are associated with a favorable prognosis, particularly in the absence of FLT3-ITD (Papaemmanuil et al., 2016). Cytogenetic and molecular data guide treatment choices and are also beginning to serve as a basis for targeted therapies. Currently, favorable-risk AML patients receive consolidation chemotherapy while poor-risk AML

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patients who achieve remission undergo allogeneic hematopoietic stem cell transplantation if feasible. Patients with intermediate-risk AML can be treated with consolidation chemotherapy or transplant. Additionally, targeted therapies are also being developed to treat AML. Initial FLT3, IDH1 and IDH2 inhibitors have been approved for treatment of AML, and additional inhibitors and other targeted therapies are in development (De Kouchkovsky & Abdul-Hay, 2016; Papaemmanuil et al., 2016). In addition to the recurring cytogenetic and molecular abnormalities that have been characterized in AML, recent discoveries have highlighted the major role of dysregulated epigenetic mechanisms (discussed in more detail later in the chapter) in AML pathogenesis. In contrast to genetic changes, epigenetic modifications are frequently reversible, which provides opportunities for targeted treatment using specific inhibitors. It is now evident that abnormal methylation patterns play a role in altering expression of genes crucial to leukemogenesis. The finding that AML subsets with the chromosome abnormalities t(8;21), inv(16), and t(v;11q23) each have distinct methylation signatures suggests that epigenetic changes in leukemia cells occur in a distinct manner in distinct AML subsets (Conway O’Brien et al., 2014; Estey, 2013b). DNA demethylating agents (discussed in more detail later in this chapter) are now utilized as a therapeutic option for older patients with AML who cannot tolerate intensive chemotherapy (Estey, 2013a, 2013b). Additionally, these drugs are used as second-line treatment for patients for whom chemotherapy has failed (Estey, 2013a). Unfortunately, response rates were suboptimal and these responses were not durable (Palumbo et al., 2013). Moreover, pre-clinically, combining DNA demethylating agents with other epigenetic drugs, such as histone deacetylase inhibitors (HDACi), has enhanced efficacy, but these drug combinations have not led to improved patient survival (Cameron, Bachman, Myohanen, Herman, & Baylin, 1999). Relapse of AML is attributable at least in part to persistence of disease-driving, chemo-resistant LSCs in specific niches in the BME (Carter et al., 2014; Tabe & Konopleva, 2015). The bone marrow (BM) cavities of long bones are the principal sites of postnatal hematopoiesis, which is sustained by a rare population of hematopoietic stem and progenitor cells (HSPCs) (Nombela-Arrieta et al., 2013). AML LSCs are quiescent and reside within the osteoblast-rich areas of the bone marrow (Rashidi & Uy, 2015). BM niches are presumed to be reservoirs for LSCs and have been shown to fuel the growth of leukemia cells and contribute to their therapy resistance (Rashidi & Uy, 2015; Tabe & Konopleva, 2015). Specifically, studies have shown that co-culturing AML blasts in the presence of stromal cells in a microenvironment

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containing soluble factors such as granulocyte-macrophage colonystimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha renders the blasts chemo-resistant (Bendall, Daniel, Kortlepel, & Gottlieb, 1994; Garrido, Appelbaum, Willman, & Banker, 2001). AML with FLT3-ITD has not responded optimally to FLT3 inhibitors (Kiyoi, 2015). In one study, Sexauer et al. showed that resistance to FLT3 inhibitors occurs by both direct stromal contact and stroma-derived soluble factors and can be achieved via ERK (extracellular-regulated kinase) activation (Sexauer et al., 2012; Yang, Sexauer, & Levis, 2014). They co-cultured AML blasts with bone marrow stromal cells or with cytokines and found that downstream activation of ERK resulted in resistance to FLT3 inhibitors. Therefore, targeting the stem cell-promoting components of the tumor microenvironment that act as a protective mechanism for AML cells is an attractive strategy for improving therapeutic outcomes in AML. Much less is known about the immune microenvironment within the BM niche in AML, but there is evidence that immune cells play a role in regulating HSCs (Greim et al., 2014; Schepers, Campbell, & Passegue, 2015; Tasian, Bornhauser, & Rutella, 2018). Moreover, the immune microenvironment in AML may also play a role in promoting treatment resistance and disease relapse. As evidence, post-allogeneic bone marrow transplant modulation of the immune system can cure patients, even those with high-risk disease (Bouchlaka, Redelman, & Murphy, 2010). These sorts of studies have encouraged many groups to start exploring the utility of novel immune therapies in AML (Lamble & Lind, 2018). Regulation of LSCs by the immune microenvironment and the role of immune therapy in AML will be discussed in more detail later in this chapter. Recently, Tsai et al. showed that demethylating agents at low concentrations can reprogram aspects of the AML epigenome, transcriptionally altering the expression of key genes in multiple cancer-related signaling pathways (Tsai et al., 2012). These epigenetic alterations may affect LSCs and the immune microenvironment in the BM (Bakker, Qattan, Mutti, Demonacos, & Krstic-Demonacos, 2016; Korn & Mendez-Ferrer, 2017; Tasian et al., 2018; Tsai et al., 2012). We will review the relationship between the BME and LSCs, and how specific epigenetic alterations interact dynamically with the microenvironment, the immune microenvironment and genes encoding surface receptors on AML cells. We will also discuss how epigenetic therapy can alter these interactions, and how changes in the microenvironment may re-sensitize AML cells to standard therapies and to novel therapies that target LSCs.

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2. Bone marrow microenvironment (BME) 2.1 Normal hematopoietic stem cells (HSCs) The association between the stem cell and its surrounding cells determines stem cell behavior and fate (Gattazzo, Urciuolo, & Bonaldo, 2014; MendezFerrer et al., 2010; Morrison & Spradling, 2008; Rashidi & Uy, 2015). The BM niche maintains normal hematopoietic stem cells in a mostly quiescent state by providing signals that inhibit cell proliferation (Chotinantakul & Leeanansaksiri, 2012; Li, 2011; Mendelson & Frenette, 2014). When a stimulating signal is received, stem cells become activated to proliferate (Tabe & Konopleva, 2015). The two distinct niches within the BM are the endosteal and vascular niches. These niches are closely related to vascular structures, arterioles and sinusoids, respectively, and work in concert (Tabe & Konopleva, 2015). The surface of the endosteum is lined by osteoblasts and osteoclasts. Osteoblasts, progenitor bone-forming cells derived from pluripotent mesenchymal stem cells (MSCs), and bone-resorbing osteoclasts work in tandem in the process of osteogenesis. The endosteal niche is heavily regulated by both osteoclasts and osteoblasts, both of which are involved in the formation and maintenance of the niche (Chen et al., 2016). Extracellular matrix (ECM) proteins, osteopontin and N-cadherin, mediate HSC localization (Tabe & Konopleva, 2015). Expansion of the HSC pool is stimulated by expression of jagged-1 (Jag-1) by osteoblasts and binding to its receptor, Notch, on HSCs (Frisch, Porter, & Calvi, 2008; Koch, Lehal, & Radtke, 2013; Lampreia, Carmelo, & Anjos-Afonso, 2017; Tabe & Konopleva, 2015). Interaction of angiopoietin-1 produced by osteoblasts with its receptor, Tie-2, on HSCs causes activation of β1-integrin and N-cadherin, resulting in enhanced adhesion between the niche cells and the HSCs, which contributes to the maintenance of stem cell quiescence (Tabe & Konopleva, 2015). Regulatory T cells (Tregs) and other cell types such as macrophages and megakaryocytes also contribute to the regulation of HSC quiescence (Riether, Schurch, & Ochsenbein, 2015). The C-X-C motif chemokine 12 (CXCL12) produced by osteoblasts is the major chemoattractant for HSCs. It plays a critical role in various cellular mechanisms including tissue homeostasis, inflammatory response, and tumor progression and metastasis. The perivascular niche is an important area where HSCs reside. Specific niche cells express multiple soluble and membrane-bound factors that

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regulate HSC self-renewal and retention, including CXCL12-abundant reticular (CAR) cells and MSCs that express nestin and leptin receptors (Tabe & Konopleva, 2015). CXCL12 (previously known as SDF1) attracts HSCs expressing its cognate receptor CXCR4 (Meads, Hazlehurst, & Dalton, 2008). CXCL12–CXCR4 signaling is involved in homing and adhesion of HSCs to BM and plays an essential role in maintaining the HSC pool (Meads et al., 2008; Rashidi & Uy, 2015; Tabe & Konopleva, 2015). It has been shown that when CXCR4 is depleted, or CXCL12 is downregulated, the HSC population decreases due to mobilization of HSCs into the peripheral blood (Mendelson & Frenette, 2014). E-selectin is a cell adhesion molecule that mediates the tethering of leukocytes to vascular endothelial cells. Genetic deletion of E-selectin has been shown to enhance HSC quiescence and self-renewal potential and improve HSC survival following treatment with chemotherapy or radiation (Winkler et al., 2012). Very late antigen-4 (VLA-4) mediates retention of HSCs within the marrow through binding of vascular cell adhesion molecule-1 (VCAM-1) and binding to fibronectin (Rashidi & Uy, 2015). These are the three main known molecular mechanisms of HSC retention in marrow (and homing/mobilization).

2.2 Immune cells in the BME and effects on HSCs The BME also serves as a lymphoid organ and reservoir for various mature immune cells, including T and B cells, plasma cells, dendritic cells (DCs), neutrophils and macrophages (Mercier, Ragu, & Scadden, 2011). These immune cells are involved in the regulation of HSC homeostasis and hematopoiesis (Mercier et al., 2011). Lymphocytes constitute an important fraction of total BM mononuclear cells, and are distributed throughout the BM parenchyma, including mature CD3+ and CD4+ T cells that play important roles in hematopoiesis and engraftment of BM. CD4+ T cells also play a role in maintaining HSC function (Zhao et al., 2012). Tregs, formerly known as suppressor T cells, represent one-third of all CD4+ T cells in the BM and suppress colony formation and myeloid differentiation of HSCs (Duggleby, Danby, Madrigal, & Saudemont, 2018; Riether, Schurch, & Ochsenbein, 2015). Additionally, imaging studies show that Tregs colocalize with HSCs in the endosteum (Zheng, Song, & Zhang, 2011). This colocalization protects HSCs from being attacked by the immune system through secretion of IL-10 by the associated Tregs (Zheng et al., 2011). Current thoughts are that Tregs provide a niche in the BM where HSCs are protected from immune destruction (Fujisaki et al., 2011; Zhao et al., 2012).

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Additionally, mononuclear phagocytes in the BM niche contribute to the regulation of HSCs in the BM. Depletion of mononuclear phagocytes using clodronate-containing liposomes increased the number of circulating HSCs (Chow et al., 2011). CD169+ macrophages in the BM secrete soluble factors that stimulate nestin+ MSCs to express HSC retention factors, including CXCL12, angiopoietin-1 and VCAM-1 (Chow et al., 2011). Monocytes and macrophages expressing high levels of α-smooth muscle actin and cyclooxygenase 2 induce prostaglandin E2 production and upregulation of CXCL12 on nestin+ MSCs (Ludin et al., 2012). These macrophages have been also identified as central regulators of HSC egress from the BM after phagocytosis of aged neutrophils (Casanova-Acebes, A-Gonza´lez, Weiss, & Hidalgo, 2014).

2.3 Leukemia stem cells (LSCs) AML is theorized to be organized as a cellular hierarchy initiated and maintained by a subpopulation of LSCs ( Jung, Dai, Gentles, Majeti, & Feinberg, 2015). Expansion of LSCs is associated with impairment of normal hematopoiesis (Tabe & Konopleva, 2015). Understanding the mechanisms by which LSCs respond to, as well as influence, the microenvironment is crucial to find better treatments for AML. Most AML LSCs are quiescent and reside within the osteoblast-rich areas of the BM (Liesveld, 2012; Rashidi & Uy, 2015). The interaction between LSCs and the BM niche is recognized as the major cause of AML relapse (Fig. 1). LSCs interact with their environment via various soluble factors and membrane receptors (Civini et al., 2013). Overexpression of the oxygen-regulated component of hypoxia-inducible factor (HIF)-1α has been demonstrated in leukemia cells in BM specimens (Dunphy, O’Malley, Perkins, & Chang, 2007). HIF-1α is involved in upregulation of the growth factor VEGF and stimulation of angiogenesis. The microvasculature is an active component of the BM microenvironment, supplying oxygen and nutrients. VEGF secreted by leukemia cells activates receptors on both leukemia and endothelial cells, contributing to the growth of leukemia cells (Ferrara, Gerber, & LeCouter, 2003; Tabe & Konopleva, 2015). HIF-1α has also been shown to regulate CXCL12 gene expression in endothelial cells, increasing migration and homing of LSCs to ischemic tissue, and hypoxia upregulates CXCR4 expression in AML cells (Tabe & Konopleva, 2015). Ineffective elimination of LSCs during treatment is considered a major cause of AML relapse. Treatments that selectively eliminate LSCs should

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Fig. 1 Schematic representation of the crosstalk between leukemia stem cells (LSCs) (A), immune system, and bone marrow mesenchymal stromal cells (BM-MSCs) (B). Growth factors and cytokines released by BM-MSCs contribute to chemoresistance in AML. Presence of CXCL8 (C), known to bind to G protein-coupled receptor, CXCR2 [D; G-protein (I)], and CXCL2 (E) to CXCR4 (F), has been associated with increased leukemia cell survival. Other LSC survival signal pathways are activated via binding of VCAM-1 (G) to VLA-4 (H). AML cells directly interact with NK cells via the Fas receptor (K), and T-cells via PD-1/PD-L1(J), the MHC1/TCR complex (L) and CTLA-4 (M)/CD86 (N) interactions to evade the immune system.

prevent AML relapse (Peng & Liu, 2015). Based on these considerations, it would be of interest to identify “druggable” targets that are critical for LSCs and LSC-promoting microenvironments, such as HIF-1α (Peng & Liu, 2015; Tabe & Konopleva, 2015). HIF-1α is involved in tumor response to changes in oxygen level and leads to downstream activation of biological processes involved in migration, proliferation and angiogenesis (Hatfield, Bedringsaas, Ryningen, Gjertsen, & Bruserud, 2010; Masoud & Li, 2015). This pathway has been associated with poor prognosis and resistance to therapy, making it an important target for novel therapeutic design (Masoud & Li, 2015). A HIF-1α inhibitor decreased expression of VEGF, which is upregulated by hypoxia, and led to anti-tumor activity (Koh et al., 2008). Together, cancer cells and host cells form a microenvironment that enables leukemia initiation and progression, and this codependence suggests that host cells could also be targeted for cancer therapy (Peng & Liu, 2015).

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3. Epigenetic deregulation in AML The term epigenetics refers to changes in gene expression that are inheritable by cell division but not caused by changes in the DNA sequence itself ( Jones & Baylin, 2007; Plass et al., 2008; Wouters & Delwel, 2016). Epigenetic regulation contributes significantly to the fidelity of biological control, and dysregulation of the epigenome has been shown to be a common feature in AML ( Jones & Baylin, 2007; Stein et al., 2016). The two main mechanisms of epigenetic regulation in the cell are DNA methylation and hydroxymethylation and posttranslational histone modifications (Fig. 2) (Conway O’Brien et al., 2014). There is also growing evidence that microRNAs are another dimension to epigenetic control of hematopoiesis and leukemogenesis contributing to the epigenetic landscape by inducing heritable changes without changing the DNA sequence (described in more detail below) (Kelly, De Carvalho, & Jones, 2010; Stein et al., 2016, 2010).

3.1 DNA modifications Cytosines in the CpG motif are methylated by enzymes called DNA methyltransferases (DNMTs). DNMT1 is the most abundant DNMT in

Fig. 2 Fundamental mechanisms of epigenetic regulation. (1) DNA methylation by the addition of a methyl group (A) to cytosine (B) at CpG sites. (2) Histone modifications via the binding of enzymes and proteins (C) to histone tails (D). (3) RNA based mechanisms modulate gene expression via non-coding RNA (E).

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mammalian cells and is considered to be the key maintenance methyltransferase in mammals. In contrast, DNMT3a and DNMT3b are responsible for de novo DNA methylation patterns, which are then copied to daughter cells during S-phase by DNMT1 (Kelly et al., 2010). DNA methylation patterns are similar in HSCs and in non-hematopoietic tissues (Issa, Baylin, & Herman, 1997). Control of gene expression is derived through methylation of cytosine residues in areas high in CpG density, termed CpG islands (Delcuve, Rastegar, & Davie, 2009; Suva`, Riggi, & Bernstein, 2013). While non-CpG island methylation is reversible, methylation of CpG islands persists through mitosis and is only physiologically reversible in the embryo. In normal cells, methylation of CpG sites is consistent; genes that are highly expressed generally have hypomethylated CpG islands, while genes whose CpG islands are hypermethylated usually are not expressed (Issa et al., 1997). DNA methylation alterations are a common feature in hematological malignancies. Hypermethylation of cytosines in CpG islands is directly associated with silencing of tumor suppressor genes (Baylin & Jones, 2011; Wouters & Delwel, 2016). The focus of DNA methylation research, particularly as it relates to cancer, has been CpG island promoter methylation. Leukemogenesis has been associated with both hypo- and hypermethylation of CpG islands at different loci and also with global methylation changes, although the pathological mechanisms remain unclear (Baylin & Jones, 2011; Conway O’Brien et al., 2014). DNMTs also play a crucial role in embryonic cell differentiation and proliferation (Li et al., 2013). Mouse studies have shown that loss of DNMT1, 3a, or 3b will result in death at the embryonic stage, or just weeks after birth (Okano, Bell, Haber, & Li, 1999). HSCs need to be capable of both differentiation and proliferation, a balance that is of key importance to maintain, and in which DNA methylation is considered to be the key regulator (Li et al., 2013). DNMTs have been implicated in both of these processes in hematopoiesis as well as hematologic malignancies (Broske et al., 2009; Vardiman et al., 2009), and thus are likely important in LSCs. Studies have shown that ablation of DNMTs leads to upregulation of genes responsible for HSC proliferation, while genes involved in differentiation showed lower expression, compared to controls (Broske et al., 2009; Challen et al., 2011). It has been suggested that DNMT3a mutations contribute to the hypomethylation of HOXB family genes, whose subsequent upregulation leads to the proliferation of HSCs (Shen et al., 2011; Yan et al., 2011). Hypomethylation has also been shown in multiple other genes commonly associated with hematologic malignancies, including AML1, MYC, and STAT1

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(Challen et al., 2011). LSCs have a similar capacity for self-renewal and differentiation to that of HSCs, and several studies have linked defects in DNMTs to LSC expansion and leukemogenesis (Broske et al., 2009; Trowbridge et al., 2012; Trowbridge, Snow, Kim, & Orkin, 2009). Together, these findings suggest that targeting DNMTs could alter methylation patterns in LSCs leading to favorable therapeutic outcomes.

3.2 Histone modifications The amino-terminal tails of histones are subjected to a variety of posttranslational modifications (Fig. 2) (Munoz, Iliou, & Esteller, 2012). These modifications are reversible to accommodate cellular requirements for gene expression (Stein et al., 2016). Histone methyltransferases (HMTs) are responsible for methylation of histones, while acetylation is catalyzed by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs) (Munoz et al., 2012). The complex interplay between HMT, DNMTs, HATs and HDACs in epigenetic pathways contributes to aberrant gene expression in cancer cells (Munoz et al., 2012). The best studied histone modifications are methylation of lysine (K) residues, which can be either activating or repressing, and acetylation of K residues (Munoz et al., 2012). Methylation of histone 3 at lysine 4 (H3K4) is frequently associated with active promoters. LSD1/KDM1 is a histone demethylase that suppresses gene expression by converting H3K4me2 to H3K4me and unmethylated H3K4 (Rotili et al., 2014; Rotili & Mai, 2011). Histone methyltransferase and demethylase enzymes are more specific than HDACs in that they target a limited number of residues, but, like HDACs, lysine and arginine methyltransferase enzymes methylate non-histone proteins as well as histone proteins (Bannister & Kouzarides, 2011). The repression mediated by the H3K27 tri-methylation mark occurs through the actions of two multi-subunit complexes, polycomb repressive complex (PRC) 1 and 2. H3K27me3 is deposited by EZH2 and is then recognized and bound by PRC1, which can further recruit additional proteins to establish a repressed chromatin configuration (Dorafshan, Kahn, & Schwartz, 2017; Jiao & Liu, 2015). Gene promoters which are marked by PRC2 (i.e., polycomb target genes) in embryonic stem cells have recently been shown to be far more likely than other genes to become methylated in cancer (Dorafshan et al., 2017). Thus alterations in chromatin structure do not always coincide with changes in gene expression, but rather DNA methylation replacement of polycomb repressive marks acts

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to “lock in” an inactive chromatin state through a process called “epigenetic switching” (Gal-Yam et al., 2008; Kelly et al., 2010). The mechanism underlying the predisposition of polycomb targets for DNA methylation is not fully understood, but some links have recently been uncovered. CBX7, a component of the PRC1 complex, can directly interact with DNMT1 and DNMT3B at polycomb target genes (Li et al., 2010). Tumor cells have been proposed to shape their microenvironment by controlling the methylation state of tumor suppressor genes. In LSCs, aberrant methylation may cause continued transcription of pathways involved in stem cell self-renewal, and contribute to LSC self-renewing ability during tumor recapitulation and/or progression (Horton & Huntly, 2012). For example, the EZH2 gene, which encodes a histone methyl transferase methylating lysine tails at the 4 and 79 position in chromatin (H3K4 and H3K79), and functions as the enzymatic component of PRC2, is responsible for the epigenetic maintenance of genes responsible for embryonic development. When EZH2 is “knocked in” using transgenic mouse models, HSCs promote myeloid expansion, demonstrating their ability to function ontogenically and in a stem cell-specific fashion (Herrera et al., 2011). In addition, upregulation of EZH2 expression in tumors can contribute to the maintenance of a reversible and undifferentiated stem-like phenotype (Burdach et al., 2009; Chang et al., 2011). Conversely, pharmacological inhibition and downregulation of EHZ2 reduces expression of stem cell markers and inhibits cancer stem cell (CSC) self-renewal in vitro, and blocks tumor-initiating capacity in vivo (Bao et al., 2012; Crea et al., 2011; Rizzo et al., 2011; Suva et al., 2009). Similarly, BMI1, a subunit of PRC1 complex previously implicated in leukemogenesis, is upregulated by the embryonic stem cell transcription factor Sall4, through increase of H3K4me3 and H3K79me2 marks on the BMI1 promoter, and this mechanism can regulate self-renewal in normal and LSCs (Yang et al., 2007). Despite lack of specificity, targeting histone modifications has been clinically successful. For example, the HDACi valproic acid induces differentiation of leukemic blasts and decreases tumor growth in vivo (Gottlicher et al., 2001). Hopefully the development of therapeutics that target-specific histone-modifying enzymes will increase their therapeutic success while decreasing side effects due to the lack of specificity. On the other hand, targeting individual histone-modifying enzymes may decrease clinical efficacy due to compensation by other histone-modifying enzymes, potentially leading to resistance. Designing personalized cocktails of inhibitors based on an individual’s tumor epigenetic profile may help overcome the potential problems of compensation and resistance.

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3.3 Other epigenetic modifiers MicroRNAs (miRNAs) are a family of small, non-coding RNAs that regulate gene expression and play an important role in hematopoietic cell fate (He & Hannon, 2004; Li et al., 2013). They can regulate multiple target genes, and modulate the expression of oncogenes as well as tumor suppressors (Li et al., 2013). There is growing evidence that microRNAs provide another dimension to epigenetic control of hematopoiesis and leukemogenesis (Stein et al., 2010). Several miRNAs implicated in development have been shown to cooperate with polycomb group (PcG) protein complexes and DNA methylation to regulate the balance between self-renewal and differentiation of CSCs (Esquela-Kerscher & Slack, 2006; Volinia et al., 2006). Let-7 is one of the most consistently and impactfully downregulated miRNAs in different types of cancers and is frequently linked to tumor malignant progression ( Johnson et al., 2005; Viswanathan et al., 2009). In contrast to let-7, the expression of miR-200 is unaffected during transformation, but is downregulated in CSCs (Wang, Du, Piazza, & Xi, 2013). In fact, miR-200b and miR-200c overexpression, mediated by targeting different subunits of PcGs complexes, strongly inhibited the proliferation of CSCs and their ability to form tumors in vivo (Iliopoulos et al., 2010; Lo et al., 2011; Shimono et al., 2009). MiR-200c repressed the expression of BMI1, while loss of miR-200b increased Suz12 expression and H3K27 methylation (Lo et al., 2011; Shimono et al., 2009). In addition, miR-200c levels are regulated by a complicated loop comprised of BMI1 and ZEB1 (Wellner et al., 2009). Thus miR-200 family members play an important role in the regulation of CSC formation and function, implicating PcG complexes in this process. In addition, miR-34a modulates CSC function in tumor growth and metastasis. Expression of miR-34a is regulated by p53, and miR-34a induces apoptosis, cell cycle arrest or senescence when introduced into cancer cells (Bommer et al., 2007; Chang et al., 2007; He et al., 2007; Raver-Shapira et al., 2007; Tarasov et al., 2007). miR-130b, which targets TP53INP1, and miR-181, which targets regulators of differentiation such as CDX2, GATA6 and NLK, are over-expressed in LSCs, as well as CSCs in different tumor types, resulting in enhanced self-renewal and tumorigenicity in vivo (Cairo et al., 2010; Garofalo & Croce, 2015; Ji et al., 2009; Ma et al., 2010; Munoz et al., 2012; Pan, Meng, Zhang, Han, & Zhou, 2014). miRNAs are interesting therapeutic targets because one miRNA alters several pathways, and restoring the function of a misregulated miRNA may be more effective than identifying different misregulated pathways

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and proteins and targeting them separately (Kelly et al., 2010). However more research is needed to better understand miRNA profiles in healthy and diseased tissues in order to develop better therapeutic strategies.

4. Dysregulation of the epigenetic microenvironment 4.1 Epigenetic dysregulation Epigenetic landscapes are deregulated in hematologic malignancies and these modifications contribute to hematopoietic transformation within the BME (Woods & Levine, 2015). Changes in the epigenome contribute to chromosomal instability, provide survival advantages to LSCs, and contribute to tumor initiation and progression (Munoz et al., 2012; Vincent & Van Seuningen, 2012). Alterations in DNA methylation, histone modifications, and miRNAs are mechanisms that have been shown to directly contribute to tumorigenesis (Munoz et al., 2012). The change of epigenetic marks over time, known as epigenetic drift, is thought to be the result of accumulation of epigenetic errors and acquired somatic mutations in epigenetic regulator genes (Oshima & Iwama, 2014). DNA methylation and specific posttranslational modifications on histone tails are key mechanisms that control chromatin compacting (Vincent & Van Seuningen, 2012). miRNAs have also been shown to trigger transcriptional silencing via chromatin remodeling in human cells (Vincent & Van Seuningen, 2012). Epigenetic regulation is mediated by DNA methylation, histone modification, and non-coding RNAs (Oshima & Iwama, 2014). These epigenetic regulators play critical roles in the maintenance of self-renewing HSCs (Oshima & Iwama, 2014). Mutations in epigenetic regulatory genes have also been identified in hematological malignancies (Oshima & Iwama, 2014). It has therefore been suggested that epigenetic dysregulation can promote transformation and maintenance of LSCs (Oshima & Iwama, 2014). Epigenetic alterations play a critical role in cell differentiation and cell fate in the hierarchical organization of leukemia (Vincent & Van Seuningen, 2012). LSCs are able to turn specific markers on and off, as evidenced by the tremendous heterogeneity within AML cell populations. Epigenetic mechanisms are likely responsible for this heterogeneity. CSC markers have been shown to be mainly regulated by DNA methylation and histone modification (Vincent & Van Seuningen, 2012). The importance of DNA methylation in LSCs, specifically, is evidenced by the observation that abrogation of DNMT1, a DNA methyltransferase responsible for the maintenance of established methylation patterns, blocked leukemia development (Trowbridge et al., 2012). If aberrant methylation leads to

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activation of a self-renewal pathway, LSCs would have a survival advantage that would lead to subsequent leukemia progression. The reversible nature of the epigenetic changes that silence genes important in hematopoiesis makes them an attractive target for therapies aimed at both restoring normal BM function and inducing clinical disease response in AML.

4.2 Dysregulation of specific microenvironmental factors LSCs can evade therapeutic agents by exploiting perturbations in the tumor microenvironment. As reviewed by Konopleva and Jordan, extrinsic factors from the BME, including adhesion molecules, hypoxia-related proteins and chemokine receptors, influence the survival of LSCs. In particular, dysregulation of the microenvironment is associated with increased hypoxia areas and overexpression of HIF-1α, a key hypoxia response protein, discussed earlier in the chapter (Konopleva & Jordan, 2011). CXCL12, formerly known as SDF-1, is upregulated during hypoxia (Benito, Zeng, Konopleva, & Wilson, 2013). LSCs home to CXCL12 positive areas, directly competing for this space with normal HSCs (Galan-Diez & Kousteni, 2017). Furthermore, the cognate receptor of CXCL12, CXCR4 is also dysregulated in AML cells under hypoxic conditions, which facilitates homing of LSCs to the BM niche. Moreover, inhibition of CXCR4 decreased leukemia burden (Benito et al., 2013). A mouse model of T-cell acute lymphoblastic leukemia (T-ALL) was used to determine the mechanism underlying leukemia expansion in the BM (Gu, Masiero, & Banham, 2016). As with other leukemias, these T-ALL model cells were able to displace normal cells within the mouse BME. Additionally, there was a reduction of osteoblasts in the marrow, correlating with decreased expression of osteoblast transcription factors such as Runx2, osterix, osteocalcin, and ostegrin, as well as CXCL12 (Camacho, McClearn, Patel, & Welner, 2017). Osteoclasts, which work in tandem with osteoblasts to maintain the HSC niche, have been shown to be increased in myeloid diseases resulting in bone destruction (Qiang et al., 2008; Tabe & Konopleva, 2015). Meanwhile, stromal cells showed increased expression of IL-6, SCF, HIF-1α, VEGF, and Jag1 during cancer progression (Camacho et al., 2017). There are four types of Notch, and many cases of T-ALL harbor a gain-of-function mutation in the intracellular domain of Notch1. Notch1 was found to bind to the promoter of the CXCL12 gene and affect its expression (Wang et al., 2016). In fact, Notch1 activation suppresses osteoblast proliferation by decreasing CXCL12, resulting in inhibition of normal hematopoiesis (Calvi et al., 2003; Wang et al., 2016). The interaction of these

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pathways in the BME suggests that niche-targeted approaches could be used to restore osteoblast function and normal hematopoiesis (Wang et al., 2016). LSCs use the factors described above in different niche environments to resist chemotherapy. LSCs have lower proliferation rates than the remainder of the AML cells in the bone marrow, which may allow them to evade the effects of treatment, resulting in resistance (Vincent & Van Seuningen, 2012). Identifying the reprogramming that occurs in LSCs and understanding the differences between LSCs and HSCs will likely help to design LSCtargeted therapies to kill these difficult-to-treat cells (Vincent & Van Seuningen, 2012). New treatments will have to target both LSCs and more differentiated leukemia cells to eliminate both the bulk of leukemia cells and the potential roots of the disease (Vincent & Van Seuningen, 2012).

4.3 Immune microenvironment and effects on LSCs In addition to serving as a primary organ for hematopoiesis, the BM is an important immunological transit point (Massberg et al., 2007; Zhao et al., 2012). LSCs utilize specific mechanisms to evade immune surveillance. They secrete various co-stimulatory molecules and suppressive cytokines (Kornblau et al., 2010) that hinder immune effector cell function. At the molecular level, LSCs exhibit unique expression profiles and antigens in relation to HSCs and to their differentiated progeny (Gentles, Plevritis, Majeti, & Alizadeh, 2010; Jung et al., 2015; Korsmeyer et al., 1983). LSCs also target metabolic pathways and create an anti-apoptotic environment that facilitates their own proliferation while inhibiting immune cells (Curti et al., 2007; Milojkovic et al., 2004). Additionally, several surface proteins common to LSCs and immune cells, including T-cell immunoglobulin mucin-3 (TIM-3), are associated with LSC self-renewal (Kikushige et al., 2015). More specifically Kikushige et al. showed that TIM-3 remains absent from the surface of normal HSCs, making it an interesting therapeutic target. The BM is also an integral site for lymphocyte circulation (Massberg et al., 2007; Zhao et al., 2012). The cytokine networks in the BM are important for HSC maturation, pluripotency, and contact with immune cells (Moudra et al., 2016). Alterations of this milieu within the BM have been implicated in expansion of LSCs, as evidenced by Moudra et al. who showed elevated interleukin levels in AML patients as compared to healthy donors (Moudra et al., 2016). The BM serves as a supportive environment for growth and development of immune cells and LSCs, and it is also the principal site for crosstalk between these cells. It is therefore necessary to tease out the mechanisms involved in cell-cell interactions and soluble factors that

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are exchanged between LSCs and immune cells in the BME (Camacho et al., 2017). Trafficking of various immune cells within this microenvironment has been observed. These immune cell populations include CD3+ T cells, Tregs, B cells, natural killer cells (NKs), neutrophils, dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) (Riether, Schurch, et al., 2015). The BM not only acts as a reservoir for immune cells but also provides necessary architectural elements to support T cell maturation (Garcia-Ojeda, Dejbakhsh-Jones, Weissman, & Strober, 1998). In addition to modulating the HSC compartment, the immune cells in BM also interact with other cells residing in the niche, causing widespread dysregulation of immune networks within the BM (Zhao et al., 2012). Immune cells play an important role during hematopoiesis, maintaining HSC homeostasis by eradicating aberrant clones, protecting HSCs from apoptosis and tightly monitoring cell proliferation (Zou et al., 2004). Tregs are key mediators in these processes, with higher frequencies in BM than in spleen and lymph nodes; they have been shown to comprise as high as 30% of CD4+ T cells in the BM (Zou et al., 2004). Tregs are a dynamic class of cells that temper the immune response throughout the body and shield the stem cell compartment from autoimmunity, excessive inflammation and apoptosis (Pandiyan, Zheng, Ishihara, Reed, & Lenardo, 2007). They suppress IL-2 cytokine production and also secrete various inhibitory cytokines, such as IL-35, to regulate T cell activity and apoptosis (Collison et al., 2007; Pandiyan et al., 2007). It has therefore been suggested that LCSs hijack Tregs to use them as protection for survival (Andersen, 2014; Camacho et al., 2017; Riether, Schurch, et al., 2015). It is likely that LSCs recruit Tregs and exploit their modulatory capacities to escape detection (Ustun, Miller, Munn, Weisdorf, & Blazar, 2011). However, the mechanisms by which LSCs recruit Tregs remain to be elucidated. In leukemia, alterations in NK cells have been shown to result in the disruption of the innate immune response and allow LSCs to escape immune surveillance (Mundy-Bosse et al., 2016). With respect to signaling, cytokine stimulation and differentiation of NK cell populations have been shown to enhance NK cell response and functionality (Wagner et al., 2017). It has also been shown that LSCs employ the CD160 signaling axis, a pathway common to NK cells and CD8+ T cells, to facilitate their expansion (Liu et al., 2010). Leukemia-initiating cells have also been associated with a mast cell signature, which activates inflammation, and studies have shown that the IL-2/CD25 axis may serve as a key regulator in leukemia cell activation (Kobayashi et al., 2014). These unique cytokine and

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chemokine expression patterns alter the communication landscape between immune cells, stem cells, and stromal cells of the hematopoietic system which lead to immune evasion and survival of LSCs. Toll-like receptors (TLRs) are critical players in innate immune signaling, important for the cooperative regulation of pro-inflammatory cytokines and chemokines and their deregulation is associated with leukemogenesis (Fracchiolla, Fattizzo, & Cortelezzi, 2017). TLR dysfunction has been implicated in progression, due to aberrant activation of signal transduction of important proliferative and apoptotic pathways (Monlish, Bhatt, & Schuettpelz, 2016). The cytokine imbalances and dysregulated activation of TLRs observed in myelodysplastic syndrome (MDS) depress immune cell function, increase inflammation, and disrupt cellular communication. Collectively, these changes contribute to the formation of the LSC niche. Under normal conditions, HSCs undergo a series of transitions as they evolve into specialized cells. The immune system plays a role in regulating these transitions. Proper expansion of normal HSCs and eradication of malignant cells is carried out under the direction of immune cells (Camacho et al., 2017). In AML, loss of immune function results in the transformation of HSCs to LSCs via disruption of the balance between self-renewal and lineage commitment (Camacho et al., 2017). Given the interdependency between immune and stem cells, an investigation of immune components is required to determine the regulatory checkpoints that become aberrant during LSC transformation.

5. Targeted epigenetic therapy 5.1 Targeting DNA methylation While cancers generally exhibit global hypomethylation patterns, they usually also experience gene silencing due to the hypermethylation of a subset of gene promoters contained within CpG islands (Sharma, Kelly, & Jones, 2010). Attempts to alter these epigenetic patterns have included preclinical and clinical testing of DNA methylation inhibitors (Yang, Lay, Han, & Jones, 2010). 5-Azacytidine (AZA) is a nucleoside analog that incorporates into RNA and is subsequently transcribed into DNA. 5-Aza-2deoxycytidine (decitabine; DAC) is the deoxy derivative of 5-Aza-CR and is incorporated directly into DNA (Diesch et al., 2016). AZA and DAC sequester and inhibit methylation enzymes after their incorporation into RNA and DNA, respectively (Tsai et al., 2012). Both AZA and DAC are approved by the United States Food and Drug Administration (FDA) to treat high-risk MDS patients, and successful clinical results have

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been reported in AML (Diesch et al., 2016; Dombret et al., 2015; Issa, 2007). However, responses in AML are often not durable, and it has been suggested that LSC resistance to AZA is responsible for the rapid relapse (Sykes, Kokkaliaris, Milsom, Levine, & Majeti, 2015). Baylin et al. propose that the clinical efficacy of the DNMT is mediated through the antagonization of epigenetic changes necessary for the survival of a cancer (Baylin & Jones, 2011; Issa, 2007). At high concentrations, these agents produce direct DNA damage and cytotoxicity, but these effects may not be the primary mechanisms responsible for clinical efficacy in patients with MDS or AML. Transient, low dose, exposure of cultured and primary leukemic and epithelial tumor cells to nanomolar concentrations of epigenetic drugs produce an anti-tumor memory response and inhibit expansion of subpopulations of cancer stem-like cells, but do not cause immediate cytotoxicity (Karpf & Jones, 2002; Palii, Van Emburgh, Sankpal, Brown, & Robertson, 2008; Tsai et al., 2012). Moreover, studies of long-term culture-initiating cell (LTC-IC), which model LSCs in vitro, used low concentrations of DNA methyltransferase inhibitors (DNMTis) also to blunt the growth of AML Kasumi-1 and KG-1 cell lines without significant cytotoxicity (Tsai et al., 2012). However, treatment of these same cell lines with the cytotoxic drug cytarabine (Ara-C) fails to reduce LTC-IC, consistent with the inability of this traditional chemotherapeutic drug to target LSCs (Guzman & Allan, 2014). Low doses of epigenetic therapies can lead to re-sensitization of AML cells to cytotoxic chemotherapy (Oronsky, Oronsky, Knox, Fanger, & Scicinski, 2014; Oronsky, Oronsky, Scicinski, et al., 2014). The most rigorous laboratory measure of AML initiation capacity is ability of stem-like cells to engraft in mice ( Jordan, Guzman, & Noble, 2006). Purified CD34+ cells from the Kasumi-1 AML cell line can engraft and initiate tumors in mice. Transient, low-dose treatment with DAC inhibits colony formation of both CD34+ and CD34
Kasumi-1 cells. This suggests efficacy of these epigenetic drugs against both stem-like and non-stem-like leukemic cells. Kasumi-1 cells pre-treated with 10 nM DAC generated markedly delayed engraftment, even without additional drug treatment of the recipient mice. DAC pretreatment also significantly decreased the percentage of CD34+ stem-like Kasumi-1 cells present in the BM after intravenous injection, demonstrating inhibition by DAC of leukemia engraftment in vivo (Tsai et al., 2012). Tsai et al. also showed that low doses of demethylating agents alter the expression of genes across multiple cancer signaling pathways in stem cells, immune cells, and the microenvironment. Various components of the BME, including stromal cells and interleukins, are reprogrammed by cancer cells to mediate homing of LSCs to the BM niche and survival there, and act

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in a protective fashion to decrease drug effects and induce drug resistance (Meads et al., 2008). While it remains unclear how to target LSC specifically, such targeted treatment would likely significantly improve patient outcome and is an important goal. Development of LSC-targeted therapies for AML has therefore become a crucial component in the endeavor to improve treatment outcomes (Fig. 3) ( Jones et al., 2016). DNA demethylating agents alone are currently insufficient to treat myeloid malignancies. However since low doses of these epigenetic drugs can lead to re-sensitization of AML cells to cytotoxic chemotherapy, combination treatments with epigenetic plus traditional drugs are promising (Oronsky, Oronsky, Knox, et al., 2014; Oronsky, Oronsky, Scicinski, et al., 2014). Since cytotoxic chemotherapy kills cells by activating pro-apoptotic genes, and

Fig. 3 Pathway analysis of expression changes following decitabine (DAC) or azacitidine (AZA) treatment. Microarray analyses post DNMTi treatment show stable changes in gene expression of multiple genes, also known as transcriptional reprograming, in cancer pathways defined by Weinberg and Hanahan. Modified from Tsai, H. C., Li, H., Van Neste, L., Cai, Y., Robert, C., Rassool, F. V., et al. (2012). Transient low doses of DNAdemethylating agents exert durable antitumor effects on hematological and epithelial tumor cells. Cancer Cell, 21(3), 430–446. https://doi.org/10.1016/j.ccr.2011.12.029.

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DNA methylation of these pro-apoptotic genes can block cell death from occurring, reactivation with DNMTis of epigenetically silenced apoptotic genes can increase the efficacy of chemotherapy (Oronsky, Oronsky, Scicinski, et al., 2014). For example, APAF1 is silenced in metastatic melanoma cells and treatment with AZA restores APAF1 expression and chemosensitivity (Soengas et al., 2001). Conversely, methylation-induced silencing of DNA repair genes can be detrimental by leading to microsatellite instability, or even beneficial by preventing the repair of genes targeted by chemotherapy, causing cells to undergo apoptosis rather than repair (Lahtz & Pfeifer, 2011). Continued work is aimed at understanding how DNMT inhibitors function, and at identifying biomarkers of response. Overall, it is possible that effective therapy may require agents that target other aspects of the epigenome, and that synergize more effectively with a variety of combination treatments. For example, there are ongoing studies testing combinations of hypomethylating agents and biological agents such as retinoic acid, and promising results have also been described for the combination of DAC with IL-2 in melanoma. Reversal of DNA methylation using DNMT can also overcome chemoresistance induced by gene silencing, and suggests additional combination options (Issa, 2007; Kelly et al., 2010). Despite the clinical successes achieved with DNA methylation inhibitors, there is still much room for improvement. Currently-available DNA methylation inhibitors block DNA methylation at the enzymatic level, leading to global DNA methylation inhibition. This is therapeutically beneficial as tumor suppressor genes are hypermethylated in cancer, but global hypomethylation may lead to activation of oncogenes and/or increased genomic instability (Issa, 2007). Furthermore, DNA methylation inhibitors act during S-phase of the cell cycle, and thus they preferentially affect dividing cells. This is advantageous when treating cancers that grow rapidly, but may be less clinically useful in treating cancers that are not characterized by rapid cell cycling (Issa, 2007). Additionally, upon DNMTi withdrawal, DNA methylation returns to pre-treatment levels, demonstrating a need for continuous DNMT inhibition (Kelly et al., 2010; Sharma et al., 2010). Thus, while DNA methylation inhibitors are clinically successful, their lack of specificity, cell cycle dependency and need for continuous administration leave room for the development of other targeted therapies (Issa et al., 1997).

5.2 Targeting histone modification As mentioned above, drugs affecting abnormal histone modification are also being developed for clinical use. Histone acetylation is regulated by HATs

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and HDACs (Bose, Dai, & Grant, 2014). While MDS and AML patients respond to HDACi treatment, the response rate is relatively low compared to the response rate reported in DNMTi trials (Woods & Levine, 2015). But as with DNMTi therapy, the mechanisms of action of HDAC inhibitors are not well-delineated, which makes understanding and interpreting patient responses very difficult (Woods & Levine, 2015). HDAC inhibition increases histone acetylation levels, but this effect is not predictive of clinical response. The different HDAC inhibitors (HDACis) under investigation have widely different effects on gene expression. For instance, vorinostat increases reactive oxygen species, while panobinostat can reduce EZH2 expression levels and selectively kill AML cells (Han Li & Chen, 2015; Jing et al., 2018). HDACis can affect acetylation of non-histone proteins, as well as histones, potentially leading to more global effects (Ververis, Hiong, Karagiannis, & Licciardi, 2013). Significant effort is underway to find new molecules that are able to selectively inhibit specific HDACs and thus avoid the side effects that occur with global HDACis (Li & Seto, 2016). The development of specific HDACis, inhibitors of HDAC6 (class II) and HDAC8 (class I), combined with a better understanding of the pathophysiology of diseases associated with alterations in HDACs will allow more rational therapy and potentially reduce side effects (Li & Seto, 2016). For example, the HDACi PCI-34051 was recently shown to selectively inhibit HDAC8 and induce apoptosis specifically in T-cell lymphomas but not in other tumor or normal cells, showing that HDAC8 plays an important role in the pathophysiology of this disease and suggesting that therapy with a HDAC8-specific inhibitor may lead to reduced side effects (Balasubramanian et al., 2008). While the identification of additional specific HDACis will increase specificity and the possibility of personalized treatments, this may also potentially limit the likelihood of success in combination regimens. A great deal of effort is underway to find drugs able to revert specific histone methylation marks or to target histone methyltransferase or histone demethylases (Kelly et al., 2010). In this regard, a new class of oligoamine analogs was found that act as potent inhibitors of lysine-specific demethylase 1 (LSD1) (Huang et al., 2009). Interestingly, LSD1 can also demethylate DNMT1, resulting in destabilization and loss of global DNA methylation maintenance (Huang et al., 2009). The ability of LSD1 to affect both histone and DNA methylation makes it a promising target for epigenetic therapy. Since LSCs have been proposed to be the major cause of relapse, research

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has been focused on eradicating the LSC population in myloid neoplasms. HDACis have been shown to eradicate the LSC population in combination with Imatinib, in patients who have become resistant to imatinib alone (Zhang et al., 2010). Similar results have been seen with HDACi AR-42 in AML, where it was shown that AR-42 was capable of targeting the LSC population (Guzman et al., 2014). Further research is required in this field with specific focus on potential combination therapies that can target both the bulk disease as well as the LSCs.

5.3 Targeting microRNA Abnormally high expression of miRNAs can be targeted using recently developed locked-nucleic-acid (LNA)-modified phosphorothioate oligonucleotide technology (Gallo Cantafio et al., 2016; Lima et al., 2018). LNA-modified oligonucleotides contain an extra bridge in their chemical composition that leads to enhanced stability compared to their unmodified counterparts. These LNA-modified phosphorothioate oligonucleotides can be used to create LNA-antimiRs, which can be delivered systemically (van Rooij & Kauppinen, 2014). LNA-antimiRs may also be used to target aberrantly expressed miRNAs in other diseases. miRNAs can alter the epigenetic machinery and can also be regulated by epigenetic alterations, creating a highly controlled feedback mechanism which is a suitable target for epigenetic therapy and possibly an epigenetic drug itself (Garzon, Marcucci, & Croce, 2010; Iorio & Croce, 2017). One unique advantage of targeting miRNAs is the ability of one miRNA to regulate several target genes and multiple cellular processes (Garzon et al., 2010). Thus, if the level of one or a few miRNAs has changed in a pathological state, alterations in several different pathways may result. Rather than trying to identify and directly target the proteins in multiple pathways, it would be more effective to restore the physiological level and functions of the misregulated miRNA(s) (Iorio & Croce, 2017). This clinical potential highlights the importance of better understanding miRNA profiles in healthy and diseased tissues in order to develop better therapeutic strategies. Furthermore, multiple miRNAs that target different steps in an over-active pathway can be combined to increase efficacy and allow for customization of therapies to individual patients (Kelly et al., 2010). While the unique composition of miRNA-based therapy provides many potential benefits, additional research is necessary to determine the best method of delivery and increase miRNA stability to ensure efficacy.

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5.4 Targeting immune factors and the microenvironment Targeting microenvironmental alterations, due to their critical role in tumor development, progression, and relapse, could potentially improve outcomes in AML. One way to do this would be to stop LSCs from homing to the BM niche by targeting migration and adhesion signals (Konopleva & Jordan, 2011). Knowing the important role that CXCR4 plays in the BME, it would seem that treatments using CXCR4 agonists would also have negative effects on HSCs, but it has been shown that this is not the case (Meads et al., 2008). In fact, anti-CXCR4 treatment caused a decrease in AML cell survival, while showing no effect on normal stem cells. Other studies of CXCR4 inhibition with plerixafor combined with conventional chemotherapy in AML have shown the importance of the microenvironment’s antitumor effects both in vitro and in vivo (Cho, Kim, & Konopleva, 2017). Previous work has identified VLA-4 as a potential drug target to reduce tumor growth and inhibit drug resistance, but preclinical studies have been limited (Meads et al., 2008). Treatment strategies that involve the use of these small molecule inhibitors and antagonistic antibodies that have shown low toxicity to normal cells may facilitate elimination of minimal residual disease (Meads et al., 2008). Integrin inhibitors, such as VLA-4 antagonists, have been shown to reduce tumor burden when combined with melphalan and concomitant treatment with Ara-C restored sensitivity to Ara-C (Meads et al., 2008). Other studies have shown the potential for targeting granulocyte colony stimulating factor (G-CSF), as this cytokine rescues AML cell lines from apoptosis induced by targeting c-KIT-induced using imatinib or nilotinib (Gordon, Dias, & Williams, 2014; Jones et al., 2016). Eradicating the founding clones that cause relapse with targeted therapy may lead to resensitization of AML to other therapies (Wouters & Delwel, 2016). The combination of multiple treatment types will likely have the best potential success in the treatment of AML, given its heterogeneity even within a single patient. Recently, the inhibition of programmed death-1 (PD-1)/PD-ligand1 (PD-L1) axis, known as immune checkpoint blockade, has been used to treat cancer by activating suppressed immune cells (Ilcus et al., 2017). Notably, Kim et al. showed that treating leukemic mice with plerixafor, antiPDL1 and Ara-C chemotherapy significantly reduced Tregs and M-MDSCs, while G-MDSCs were significantly increased in the BM (Kim et al., 2017). In addition, PD-1 expression in both BM and spleen was relatively low compared to those in control subgroups. These findings

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implied that modulation of immune status by treatment with plerixafor, anti-PD-L1 and Ara-C could lead to more effective elimination of leukemic blasts through activation of CD4+ and CD8+ T cells and possibly suppression of Tregs and/or M-MDSCs in AML (Kim et al., 2017). LSCs have been shown to have increased PD-L1 expression, making them an attractive target for anti-PD-L1 therapy (Riether, Gschwend, Huguenin, Schurch, & Ochsenbein, 2015; Riether, Schurch, et al., 2015). Additional studies integrating responses of these immune cell subsets should be pursued in AML (Kim et al., 2017). Methylation-induced silencing of cancer-testis antigens, such as NY-ESO-1, can protect cancer cells from being recognized by T cells, which can be antagonized by the treatment with demethylating agents and engineered lymphocytes (Wargo et al., 2009). These data support an interaction between epigenetic states and the immune system, and provide a rationale for the combination of epigenetic therapy with immunotherapy (Fig. 4).

Fig. 4 Immune and targeted therapy approaches in AML. Targeted therapies display immunomodulatory effects by: (1) acting on the leukemia cell by epigenetically changing global gene expression levels, (2) inhibiting interactions between immune and AML cells, or (3) altering microenvironmental factors affecting leukemia cell survival. HDACi, Histone deacetylase inhibitors; DNMTi, DNA methyltransfersae inhibitor; LSD1i, Lysinespecific demethylase 1 inhibitor; BETi, Bromodomain and extra-terminal protein inhibitor; Anti CTLA-4 antibody; Anti PD-1/PD-L1 antibody.

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6. Conclusions and future directions Current studies are investigating LSC development in disease-specific BMEs, as well as the effects of targeting these disease specific features. Low-dose epigenetic therapies have the ability to reprogram multiple pathogenic signaling pathways, including in LSCs, the BME and the immune environment (Tsai et al., 2012). The recent explosion of interest in immunotherapy, especially immune checkpoint blockade, is a result of discoveries involving the fundamental ligand-receptor interactions that occur between immune and AML cells within the tumor microenvironment (Lamble & Lind, 2018). Distinct ligands expressed by cancer cells, such as PD-L1, engage with cell surface receptors on immune cells, triggering inhibitory pathways which help cancer cells evade the immune system (Dunn & Rao, 2017). Importantly, recent studies on the role of epigenetics in immune evasion have exposed a key role for epigenetic modulators in augmenting the tumor microenvironment and restoring immune recognition and immunogenicity (Chiappinelli et al., 2015; Topper et al., 2017). Epigenetic drugs such as DNMTis and HDACis can reverse immune suppression by enhancing expression of tumor-associated antigens, components of the antigen processing and presenting machinery pathways, immune checkpoint inhibitors, chemokines, and other immune-related genes (Chiappinelli et al., 2015; Topper et al., 2017). Notably, a key component of this signaling is an interferon response induced through the activation of endogenous retrovirus transcription and a cytoplasmic double-stranded RNA (dsRNA) response (Chiappinelli et al., 2015; Topper et al., 2017). This has been termed DNMTi-induced viral mimicry (Chiappinelli et al., 2015; Roulois et al., 2015). Thus, there is a scientific rationale for studies combining epigenetic targeting and immune checkpoint inhibition in AML. Moreover, these therapies may synergize with chemotherapy, and targeting specific factors in the BME may also be a promising strategy for increasing treatment efficacy. These therapeutic strategies may be particularly suited for chemotherapy-resistant AML patients. Elucidation of the precise mechanisms of interactions between AML LSCs and their surrounding niche in the BM will help develop novel therapeutic strategies that can target AML cells as well as their surrounding BME. Ultimately, cure of leukemia implies the elimination of LSCs, and a better understanding of the “immune niche” and its interactions with LSCs in the context of the BM microenvironment may help to develop specific therapies targeting leukemia at the level of the LSC.

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