Epigenetic Mechanisms in Leukemias and Lymphomas

Abstract

Although we are just beginning to understand the mechanisms that regulate the epigenome, aberrant epigenetic programming has already emerged as a hallmark of hematologic malignancies including acute myeloid leukemia (AML) and B-cell lymphomas. Although these diseases arise from the hematopoietic system, the epigenetic mechanisms that drive these malignancies are quite different. Yet, in all of these tumors, somatic mutations in transcription factors and epigenetic modifiers are the most commonly mutated set of genes and result in multilayered disruption of the epigenome. Myeloid and lymphoid neoplasms generally manifest epigenetic allele diversity, which contributes to tumor cell population fitness regardless of the underlying genetics. Epigenetic therapies are emerging as one of the most promising new approaches for these patients. However, effective targeting of the epigenome must consider the need to restore the various layers of epigenetic marks, appropriate biological end points, and specificity of therapeutic agents to truly realize the potential of this modality.

Figure 1.

Frequent mutations of epigenetic regulators in acute myeloid leukemia (AML). Upper panel illustrates epigenetic modifications on the DNA and histone layer of the epigenome. Lower panel shows gene mutations that impact DNA and/or histone modifications in at least 1% of AML cases. Ranges of reported mutation frequency are indicated in parentheses (Abbas et al. 2010; Marcucci et al. 2010, 2012; Paschka et al. 2010; Hollink et al. 2011; Shen et al. 2011; Gaidzik et al. 2012; Patel et al. 2012; Weissmann et al. 2012; Cancer Genome Atlas Research 2013; Gao et al. 2013; Metzeler et al. 2016; Papaemmanuil et al. 2016; Terada et al. 2018). The asterisk indicates frequency in adult AML. KMT, lysine methyltransferase; KDM, lysine demethylase; 2OG, 2-oxoglutarate, also called α-ketoglutarate (αKG).

Figure 2.

Epigenetic features and perturbations in acute myeloid leukemia (AML). (A) Diagram showing the structure of methylated cytosine (5mC) and oxidized mCs (5hmC, 5fC, 5caC) as well as their transcriptional association at promoters. Active DNA demethylation is indicated by black arrows below modified cytosines. 5fC and 5caC can be excised directly by the thymine DNA glycosylase (TDG), resulting in an abasic site that is eventually replaced with unmethylated cytosine by the base excision repair (BER) machinery. Activation-induced cytidine deaminase (AID) or APOBEC can deaminated 5mC to thymine that is subsequently removed by TDG and repaired by BER. On the other hand, passive demethylation (brown arrows) is an alternative process that can occur through dilution following cell replication. DNMT1 can directly replicate the 5mC pattern onto the newly synthesized daughter strand unlike the pattern of oxidized mCs. (For review, see Rasmussen and Helin 2016.) (B) Enzymatic processes involved in cytosine methylation and oxidation of 5mC. DNMTs catalyze the addition of a methyl group to cytosine using S-adenosyl methionine (SAM) as methyl donor. Ten-eleven translocation (TET) proteins oxidize methylated cytosines to 5hmC, 5fC, and 5caC in an iterative manner using 2OG (αKG) as co-substrate. Mutant IDH1/2 generates 2HG that inhibits competitively the enzymatic activity of TET proteins. (C) Nucleosome model shows lysine substrate residues on histone H3 for writers (KMTs) and erasers (KDMs) that are found mutated in AML. Chromosomal translocations involving KMT2A (KMT2A-re) often result in fusion proteins that associate with the H3K79 histone methyltransferase DOT1L. (D) Enzymatic processes of lysine (de)methylation for selected KMTs. Besides TET proteins, 2HG can also impair the enzymatic activity Jumonji domain (JMJD)-containing KDMs.

Figure 3.

Epigenetic therapy and combination therapy targeting cooperating layers of the epigenome in acute myeloid leukemia (AML). (A) Scenario illustrating the effect of distinct epigenetic mutations (indicated in bold letters) in leukemia cells versus normal differentiated cells derived from hematopoietic stem cells (HSCs). Epigenetic therapy and strategies targeting aberrant DNA methylation are shown by green lines. Loss of DNA methylation at self-renewal genes in DNMT3A^mut AML may be potentially reversible by increasing intracellular levels of SAM (methyl donor). Loss of TET2 function can be compensated with vitamin C treatment. Specific inhibitors (AG120, AG221) against mutant IDH1/2 can block production of the TET inhibitor 2HG. Direct DNA hypermethylation (mutant TET2 or IDH1/2) or indirect hypermethylation due to secondary effects from other disease-driving mutations can be treated with DNMT inhibitors like 5-azacytidine or guadecitabine/decitabine (Issa et al. 2015; Gardin and Dombret 2017). (B) Drugs to target histone-modifying enzymes (writers+erasers) as well as readers (e.g., BET inhibitors) that are critical for AML maintenance and present vulnerabilities of the disease. Specific LSD1 inhibitors include GSK-LSD1 and ORY-1001 (Mohammad et al. 2015; Maes et al. 2018). Although PRC2 is deleted in a subset of AML, other AML subtypes such as KMT2A-re (MLL-rearranged) leukemias depend on functional PRC2. UNC1999, a dual inhibitor of EZH1/2 EZH1/2, impaired the growth of KMT2A-re in preclinical studies (Xu et al. 2015). DOT1L inhibitors (e.g., EPZ-5676) were also developed to target KMT2A(MLL)-re AML (Chen et al. 2016). The BRD 2/3/4 inhibitor OTX015 induces apoptosis in a variety of AMLs (Coudé et al. 2015). Inhibition of PRMT1 and PRMT5 show an antileukemia effect in distinct AMLs (Shia et al. 2012; Tarighat et al. 2016; Fedoriw et al. 2019). (C) Scheme illustrating the disruption of multiple layers of the epigenome in TET2^mut cells. Loss of TET2 facilitates recruitment of the H3K4me1/2 histone demethylase LSD1 that inactivates enhancers at target genes. In addition, loss of TET2 results in promoter methylation at target genes such as GATA2. (D) Concept of targeting cooperating layers of the epigenome at enhancers and promoters in TET2^mut AML to reconstitute enhancer–promoter interactions. Removal of 5mC promoter methylation by 5Aza treatment combined with LSD1 inhibition (GSK-LSD1) facilitates interactions of the LSD1-occupied enhancer and its target promoter, resulting in up-regulation of target genes like GATA2.

Figure 4.

Frequent mutations during B-cell development. In the bone marrow, rearrangements of the immunoglobulin genes of B-cell precursors to form a B-cell receptor (BCR) generate DNA breaks that are occasionally resolved aberrantly, leading to chromosomal translocations (Fugmann et al. 2000). These are the most common genetic alterations in B-precursor acute lymphoblastic leukemia (B-ALL) (Mullighan 2012). The most frequent mature B-cell neoplasms that have their origin outside the germinal center (GC) are B-cell chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and mucosa-associated lymphoid tissue (MALT) lymphoma. B-CLL and MCL differ in their molecular pathways, genomic alterations, and clinical behavior, being more aggressive in naive-like- than memory-like-derived tumors. The pathogenesis of the two malignancies involves the BCR signaling, tumor cell microenvironment interactions, genomic alterations, and epigenome modifications (Zhang et al. 2014; Landau et al. 2015). MALT lymphoma is the commonest MZL type and presents recurrent chromosomal translocations, which usually lead to activation of the NF-κB pathway. Nodal and splenic MZLs share recurrent mutations affecting the Notch pathway and the transcription factor KLF2, but differ for the inactivation of two tumor-suppressor genes, detected exclusively (PTPRD) or much more commonly (KMT2D/MLL2) in the nodal type (Rossi et al. 2012; Spina et al. 2016). The presence of immunoglobulin mutations is evidence that the cell of origin of the tumor passed through the GC microenvironment. Follicular lymphomas, Burkitt lymphomas, and DLBCLs express GC B cell signature genes. In the GC, two molecular processes remodel DNA: immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM), mechanisms that predispose to chromosomal translocations and mutations (Muramatsu et al. 2000). DLBCL is a clinically and genetically heterogeneous disease and accounts for 35% of non-Hodgkin lymphomas. Based on transcriptional profiles, DLBCL is further classified into activated B-cell (ABC) and germinal center B-cell (GCB) subtypes (Alizadeh et al. 2000; Rosenwald et al. 2002). ABC-DLBCLs derive from B cells that are committed to plasmablastic differentiation (Victora et al. 2012). These tumors have increased NF-κB activity, genetic alterations in NF-κB modifiers and components of the BCR pathway, and perturbed terminal B-cell differentiation (Lenz et al. 2008; Ngo et al. 2011). GCB-DLBCLs originate from light-zone GC B cells (Alizadeh et al. 2000; Victora et al. 2012). These tumors have frequent alterations in chromatin-modifying enzymes, PI3 K signaling, and genetic alterations of BCL2 (Pfeifer et al. 2013; Basso and Dalla-Favera 2015). Modifications in these pathways could favor epigenetic reprogramming and escape from cellular immunity. Recent genomic profiles have identified sub-ABC and GCB-DLBCL clusters: C1-C5 in one study (of which two are GCB-, two are ABC-subtypes, and the fifth is mostly characterized by genomic instability and TP53 mutations) (Chapuy et al. 2018), BN2, MCD, N1 (mostly ABC), and EZB (mostly GCB) in a different study (Schmitz et al. 2018). Follicular lymphoma (FL) is characterized by a unique histology in which tumor B cells form follicle-like structures with large numbers of nonmalignant immune cells infiltrating within the follicular and interfollicular regions (Kridel et al. 2012). The most frequent genetic event is the t(14;18) translocation that places BCL2 under control of the immunoglobulin heavy-chain enhancer, which occurs in 90% of FL patients. Mutations in epigenetic modifiers (KMT2D, CREBBP, and EZH2) are also a hallmark of FL (Green 2018). These mutations result in altering normal B-cell differentiation programs and impeding GC exit (Green et al. 2015). Burkitt lymphoma is characterized by deregulation of the MYC gene through its translocation to one of the immunoglobulin loci (Love et al. 2012). LPL, lymphoplasmacytic lymphoma; WM, Waldenstrom macroglobulinemia.

Figure 5.

Vulnerable points of germinal center (GC) B cells that give advantage to lymphomagenesis. (A) Chromatin-based epigenetic switches transiently poise the active plasma/memory B-cell program to enable the GC phenotype to emerge in a reversible manner. Cell fate decisions during a normal GC reaction: (1) negatively selected centrocytes (CC) undergo apoptosis; positively selected centrocytes may (2) recycle to centroblast (CB) and reenter the dark zone, or (3) differentiate to (3a) plasma cells (PC) or (3b) memory B cells (MBC). (B) GC B cells feature the typical hallmarks of transformed cells through epigenetic mechanisms, without requiring somatic mutations. This “pseudo-malignant” state can be reversed to normal state also through epigenetic switching mechanisms. (Sketch adapted from Hanahan and Weinberg 2011.) An immunohistochemistry (IHC) picture of GCs identified with peanut agglutinin (PNA) stain in a murine splenic section is shown in the middle. (C) Mutations in epigenetic modifiers maintain B cells in the GC phenotype, allowing the development of GC-derived B-cell lymphomas. Although mutations of one or more chromatin modifier genes occur within 96% of follicular lymphoma (FL) and ∼70% diffuse B-cell lymphoma (DLBCL) patients, 76% FL, and ∼40% DLBCL cases feature at least two mutations in epigenetic regulators (Green et al. 2015; Ortega-Molina et al. 2015; Reddy et al. 2017; Chapuy et al. 2018; Schmitz et al. 2018).

Figure 6.

The immune synapse and epigenetic therapy. Like germinal center (GC) B cells, the microenvironment in B-cell malignancies is crucial for the provision of survival and proliferation signals. (A) In both cases, normal GC B cells and malignant B cells interact with T cells, dendritic cells, macrophages, and lymphoid stromal cells (follicular dendritic cells) (Papa and Vinuesa 2018). (B) Mutations in epigenetic regulators and oncogenes such as BCL2 allow low-affinity B cells to survive, leading to the initiation of a prosurvival, immunosuppressive microenvironment in the lymphoid tissue. (C) Although epigenetic therapy can overcome the effect of founder mutations, the microenvironment makes critical contributions to both disease progression and drug resistance/disease relapse. The combination of epigenetic therapy with checkpoint inhibitor therapy can lead to potent antilymphoma effects.