Abstract

Multiple myeloma is a common hematologic malignancy with an incidence of 1 per 100,000 population and is characterized by a nearly 100% risk of relapse, necessitating treatment with newer therapeutic agents at each instance of progression. However, use of newer agents is often precluded by cost and accessibility in a resource-constrained setting. Description of newer pathways of disease pathogenesis potentially provides opportunities for identification of therapeutic targets and a better understanding of disease biology. Identification of epigenetic changes in myeloma is an emerging premise, with several pathways contributing to pathogenesis and progression of disease. Greater understanding of epigenetic alterations provides opportunities to detect several targetable enzymes or pathways that can be of clinical use.

Introduction

Epigenetics is defined as heritable changes in genetic function that occur without alteration of the underlying genetic code. Since its original description, it is now clear that epigenetic mechanisms are vital for cellular growth, development, and establishment of a distinct cellular identity.[1] The term ?epigenome? describes the epigenetic regulatory mechanisms across the entire genome; and unlike the genome, it varies across different cell types. Abnormalities in epigenetic control can lead to a wide variety of cellular dysfunction, and have been specifically found to contribute to carcinogenesis.[2] Epigenetic alterations are early events in the pathogenesis of certain cancers and contribute to disease progression, analogous to clonal cytogenetic evolution.[3] Epigenetic modifications are of interest in several malignancies, and have been utilized most notably for the treatment of acute myeloid leukemia in the form of hypomethylating agents. Significant molecular data are also emerging on the role of epigenetics for several other malignancies, and is of specific importance in multiple myeloma (MM).

MM is a common hematologic malignancy with a universal risk of relapse and has undergone advances in survival over the past two decades.[4] However, it still carries a nearly 100% risk of relapse, necessitating newer agents to be initiated at each instance of progression.[5] As a result, no functional cure is defined, and therapy must continue indefinitely. Although several newer novel agents have been introduced in the past 5 years, costs continue to be prohibitive, especially in resource constrained settings.[6] Thus, there is an ongoing need to identify newer cellular pathways and targetable genetic lesions to ensure the availability of treatment options at each relapse.

Epigenetic mechanisms have recently emerged as critical mediators of disease pathogenesis and progression in myeloma. Several mechanisms are linked to drug responsiveness and known to modify survival. As seen in the case of acute myeloid leukemia (AML), several components of epigenetic mechanisms are eminently targetable and will likely offer an opportunity for a new approach to drug development. An added impetus for studying epigenetic pathways would be definition of differences in disease biology based on geographic variation. For instance, significant differences in age of incidence of myeloma have been noted in Indian patients compared with Western data, with higher likelihood of end organ damage at diagnosis.[7]

We provide a succinct snapshot of the current role of epigenetic mechanisms in disease pathogenesis in MM, and highlight potential therapeutic applications. We also provide a snapshot of potential drug targets and current ongoing trials targeting epigenetic pathways in MM.

Summary of Basic Mechanisms of Epigenetic Control

Epigenetic control is facilitated by several biochemical processes, of which the best understood mechanisms are (a) DNA methylation, (b) histone modification, and (c) microRNA (miRNA) expression. These three processes are most widely described and are targets of several investigational approaches to treatment.

DNA Methylation

DNA methylation consists of addition of a methyl group to the 5? carbon of cytosine on DNA. The cytosine bases that are involved in methylation are usually located in regulatory (enhancer or repressor) sequences, and can recruit other proteins to functionally silence downstream genes.[8] DNA methylation of promotor genes, therefore, leads to silencing of the downstream genes regulated with the same. DNA methylation is performed by enzymes called DNA methyl transferases (DNMT) and typically occurs at sites of CpG (cytosine-phosphate-guanine) dinucleotides, which are concentrated in sites known as CpG islands, mostly at 5? ends of DNA.[8] There are ~28 million CpGs in the human genome, and ~60% to 80% are methylated.

Histone Modification

Intracellular DNA is tightly wound around proteins called histones, which are octamers consisting of eight subunits. This process is best summarized by its original definition given in the 1960s, ?dynamic and reversible mechanism for activation as well as repression of RNA synthesis.?[9] It is known that modification of histone proteins by biochemical changes enables regulation of DNA expression by increasing or decreasing chromatin accessibility. The first mechanism to be elucidated was histone acetylation and deacetylation, following which several other mechanisms have been described over the last decade.[10] As genetic regulation needs to be reversible and dynamic, most of the processes are reversible and involve reciprocal ?effector? and ?eraser? enzymes. These include acetylation, methylation, and phosphorylation, and the use of so-called ?erasers,? or enzymes with reciprocal activity to the ones above. [Fig. 1] summarizes the basic process of histone modification.

|?Fig. 1?Histone modifications in DNA. Histone proteins (dotted blue) are tightly wound to DNA (green), preventing access of transcriptional machinery. Reversible binding of covalent modifiers can occur either on the histone protein core or on one of the histone tails (red). Binding or a modifier, for example, an acetyl group (blue), leads to a conformational change, opening the chromatin structure and enabling access to transcriptional machinery.|

Noncoding RNA Expression

Noncoding (ncRNAs) are RNAs that are not coded into proteins. They are classified by their size into small and large ncRNAs. The small ncRNAs include miRNA, piwi interacting RNA, transfer RNA, and small nucleolar RNA.[11] They are involved in regulation of DNA expression by several mechanisms, out of which epigenetic alteration is one of them. [Fig. 2] summarizes mechanisms of miRNA control with potential therapeutic interest.

|?Fig. 2?Summary of micro-RNA (miRNA)-mediated control of genetic expression. Starting from lower left, RNA polymerase II encodes primary miRNA (pri-miRNA) that is converted to pre-miRNA and exported out of the nucleus by Exportin. After cleavage by the Dicer (helicase with RNase motif) complex, the miRNA forms an RNA-induced silencing complex (RISC) that binds to complementary miRNA, silencing its expression. This repressor function of miRNA against has demonstrated antimyeloma activity in vitro.|

|?Fig. 2?Summary of micro-RNA (miRNA)-mediated control of genetic expression. Starting from lower left, RNA polymerase II encodes primary miRNA (pri-miRNA) that is converted to pre-miRNA and exported out of the nucleus by Exportin. After cleavage by the Dicer (helicase with RNase motif) complex, the miRNA forms an RNA-induced silencing complex (RISC) that binds to complementary miRNA, silencing its expression. This repressor function of miRNA against has demonstrated antimyeloma activity in vitro.

Genetic Changes in Myeloma

Plasma cell (PC) clones in MM acquire several genetic abnormalities while progressing from the stage of monoclonal gammopathy of undetermined significance (MGUS) to symptomatic disease. Broadly, these are categorized as primary and secondary cytogenetic abnormalities. Primary cytogenetic abnormalities arise at the stage of MGUS and have a significant impact on disease phenotype, organ damage, and rate of progression. These are mutually exclusive, with each clone having only one primary cytogenetic anomaly.[12] Common primary genetic changes include hyperdiploidy and immunoglobulin heavy chain promotor (IgH) translocations. Hyperploidy of at least one odd numbered chromosome is noted in a majority of patients with myeloma, commonly presenting as trisomy.[13] IgH translocations are usually a result of errors in DNA modification as a part of physiological processes of V(D)J recombination and class switching. These translocations commonly place an oncogene under control of the highly active IgH locus.[14] Both these changes can be observed in PCs at all stages of disease, indicating the fundamental nature of these changes.

Recent advances in next-generation sequencing (NGS) have also identified several patterns of single gene mutations in patients with newly diagnosed myeloma. Mutations in BRAF, TP53, KRAS, NRAS, and DIS3 are some of the commonest recurrently mutated genes. Single gene mutations have already been used to classify patients into groups with distinct phenotypes and open up avenues for individualized medicine.[15]

On the other hand, secondary cytogenetic abnormalities arise at the time of disease progression and clonal evolution, with each cell possibly having multiple secondary abnormalities. In routine practice, either of these can be detected in ~50% of patients by conventional karyotyping and 40 to 50% by fluorescence in situ hybridization.[16] Commonly described secondary cytogenetic abnormalities include del(17p), gain (1q), and myc translocations.

Summary of Epigenetic Changes in Myeloma

All three epigenetic processes, namely DNA methylation, histone modification, and miRNA processing, are active in MM. The following text is a summary of each of these processes.

DNA Methylation

DNA Methylation in Myeloma Is Heterogeneous

The potential role for DNA methylation in MM was elucidated in 2004, when it was shown that methylation of promoter regions of tumor suppressor genes (leading to inactivation) was increased in patients with MM.[17] Subsequently, it has been observed that the pattern of genome methylation in myeloma is not uniform, and consists of specific?hypo-?and?hypermethylated regions. Several in vitro studies have highlighted differential effects of methylation at different stages in the pathogenesis of myeloma. As with several solid organ malignancies, the overall methylation pattern in initial phase consists of hypermethylation, with advanced myeloma demonstrating global DNA?hypomethylation, reflecting genomic instability.[18] [19] DNA hypomethylation is postulated to activate several transcriptional units or oncogenes that are usually repressed and provide a survival advantage.[20] Hypomethylation is frequently observed on repetitive DNA elements and progressively increases while moving from healthy controls to myeloma to PC leukemia.[21] It is essential to note that global hypomethylation is not uniform, and several loci are hypermethylated in advanced or relapsed disease.[22] Interestingly, DNA methylation in MM and MGUS has been seen to occur at sites distant from CpG rich promoters, specifically in areas known for binding to B cell promotors.[18] This presents a new area of potential research, as these promotors may have a role in driving myeloma cell proliferation and may be targetable by newer therapies.

Unlike methylation, no single?mechanism?describes the process of hypomethylation. The simplest method described is lack of maintenance of methylation, which occurs with downregulation of DNMT. These changes mirror alterations in enzymes mediating DNA methylation, that is, DNMT1, which is shown to increase in expression when moving from MGUS to symptomatic disease.[21] Expression of DNMT3a has been shown to be downregulated in patients with MGUS and MM, reducing baseline maintenance methylation.[23] Putative biochemical mechanisms of DNA hypomethylation are still not clear and are summarized in this excellent review.[24] These mechanisms are clinically relevant as myeloma cell lines are susceptible to DNMT inhibition with currently available hypomethylating agents, providing a potential therapeutic target.

MicroRNA

Diverse Changes in miRNA Expression Characterize Myeloma

miRNAs were first identified in 1993, defined as small molecules with antisense 3? UTR specificity.[25] It was later identified that miRNAs are small, ncRNAs that regulate gene expression at the posttranslational level. Depending on their location, miRNAs are of two major subtypes, that is, intragenic (intronic) and intergenic, with most showing clustering with a common promotor. The major mechanism of gene regulation is at the posttranslational level, by binding to the corresponding miRNAs, typically resulting in repression. The entire process can be summed up as miRNAs are transcribed by RNA polymerase II, which creates a pri-miRNA, which is converted to pre-miRNA and finally exported as miRNA into the cytoplasm. This final miRNA forms a complex called RNA-induced silencing complex, which leads to binding and inhibition of target miRNAs.[26] Deregulation of miRNA function, both through genomic and epigenomic mechanisms, is increasingly seen to occur in several malignancies, including myeloma. In MM, deregulation of miRNA function can be noted at all disease stages, from MGUS to MM. Multiple studies have demonstrated deranged miRNA function in PCs from MGUS and MM cells compared with normal PCs.[27] Remarkably, the changes seen in a particular miRNA are seen to proceed in the same trend with disease progression, raising the possibility that specific changes have a part to play in pathogenesis and disease progression.[28]

Histone Modification in Myeloma Is a Complex Multistep Process

Histone proteins are subject to several covalent posttranslational modifications, of which over 15 have been described in detail.[10] Histone modification is reversible, and the processes involved have effector and eraser functions with a set of reciprocally active enzymes. Acetylation, phosphorylation, and methylation are the most well described, with the first two being clear markers of active transcription[29] Methylation, on the other hand, is more complex, as the final effects of methylation depend on the amino acids modified and the degree of methylation (mono, di, or tri).[30] Histone methylation is seen to occur at basic amino acids, namely arginine, histidine, and lysine, and may have a variable effect on transcription depending on context (i.e., type and site of amino acid that is methylated).[31] A common nomenclature is used to define histone modifications, comprising the name of histone protein (e.g., H2), abbreviation and position of amino acid (K or M), chemical group added (e.g., methyl = Me or Acetyl = Ac), and copies of the modifier added (1, 2, or 3). Therefore, addition of a dimethyl group to a lysine residue in position 36 on histone protein H3 is abbreviated as H3K36Me2. This process is summarized in [Fig. 3].

|?Fig. 3?Patterns of histone methylation. Methylation occurs on specific amino acids in the histone core protein and is mediated by histone methyl transferases. Depending on the number of methyl groups added, it can either occur as mono-, di-, or trimethylation.|

Histone Dimethylation

Histone dimethylation is catalyzed by enzymes containing the SET domain with methyltransferase activity. In this well-characterized group, mutations in MMSET, NSD1, and NSD2 are found in several patients at diagnosis and relapse. MMSET is one of the most well defined, and catalyzes the addition of H3K36me2, associated with active transcription.[32] It is seen to be associated with the t(4;14) translocation, leading to transcriptional activation and is associated with a poorer outcome in patients with MM.[33] Over the past decade, there has been an increasing recognition of the biological contribution of MMSET to pathogenesis of myeloma. MMSET is shown to activate the MAF gene through the MAP kinase pathway, indicating indirect activation of an oncogene. MMSET is also implicated in activation of several downstream mitogenic pathways, including c-myc and IRF4.[34]

Histone Trimethylation

Histone trimethylation is brought about by enzymatic units of enhancer of Zeste homologue 2 (EZH1/2) complexes, which deposit H3K27me3 on target genes. EZH2 is part of the polycomb repressor complex (PRC), which silences several downstream genes. The role of EZH2 varies in different malignancies, and in MM it is shown to be upregulated and correlates with a poor outcome.[35] Similar to MMSET, EZH2 overexpression induces disease progression through a variety of mechanisms. EZH2 is shown to induce proliferation of MM cells independent of interleukin 6 (IL-6). It is also shown to control the expression of tumor suppressor miRNAs that target c-myc, BLIMP-1, and IRF4.[36]

Histone Monomethylation

Histone monomethylation at lysine sites deposits H3K9me and is essential for gene repression at promoter regions. H3K9 transferring enzymes are elevated in MM patients. KDM3A leads to IRF4 supported cell growth in MM.[37]

Histone Acetylation

Histone acetylation is seen to occur at lysine residues, which opens up chromatin and makes it transcriptionally active. This process is reversibly regulated by lysine transferases (KATs) and deacetylases (histone deacetylase inhibitors [HDACs]).[38] Mutated KAT genes are found in several patients with MM, especially at relapse, possibly loss of function mutations. HDACs are overexpressed in myeloma and are associated with poor prognosis.[39]

Role of Epigenetic Mechanisms in Disease Progression and Drug Resistance

Epigenetic processes have been found to play a significant role in two important processes mediating disease progression, that is, PC plasticity and drug resistance.

Analysis of malignant PCs in myeloma reveals a heterogeneous population comprising PCs (CD138+/CD19-), preplasma cells (pre-PCs: CD19-/CD138-), and plasmablasts (CD19+). A vast majority of the cells are CD19-/CD138+ and predominant mediators of disease phenotype, including immunoglobulin secretion and organ damage. A clone of pre-PCs with a CD19-/CD138- represents a small minority, representing a quiescent population. This clone confers resistance to therapy and is in dynamic equilibrium with CD138+ PCs.[40]

Several characteristics of this process indicate a significant role played by epigenetic processes. Pre-PC populations demonstrate high concentrations of several epigenetic regulators, including PRC, MLL transcriptional activating complex, demethylases, HDACS and KDM5C/D, indicating epigenetic control. Additionally, the absence of irreversible genetic mutations or change in CD138 miRNA levels makes genetic level (transcriptional/ translational) control unlikely.[41] These cells are intrinsically resistant to proteasome inhibitors, and may indicate a reservoir that can proliferate at relapse.

Additionally, epigenetic mechanisms are observed to mediate many aspects of drug resistance in patients with myeloma. Epigenetic inactivation of RASD1 is essential in mediating glucocorticoid resistance. Several miRNAs have been found to mediate drug resistance through diverse mechanisms, including modulation of target genes, upregulation of oncogenes, and modification of drug efflux mechanisms. A review by Rastgoo et al provides a succinct overview of drug resistance mediated by miRNA.[42] The control of cell adhesion-mediated drug resistance by altering microenvironment interactions and modification of antiapoptotic signals also have significant epigenetic control. The description of all allied mechanisms is out of scope of this review and is summarized elegantly by Furukawa et al.[43]

Therapeutic Applications of Epigenetic Modifications in Myeloma

All the mechanisms described above provide potential therapeutic targets. The following discussion summarizes salient features of some of the commonly described mechanisms. Many mechanisms have been translated to clinical applications, and a summary of recent or ongoing trials utilizing the same is provided in [Table 1].

Table 4

A summary of studies describing the prognostic impact of epigenetic modifications in myeloma

Abbreviations: HDAC, histone deacetylase inhibitor; OS, overall survival; PFS, progression-free survival.

Conclusions

Epigenetic pathways are being observed to play an increasingly important role in pathogenesis and disease progression in myeloma and have a significant prognostic impact. There are two critically important clinical correlates of the above advances in basic science. First, these pathways provide a new approach to therapeutic intervention in MM, and can be targeted by multiple approaches. Considering the incurable nature of disease and paradigm for continuous therapy, identifying a newer therapeutic approach is an important step forward. Second, epigenetic mechanisms can explain geographic and ethnic differences in disease phenotype and outcomes not explained by conventional cytogenetics, improving our understanding of disease biology. The current review provides a short overview of the above, with clinical correlates, and will be a useful primer for better understanding of this intriguing approach.

Conflict of Interest

None declared.

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Study	Parameters	Specific genes	Salient findings
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		RBP1	23.9 vs. 47.7 mo, p < 0>
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		TGFB1	25.7 vs. 50.9 mo, p < 0>
Barwick et al[97]	Global DNA methylome		Low vs. high methylation status, median OS: 2 y vs. not reached, p = 8.7e-8
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