MMSET Contributes to Multiple Myeloma Oncogenesis Through Induction of Global Epigenetic Changes and Alteration of the DNA Damage Response,

Abstract 3475

Multiple myeloma (MM) is associated with recurrent chromosomal translocations that lead to overexpression of known and putative oncogenes. The MMSET (Multiple Myeloma SET domain) protein is overexpressed in multiple myeloma patients with the translocation t(4;14) and is believed to be the driving factor in the pathogenesis of this subtype of MM. MMSET contains several domains commonly found in chromatin regulators including the PHD domain, PWWP domain and a SET domain responsible for histone methyl transferase (HMT) activity for lysine 36 on histone H3 (H3K36). Our initial study identified MMSET as a major regulator of the epigenetic landscape and chromatin structure in t(4;14)+ myeloma cells. Overexpression of MMSET induces global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. These changes cause physical loosening of the chromatin structure, and increased micrococcal nuclease accessibility, leading to altered gene expression. Pathways affected by MMSET overexpression include cell cycle, apoptosis and response to DNA damage. Here, to study the mechanism by which MMSET induces global chromatin changes, we used a t(4;14)+ cell line, KMS11, in which the overexpressed MMSET allele was disrupted by homologous recombination (TKO). These cells were stably repleted with wild type MMSET or forms of MMSET deleted for putative chromatin interaction domains. Wild type MMSET increases H3K36 methylation and leads to a loss of H3K27 methylation. The complete epigenetic switch requires all four PHD fingers, the second PWWP domain and the catalytically active SET domain. Furthermore, these domains are also required for to increase cell proliferation and stimulate aberrant gene expression. Full length MMSET binds a number of peptides representing unmethylated and methylated histone tails. Loss of the fourth PHD domain severely impairs binding to histone peptides. Furthermore in cells, loss of the PHD4 domain leads to accumulation of K36 dimethylation without the complete loss of K27 trimethyl mark. MMSET deleted for the second PWWP domain fails to bind an H3 peptide methylated on lysine 27 and is also unable to methylate the H3K36 residue. These data suggest that MMSET reads the H3K27 methyl mark, removes it through recruitment of a demethylase and methylates lysine 36 through its SET domain. In accordance with this hypothesis, we found that the wild type MMSET can interact with one such demethylase, JMJD3, but not UTX. To identify potential direct transcriptional targets of MMSET, we performed chromatin immunoprecipitation followed by next generations sequencing using MMSET specific antibody. MMSET binds across genome with a preference towards gene rich regions (introns, exons, and promoters). At some loci, binding of MMSET is associated with a striking change in chromatin modifications.

Recently, it was shown that double stranded DNA breaks lead to unwinding of chromatin in a manner regulated by the DNA damage response (DDR). Considering this and the global changes in chromatin structure induced by MMSET, we hypothesized that overexpression of MMSET could affect the DDR. Cells overexpressing MMSET display more DNA damage at baseline as measured by alkaline electrophoresis comet assay and had higher levels of phosphorylated H2AX, a common DNA damage marker. To try to explain the observed resistance of t(4;14)+ myelomas to chemotherapy, we incubated KMS11 cells with melphalan. Paradoxically, despite the higher baseline level of phosphorylated H2AX and higher levels of single- and double-strand breaks of DNA upon melphalan treatment, MMSET overexpressing cells show better survival and less apoptosis in response to the drug. Furthermore, MMSET overexpressing cells fail to undergo cell cycle arrest in response to melphalan. Our data suggest that specific domains within MMSET serve as readers and writers of the histone code. MMSET regulates chromatin structure, gene expression and cell cycle. Targeting various MMSET-affected pathways may provide new opportunities for therapeutic intervention in t(4;14)+ myelomas. Additionally, overexpression of MMSET alters cellular response to DNA damaging agents, potentially explaining the lack of durable therapeutic response observed in this patient population.