Aberrant Histone Methylation in Myeloma: What Are the Rules?

Abstract SCI-5

Aberrant regulation of histone methylation is a recurrent theme in multiple myeloma, lymphoma, and other B-cell malignancies. MMSET Multiple myeloma SET domain (MMSET) is a histone methyltransferase (HMT) overexpressed as a result of the translocation t(4;14) and is present in about 15 percent of multiple myeloma patients. MMSET is a nuclear protein with multiple domains critical for gene regulation, including the SET domain, which encodes histone methyltransferase activity, and protein and DNA interaction domains, including PHD and PWWP domains. Overexpression of MMSET induces a global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. Kinetic studies using isotopic labeling and mass spectroscopy demonstrate that this change in methylation is due to both an increase in the rate of methylation of H3K36 and an increase in the demethylation of H3K27. These changes cause physical loosening of the chromatin structure, demonstrated by an increase in micrococcal nuclease accessibility, changes in DNA damage response, and aberrant gene expression. The HMT activity of MMSET is essential for growth stimulation by MMSET, as shown by the fact that reexpression of MMSET in a t(4;14) myeloma cell line, in which the rearranged MMSET allele was disrupted by homologous recombination (KMS11-TKO), rescued growth only when the HMT activity of the protein was intact. The complete H3K36/H3K27 switch mediated by MMSET requires all PHD finger domains of the protein, the second PWWP domain, and the functional SET domain. For example, a single point mutation in one PHD domain abrogated chromatin binding, histone methylation, and growth stimulation by the protein. Furthermore, deletion of the PHD domain 4 was able to increase H3K36 methylation but unable to reduce H3K37 methylation, leading to only partial growth stimulation. Despite the global change in histone methylation in response to MMSET, microarray and RNA-Seq analysis showed that only ∼1000 genes are appreciably changed in response to MMSET. The basis of the specificity of differential gene expression is under investigation. For example, many genes activated by MMSET display a peak of H3K27me3 near the transcription start site in MMSET-low cells, which is absent in MMSET-overexpressing cells, displaced by a broad pattern of H3K36me2 modification. We also found a subset of genes repressed in response to MMSET overexpression. While H3K27 methylation is decreased on a genome-wide basis in MMSET-overexpressing cells, H3K27me3 levels at repressed genes were increased in association with increased occupancy by EZH2. These regions did not show an increase in H3K36 methylation and are enriched with GC-rich elements, representing putative polycomb complex recruitment sites. We hypothesize that the global increase in H3K36me2 and drop of H3K27me3 levels on many genes leads to the displacement of the PRC2 complex from lower-affinity sites to such higher-affinity loci. These modes of action likely considerably diverge from the normal role of MMSET and EZH2 in gene regulation. Similarly, EZH2 point mutations in lymphoma lead to global chromatin dysfunction and aberrant regulation of specific sets of genes, only some of which represent previously identified EZH2 targets. Collectively, oncogenic lesions in histone-modifying enzymes in myeloma and other lymphoid neoplasms need to be understood on their own terms, as the lessons learned from the normal function of these enzymes may not predict their activity in malignancy.