Relja Popovic

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells

The multiple myeloma SET domain (MMSET) protein is overexpressed in multiple myeloma (MM) patients with the translocation t(4;14). Although studies have shown the involvement of MMSET/Wolf-Hirschhorn syndrome candidate 1 in development, its mode of action in the pathogenesis of MM is largely unknown. We found that MMSET is a major regulator of chromatin structure and transcription in t(4;14) MM cells. High levels of MMSET correlate with an increase in lysine 36 methylation of histone H3 and a decrease in lysine 27 methylation across the genome, leading to a more open structural state of the chromatin. Loss of MMSET expression alters adhesion properties, suppresses growth, and induces apoptosis in MM cells. Consequently, genes affected by high levels of MMSET are implicated in the p53 pathway, cell cycle regulation, and integrin signaling. Regulation of many of these genes required functional histone methyl-transferase activity of MMSET. These results implicate MMSET as a major epigenetic regulator in t(4;14)+ MM.

Emerging Epigenetic Targets and Therapies in Cancer Medicine

UNLABELLED Abnormalities in the epigenetic regulation of chromatin structure and function can lead to aberrant gene expression and cancer development. Consequently, epigenetic therapies aim to restore normal chromatin modification patterns through the inhibition of various components of the epigenetic machinery. Histone deacetylase and DNA methyltransferase inhibitors represent the first putative epigenetic therapies; however, these agents have pleiotropic effects and it remains unclear how they lead to therapeutic responses. More recently, drugs that inhibit histone methyltransferases were developed, perhaps representing more specific agents. We review emerging epigenetic targets in cancer and present recent models of promising epigenetic therapies. SIGNIFICANCE The use of DNA methyltransferase and histone deacetylase inhibitors in patients has validated the use of drugs targeted to epigenetic enzymes and strengthened the need for development of additional therapies. In this review, we summarize recently discovered epigenetic abnormalities, their implications for cancer, and the approaches taken for discovering small-molecule inhibitors targeting various properties of the epigenetic machinery.

Histone Methyltransferase MMSET/NSD2 Alters EZH2 Binding and Reprograms the Myeloma Epigenome through Global and Focal Changes in H3K36 and H3K27 Methylation

Overexpression of the histone methyltransferase MMSET in t(4;14)+ multiple myeloma patients is believed to be the driving factor in the pathogenesis of this subtype of myeloma. MMSET catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2), and its overexpression causes a global increase in H3K36me2, redistributing this mark in a broad, elevated level across the genome. Here, we demonstrate that an increased level of MMSET also induces a global reduction of lysine 27 trimethylation on histone H3 (H3K27me3). Despite the net decrease in H3K27 methylation, specific genomic loci exhibit enhanced recruitment of the EZH2 histone methyltransferase and become hypermethylated on this residue. These effects likely contribute to the myeloma phenotype since MMSET-overexpressing cells displayed increased sensitivity to EZH2 inhibition. Furthermore, we demonstrate that such MMSET-mediated epigenetic changes require a number of functional domains within the protein, including PHD domains that mediate MMSET recruitment to chromatin. In vivo, targeting of MMSET by an inducible shRNA reversed histone methylation changes and led to regression of established tumors in athymic mice. Together, our work elucidates previously unrecognized interplay between MMSET and EZH2 in myeloma oncogenesis and identifies domains to be considered when designing inhibitors of MMSET function.

The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial–mesenchymal transition and invasive properties of prostate cancer

Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial–mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

Point mutation E1099K in MMSET/NSD2 enhances its methyltranferase activity and leads to altered global chromatin methylation in lymphoid malignancies

Point mutation E1099K in MMSET/NSD2 enhances its methyltranferase activity and leads to altered global chromatin methylation in lymphoid malignancies

Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.

MMSET stimulates myeloma cell growth through microRNA-mediated modulation of c-MYC

Multiple myeloma (MM) represents the malignant proliferation of terminally differentiated B cells, which, in many cases, is associated with the maintenance of high levels of the oncoprotein c-MYC. Overexpression of the histone methyltransferase MMSET (WHSC1/NSD2), due to t(4;14) chromosomal translocation, promotes the proliferation of MM cells along with global changes in chromatin; nevertheless, the precise mechanisms by which MMSET stimulates neoplasia remain incompletely understood. We found that MMSET enhances the proliferation of MM cells by stimulating the expression of c-MYC at the post-transcriptional level. A microRNA (miRNA) profiling experiment in t(4;14) MM cells identified miR-126* as an MMSET-regulated miRNA predicted to target c-MYC mRNA. We show that miR-126* specifically targets the 3′-untranslated region (3′-UTR) of c-MYC, inhibiting its translation and leading to decreased c-MYC protein levels. Moreover, the expression of this miRNA was sufficient to decrease the proliferation rate of t(4;14) MM cells. Chromatin immunoprecipitation analysis showed that MMSET binds to the miR-126* promoter along with the KAP1 corepressor and histone deacetylases, and is associated with heterochromatic modifications, characterized by increased trimethylation of H3K9 and decreased H3 acetylation, leading to miR-126* repression. Collectively, this study shows a novel mechanism that leads to increased c-MYC levels and enhanced proliferation of t(4;14) MM, and potentially other cancers with high MMSET expression.

GLI2-dependent c-MYC upregulation mediates resistance of pancreatic cancer cells to the BET bromodomain inhibitor JQ1

JQ1 and I-BET151 are selective inhibitors of BET bromodomain proteins that have efficacy against a number of different cancers. Since the effectiveness of targeted therapies is often limited by development of resistance, we examined whether it was possible for cancer cells to develop resistance to the BET inhibitor JQ1. Here we show that pancreatic cancer cells developing resistance to JQ1 demonstrate cross-resistance to I-BET151 and insensitivity to BRD4 downregulation. The resistant cells maintain expression of c-MYC, increase expression of JQ1-target genes FOSL1 and HMGA2, and demonstrate evidence of epithelial-mesenchymal transition (EMT). However, reverting EMT fails to sensitize the resistant cells to JQ1 treatment. Importantly, the JQ1-resistant cells remain dependent on c-MYC that now becomes co-regulated by high levels of GLI2. Furthermore, downregulating GLI2 re-sensitizes the resistant cells to JQ1. Overall, these results identify a mechanism by which cancer cells develop resistance to BET inhibitors.

Epigenetic therapy of hematological malignancies: where are we now?

A growing amount of evidence points towards alterations in epigenetic machinery as a leading cause in disease initiation and progression. Like genetic alterations, misregulation of the epigenetic regulators can lead to abnormal gene expression. However, unlike genetic events, the epigenetic machinery may be targeted pharmacologically, potentially resulting in the reversal of a particular epigenetic state. The success of DNA methyltransferase and histone deacetylase inhibitors represents a proof of concept for the use of therapies intended to target the epigenome in the treatment of hematological malignancies. Nevertheless, the molecular mechanisms underlying the efficacy of these agents have not been completely elucidated. Recently, a large number of studies sequencing cancer cell genomes identified recurring mutations of epigenetic regulators, providing new insights into the molecular underpinnings of cancer. Consequently, the efforts to identify specific epigenetic inhibitors have been expanded in order to target particular subsets of patients. This review will summarize the progress made using the currently available epigenetic therapies and discuss some of the more recently identified targets whose inhibition may present potential avenues for the treatment of hematologic malignancies.