Eva Martinez-Garcia

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.

The proto-oncometabolite fumarate binds glutathione to amplify ROS dependent signaling

The tricarboxylic acid cycle enzyme fumarate hydratase (FH) has been identified as a tumor suppressor in a subset of human renal cell carcinomas. Human FH-deficient cancer cells display high fumarate concentration and ROS levels along with activation of HIF-1. The underlying mechanisms by which FH loss increases ROS and HIF-1 are not fully understood. Here, we report that glutamine-dependent oxidative citric acid cycle metabolism is required to generate fumarate and increase ROS and HIF-1 levels. Accumulated fumarate directly bonds the antioxidant glutathione in vitro and in vivo to produce the metabolite succinated glutathione (GSF). GSF acts as an alternative substrate to glutathione reductase to decrease NADPH levels and enhance mitochondrial ROS and HIF-1 activation. Increased ROS also correlates with hypermethylation of histones in these cells. Thus, fumarate serves as a proto-oncometabolite by binding to glutathione which results in the accumulation of ROS.

Deregulation of H3K27 methylation in cancer

A new study now reports recurrent somatic mutation of EZH2, a histone methyltransferase that modifies H3K27, in diffuse large B-cell lymphoma (DLBCL). There is now evidence for both increased and decreased activity of enzymes controlling H3K27 methylation in cancer, suggesting that a precise balance of this methylation is critical for normal cell growth.

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.

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.

MMSET/WHSC1 enhances DNA damage repair leading to an increase in resistance to chemotherapeutic agents.

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, believed to be the driving factor in the pathogenesis of this MM subtype. MMSET overexpression in MM leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3), as well as changes in proliferation, gene expression and chromatin accessibility. Prior work linked methylation of histones to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET may play a role in DNA damage repair and response. In U2OS cells, we found that MMSET is required for efficient non-homologous end joining as well as homologous recombination. Loss of MMSET led to loss of expression of several DNA repair proteins, as well as decreased recruitment of DNA repair proteins to sites of DNA double-strand breaks (DSBs). By using genetically matched MM cell lines that had either high (pathological) or low (physiological) expression of MMSET, we found that MMSET-high cells had increased damage at baseline. Upon addition of a DNA-damaging agent, MMSET-high cells repaired DNA damage at an enhanced rate and continued to proliferate, whereas MMSET-low cells accumulated DNA damage and entered cell cycle arrest. In a murine xenograft model using t(4;14)+ KMS11 MM cells harboring an inducible MMSET shRNA, depletion of MMSET enhanced the efficacy of chemotherapy, inhibiting tumor growth and extending survival. These findings help explain the poorer prognosis of t(4;14) MM and further validate MMSET as a potential therapeutic target in MM and other cancers.

Abstract PL05-02: MMSET: A pathogenic factor and therapeutic target in multiple myeloma.

Multiple Myeloma (MM), a neoplasm of plasma cells accounts for up to 10% of hematological malignancies. The MMSET (Multiple Myeloma SET domain) protein is a histone methyl transferase (HMT) overexpressed as a result of the translocation t(4;14), present in about 15% of multiple myeloma patients. MMSET is a nuclear protein with multiple domains critical for gene regulation including the SET domain, which encodes histone methyl transferase activity, and protein interaction domain including PHD, and PWWP domains. Although studies have shown the involvement of MMSET in heart development and tumor progression, its mode of action in the pathogenesis of MM is largely unknown. We found that overexpression of MMSET induces global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. This is also accompanied by significant changes in over DNA methylation in these cells. These changes cause physical loosening of the chromatin structure, increased micrococcal nuclease accessibility, leading to altered gene expression. Pathways affected by MMSET overexpression include cell cycle, apoptosis and response to DNA damage. The HMT activity of MMSET is important for growth stimulation by MMSET since reexpression of MMSET in a t(4;14) myeloma cell line in which the rearranged MMSET allele was disrupted by homologous recombination (KMS11-KO) rescued growth only when the HMT activity of the protein was intact. Conversely shRNA-mediated deletion of MSMET in t(4;14)+ cell lines leads to growth arrest and cell death, together suggesting that MMSET is a therapeutic target in multiple myeloma. We investigated the mechanism by which MMSET induces global chromatin changes. KMS-11-KO cells were stably repleted with wild-type MMSET or deletion constructs. The complete H3K36/H3K27 switch mediated by MMSET requires all 4 PHD finger domains of the protein, the second PWWP domain and the functional SET domain. Furthermore, these domains are also required for the increased proliferation as well as the aberrant gene expression caused by overexpression of the wild type MMSET. We also found that the PHD and PWWP domains of MMSET were required for its ability to bind to specific chromatin marks and likely play a role in firmly binding MMSET to chromatin. Furthermore deletion of PHD domain 4 yielded a form of MMSET able to increase H3K36 methylation but unable to reduce H3K37 methylation. Wild-type MMSET but not the PHD4 deletion mutant could bind to the H3K27 histone demethyalse JMJD3. This further implicates a histone demethylase as a possible target in this form of myeloma. Recently, it has been 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 induced by MMSET in chromatin structure, we hypothesized that MMSET could affect the DDR by inducing global changes in chromatin structure. We observed that cells overexpressing MMSET displayed 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 cells in the presence of melphalan, an alkylating agent currently used in myeloma treatment. 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 in response to the drug. The cells with high MMSET levels were more resistant to the action of melphalan and displayed less apoptosis after the treatment. After repleting MMSET expression in the KMS11-KO cells, we observed that the SET domain of the protein is important for these effects as KO cells expressing the HMT inactive mutant grew more slowly and formed fewer colonies in the presence of melphalan than KO cells expressing wild type MMSET. We also profiled gene expression in cells with high and low MMSET levels using Illumina HumanWG-6 v3.0 expression arrays and performed functional annotations using Ingenuity Pathway analysis. There was an enrichment of genes implicated in cell cycle regulation (CCNE2, E2F2, TP53INP1, CDC25A) and DNA damage-repair (DNA-PK, GADD45G). We then studied the cell cycle profile of KMS11-KO treated with high dose melphalan for two hours. We performed cell cycle profile with BrdU staining two days after the release from the drug. We observed that cells with high MMSET levels could cycle normally and overcame the cell cycle arrest but cells with low MMSET were arrested in G0-G1 and G2-M phases. Collectively this information suggests that the normal role of MMSET in DNA repair is dysregulated by its overexpression in myeloma. An underlying paradox of MM is the continued proliferation and self-renewal of a highly differentiated cell. Plasma cell development requires the BLIMP1 transcription factor, which stimulates terminal B cell differentiation and represses c-MYC, a master regulator of cell growth. By contrast, malignant plasma cells express c-MYC and proliferate while maintaining the specialized machinery required for immunoglobulin production. We found that MMSET overexpression was associated with increased c-MYC protein but not mRNA expression and no change in the half-life of the MYC proteins. miRNA profiling of t(4;14) myeloma cells before and after depletion of MMSET showed that MMSET repressed expression of miR-126*, which was predicted to target c-MYC. miR126* but not a point mutant of mir126* inhibited translation of a c-MYC-UTR-luciferase reporter. Overexpression of miR126* in t(4;14) myeloma cells suppressed c-MYC expression and cell growth, while growth suppression and inhibition of c-MYC expression in myeloma cells after MMSET knock-down was partially blocked by an antagomir directed against mir126*. Growth suppression mediated by MMSET knockdown was reversed by expression of exogenous miRNA-resistant form of c-MYC. MMSET was recruited to the miR126* promoter along with a co-repressor complex and in this case repressed miR126* expression. To model the effects of an MMSET inhibitor in combination with histone deactylase (HDAC) inhibitors, cells were subjected to trichostatin A, MMSET depletion or both. HDAC inhibitor treatment on its own elevated mir126* levels and decreased c-MYC expression while an HDAC inhibitor combined with MMSET depletion led to complete silencing of c-MYC expression and increased cell death. These studies motivate the development of MMSET inhibitors. We assayed MMSET in vitro using bacterially purified enzyme and recombinant histones. In vitro, MMSET is a promiscuous HMT altering H3K36 and H3K27 residues and methylating itself on C-terminal lysine residues. Previously identified HMT inhibitors including chaetocin and BIX-01294 inhibited MMSET activity in vitro. We developed a screen for additional MMSET inhibitors using a high throughput mass spectroscopic assay in which the HMT activity of MMSET was detected by the presence of a 14Da shift in molecular weight of a small peptide representing the H3K36 methylation site of histone H3. Of 10,000 compounds screened, 7 showed >50% inhibition, none of these structurally similar to known HMT inhibitors. Secondary assays confirmed activity of at least three of these compounds that are undergoing further testing in vitro and in cell culture. In conclusion MMSET mediates extensive changes in chromatin structure and function that drive malignancy. MMSET inhibition, alone in combination with chemotherapy or other agents that affect chromatin such as HDAC inhibitors may represent future therapies for t(4;14)-associated multiple myeloma Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL05-02.