Teresa Ezponda

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.

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

The Oncoprotein SF2/ASF Promotes Non–Small Cell Lung Cancer Survival by Enhancing Survivin Expression

Purpose: SF2/ASF is a splicing factor recently described as an oncoprotein. In the present work, we examined the role of SF2/ASF in human non–small cell lung cancer (NSCLC) and analyzed the molecular mechanisms involved in SF2/ASF-related carcinogenesis. Experimental Design: SF2/ASF protein levels were analyzed in 81 NSCLC patients by immunohistochemistry. SF2/ASF downregulation cellular models were generated using small interfering RNAs, and the effects on proliferation and apoptosis were evaluated. Survivin and SF2/ASF expression in lung tumors was analyzed by Western blot and immunohistochemistry. Survival curves and log-rank test were used to identify the association between the expression of the proteins and time to progression. Results: Overexpression of SF2/ASF was found in most human primary NSCLC tumors. In vitro downregulation of SF2/ASF induced apoptosis in NSCLC cell lines. This effect was associated with a reduction in the expression of survivin, an antiapoptotic protein widely upregulated in cancer. In fact, SF2/ASF specifically bound survivin mRNA and enhanced its translation, via a mammalian target of rapamycin complex 1 (mTORC1) pathway-dependent mechanism, through the phosphorylation and inactivation of the translational repressor 4E-BP1. Moreover, SF2/ASF promoted the stability of survivin mRNA. A strong correlation was observed between the expression of SF2/ASF and survivin in tumor biopsies from NSCLC patients, supporting the concept that survivin expression levels are controlled by SF2/ASF. Furthermore, combined expression of these proteins was associated with prognosis. Conclusion: This study provides novel data on the mTORC1- and survivin-dependent mechanisms of SF2/ASF-related carcinogenic potential, and shows that SF2/ASF and survivin expression is involved in NSCLC progression. Clin Cancer Res; 16(16); 4113–25. ©2010 AACR.

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.

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

BackgroundMicroarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events.ResultsThe array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer.ConclusionsThis methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

Molecular Pathways: Deregulation of Histone H3 Lysine 27 Methylation in Cancer—Different Paths, Same Destination

Methylation of lysine 27 on histone H3 (H3K27me), a modification associated with gene repression, plays a critical role in regulating the expression of genes that determine the balance between cell differentiation and proliferation. Alteration of the level of this histone modification has emerged as a recurrent theme in many types of cancer, demonstrating that either excess or lack of H3K27 methylation can have oncogenic effects. Cancer genome sequencing has revealed the genetic basis of H3K27me deregulation, including mutations of the components of the H3K27 methyltransferase complex PRC2 and accessory proteins, and deletions and inactivating mutations of the H3K27 demethylase UTX in a wide variety of neoplasms. More recently, mutations of lysine 27 on histone H3 itself were shown to prevent H3K27me in pediatric glioblastomas. Aberrant expression or mutations in proteins that recognize H3K27me3 also occur in cancer and may result in misinterpretation of this mark. In addition, due to the cross-talk between different epigenetic modifications, alterations of chromatin modifiers controlling H3K36me, or even mutations of this residue, can ultimately regulate H3K27me levels and distribution across the genome. The significance of mutations altering H3K27me is underscored by the fact that many tumors harboring such lesions often have a poor clinical outcome. New therapeutic approaches targeting aberrant H3K27 methylation include small molecules that block the action of mutant EZH2 in germinal center-derived lymphoma. Understanding the biologic consequences and gene expression pathways affected by aberrant H3K27 methylation may also lead to other new therapeutic strategies. Clin Cancer Res; 20(19); 5001–8. ©2014 AACR.

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.