Dong-Joon Min

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.

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.

Transcriptional Profiling of Polycythemia Vera Identifies Gene Expression Patterns Both Dependent and Independent From the Action of JAK2V617F

Purpose: To understand the changes in gene expression in polycythemia vera (PV) progenitor cells and their relationship to JAK2V617F. Experimental Design: Messenger RNA isolated from CD34+ cells from nine PV patients and normal controls was profiled using Affymetrix arrays. Gene expression change mediated by JAK2V617F was determined by profiling CD34+ cells transduced with the kinase and by analysis of leukemia cell lines harboring JAK2V617F, treated with an inhibitor. Results: A PV expression signature was enriched for genes involved in hematopoietic development, inflammatory responses, and cell proliferation. By quantitative reverse transcription-PCR, 23 genes were consistently deregulated in all patient samples. Several of these genes such as WT1 and KLF4 were regulated by JAK2, whereas others such as NFIB and EVI1 seemed to be deregulated in PV by a JAK2-independent mechanism. Using cell line models and comparing gene expression profiles of cell lines and PV CD34+ PV specimens, we have identified panels of 14 JAK2-dependent genes and 12 JAK2-independent genes. These two 14- and 12-gene sets could separate not only PV from normal CD34+ specimens, but also other MPN such as essential thrombocytosis and primary myelofibrosis from their normal counterparts. Conclusions: A subset of the aberrant gene expression in PV progenitor cells can be attributed to the action of the mutant kinase, but there remain a significant number of genes characteristic of the disease but deregulated by as yet unknown mechanisms. Genes deregulated in PV as a result of the action of JAK2V617F or independent of the kinase may represent other targets for therapy. Clin Cancer Res; 16(17); 4339–52. ©2010 AACR.

Functional characterization of Wilms tumor-suppressor WTX and tumor-associated mutants

The WTX, Wilms tumor-associated tumor-suppressor gene, is present on the X chromosome and a single WTX mutation may be sufficient to promote carcinogenesis. Unlike the WT1 tumor suppressor, a transcription factor, WTX lacks conserved functional protein domains. To study the function of WTX, we constructed inducible cell lines expressing WTX and tumor-associated WTX mutants. Induction of WTX inhibited cell growth and caused G1/G0 arrest. In contrast, a short, tumor-associated truncation mutant of WTX358 only slightly inhibited cell growth without a significant cell-cycle arrest, although expression of a longer truncation mutant WTX565 led to the growth inhibition and cell-cycle arrest to a similar extent as wild-type WTX. Like WT1, WTX slowed growth and caused cell-cycle arrest through p21 induction. Gene expression profiling showed that these two tumor-suppressors regulated genes in similar pathways, including those implicated in control of the cellular growth, cell cycle, cell death, cancer and cardiovascular system development. When gene expression changes mediated by wild-type WTX were compared with those affected by mutant forms, WTX565 showed a 55% overlap (228 genes) in differentially regulated genes, whereas WTX358 regulated only two genes affected by wild-type WTX, implying that amino-acid residues 358–561 are critical for WTX function.

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.

Abstract 1046: The histone methyl transferase MMSET/WHSC1 promotes an epithelial-mesenchymal transition and invasive properties of prostate cancer

Prostate cancer (PCa) remains the most common neoplasia diagnosed among men in the USA, and the second leading cause of cancer deaths. Recent evidence has indicated that epigenetic alterations contribute to the initiation and progression of PCa; however, the mechanisms by which chromatin modifiers influence this disease are still poorly understood. The histone methyl transferase MMSET (Multiple Myeloma Set Domain) is overexpressed in a number of different tumor types, including PCa. While the precise mechanism of MMSET action is still not well defined, it is believed to involve alteration of chromatin structure through dimethylation of lysine 36 on histone H3 (H3K36me2). Analysis of microarray databases indicates that MMSET expression rises in the progression from benign prostatic epithelium to localized and metastatic PCa. We found that PCa cell lines express significantly higher levels of MMSET compared to immortalized but non-transformed prostate cells. In t(4;14)+ myelomas, MMSET overexpression changes global levels of histone modifications, promoting H3K36me2, and repressing trimethylation of lysine 27 (H3K27me3). Similarly, H3K36me2 and H3K27me3 levels in PCa cell lines depended on the expression of MMSET, while depletion of MMSET in benign prostatic epithelial cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased soft agar colony formation and cell proliferation. Furthermore, inhibition of MMSET expression strikingly decreased cell migration and invasion, while overexpression of MMSET in immortalized non-transformed RWPE-1 cells promoted migratory and invasive properties. These findings suggest that high levels of MMSET play a pathogenic role in PCa progression. Overexpression of MMSET in RWPE-1 cells was accompanied by an epithelial to mesenchymal transition (EMT), characterized by a change in cell morphology and increased expression of mesenchymal markers vimentin and N-cadherin. Among a panel of EMT promoting genes analyzed, Twist1 expression was strongly regulated by MMSET both in the knockdown and the overexpression models. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the Twist1 locus, leading to an increase in H3K36me2 and a decrease in the repressive mark H3K27me3, suggesting a direct role of MMSET in regulation of Twist1. siRNA depletion of Twist1 in MMSET-overexpressing RWPE-1 cells blocked invasion, indicating that Twist1 was a critical target of MMSET responsible for the acquisition of EMT properties. Intriguingly, Twist1 expression was also elevated in a panel of myeloma cell lines overexpressing MMSET due to chromosomal translocation t(4;14). Collectively, these data suggest that MMSET has a critical role in cancer pathogenesis and progression through epigenetic regulation of metastasis-related genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1046. doi:1538-7445.AM2012-1046