Methyltransferase KMT2D Research Articles

Dynamic Changes in Gene Mutational Landscape With Preservation of Core Mutations in Mantle Cell Lymphoma Cells.

While studies have identified a number of mutations in mantle cell lymphoma (MCL), the list may still be incomplete and contribution to the pathogenesis remains unclear. We analyzed the mutational landscape of four mantle cell lymphoma biopsies obtained during an 8-year period from the same patient with his normal cells serving as control; we also established a cell line from the final stage of the disease. Numerous mutations with high allelic burden have been identified in all four biopsies. While a large subset of mutations was seen only in individual biopsies, the core of 21 mutations persisted throughout the disease. This mutational core is also maintained in the cell line that also displays DNA-methylation and cytokine secretion profiles of the primary mantle cell lymphoma cells. This cell line is uniquely sensitive to clinically relevant inhibitors of Bruton's Tyrosine Kinase. The response to Bruton Tyrosine Kinase's inhibition is enhanced by inhibitors of CDK4/6 and mTOR. Among the mutations seen in the primary and cultured MCL cells, mutations of three genes are involved in the control of H3K4 methylation: demethylase KDM5C, present already in the early disease, and methyltransferase KMT2D and cofactor BCOR, both of which are seen late in the disease and are novel and predicted to be pathogenic. The presence of these mutations was associated with hypermethylation of H3K4. Restoration of KDM5C expression affected expression of numerous genes involved in cell proliferation, adherence/movement, and invasiveness.

Loss of KMT2D induces prostate cancer ROS-mediated DNA damage by suppressing the enhancer activity and DNA binding of antioxidant transcription factor FOXO3

ABSTRACTHistone methyltransferase KMT2D has diverse functions and distinct mechanisms in different cancers. Although we have previously found KMT2D serves as an oncogene that promotes tumor growth and metastasis in prostate cancer (PCa), the functions and mechanisms of KMT2D are complicated and most remain undefined. Here, the function of KMT2D regarding DNA damage in PCa and the underlying mechanisms of KMT2D in epigenetic regulation were explored in a series of studies. Knockdown of KMT2D sensitized cells to DNA damage through the disturbance of antioxidative gene expression and increased levels of intracellular reactive oxygen species, which led to cell apoptosis and senescence. The loss of KMT2D reduced the abundance of enhancer activity markers H3K4me1 and H3K27ac, which blocked the DNA binding of FOXO3, a critical mediator of the cellular response to oxidative stress, and suppressed antioxidative gene transcription. Moreover, KMT2D deletion in PCa cells also increased their sensitivity to genotoxic anticancer drugs and a PARP inhibitor, which suggested that lower levels of KMT2D may mediate the response of PCa to particular treatments. These findings further highlighted the important role of KMT2D in PCa progression and suggested that targeting KMT2D might be therapeutically beneficial for advanced PCa treatment.

Loss of function of Kmt2d, a gene mutated in Kabuki syndrome, affects heart development in Xenopus laevis.

Kabuki syndrome is a haploinsufficient congenital multi-organ malformation syndrome, which frequently includes severe heart defects. Mutations in the histone H3K4 methyltransferase KMT2D have been identified as the main cause of Kabuki syndrome, however, the role of KMT2D in heart development remains to be characterized. Here we analyze the function of Kmt2d at different stages of Xenopus heart development. Xenopus Kmt2d is ubiquitously expressed at early stages of cardiogenesis, with enrichment in the anterior region including the cardiac precursor cells. Morpholino-mediated knockdown of Kmt2d led to hypoplastic hearts lacking the three-chambered structure. Analyzing different stages of cardiogenesis revealed that development of the first and second heart fields as well as cardiac differentiation were severely affected by loss of Kmt2d function. Kmt2d loss of function in Xenopus recapitulates the hypoplastic heart defects observed in Kabuki syndrome patients and shows that Kmt2d function is required for the establishment of the primary and secondary heart fields. Thus, Xenopus Kmt2d morphants can be a valuable tool to elucidate the etiology of the congenital heart defects associated with Kabuki syndrome.

Genetic mutational status of genes regulating epigenetics: Role of the histone methyltransferase KMT2D in triple negative breast tumors.

PurposeEpigenetic regulating proteins like histone methyltransferases produce variations in several functions, some of them associated with the generation of oncogenic processes. Mutations of genes involved in these functions have been recently associated with cancer, and strategies to modulate their activity are currently in clinical development.MethodsBy using data extracted from the METABRIC study, we searched for mutated genes linked with detrimental outcome in invasive breast carcinoma (n = 772). Then, we used downstream signatures for each mutated gene to associate that signature with clinical prognosis using the online tool “Genotype-2-Outcome” (http://www.g-2-o.com). Next, we performed functional annotation analyses to classify genes by functions, and focused on those associated with the epigenetic machinery.ResultsWe identified KMT2D, SETD1A and SETD2, included in the lysine methyltransferase activity function, as linked with poor prognosis in invasive breast cancer. KMT2D which codes for a histone methyltransferase that acts as a transcriptional regulator was mutated in 6% of triple negative breast tumors and found to be linked to poor survival. Genes regulated by KMT2D included RAC3, KRT23, or KRT14, among others, which are involved in cell communication and signal transduction. Finally, low expression of KMT2D at the transcriptomic level, which mirror what happens when KMT2D is mutated and functionally inactive, confirmed its prognostic value.ConclusionIn the present work, we describe epigenetic modulating genes which are found to be mutated in breast cancer. We identify the histone methyltransferase KMT2D, which is mutated in 6% of triple negative tumors and linked with poor survival.

PI3K Inhibition Activates SGK1 via a Feedback Loop to Promote Chromatin-Based Regulation of ER-Dependent Gene Expression

The PI3K pathway integrates extracellular stimuli to phosphorylate effectors such as AKT and serum-and-glucocorticoid-regulated kinase (SGK1). We have previously reported that the PI3K pathway regulates estrogen receptor (ER)-dependent transcription in breast cancer through the phosphorylation of the lysine methyltransferase KMT2D by AKT. Here, we show that PI3Kα inhibition, via a negative-feedback loop, activates SGK1 to promote chromatin-based regulation of ER-dependent transcription. PI3K/AKT inhibitors activate ER, which promotes SGK1 transcription through direct binding to its promoter. Elevated SGK1, in turn, phosphorylates KMT2D, suppressing its function, leading to a loss of methylation of lysine 4 on histone H3 (H3K4) and a repressive chromatin state at ER loci to attenuate ER activity. Thus, SGK1 regulates the chromatin landscape and ER-dependent transcription via the direct phosphorylation of KMT2D. These findings reveal an ER-SGK1-KMT2D signaling circuit aimed to attenuate ER response through a role for SGK1to program chromatin and ER transcriptional output.

Grainyhead-like-2 confers NK-sensitivity through interactions with epigenetic modifiers

Natural Killer (NK) cells suppress tumor initiation and metastasis. Most carcinomas are heterogeneous mixtures of epithelial, mesenchymal and hybrid tumor cells, but the relationships of these phenotypes to NK susceptibility are understood incompletely. Grainyhead-like-2 (GRHL2) is a master programmer of the epithelial phenotype, that is obligatorily down-regulated during experimentally induced Epithelial-Mesenchymal Transition (EMT). Here, we utilize GRHL2 re-expression to discover unifying molecular mechanisms that link the epithelial phenotype with NK-sensitivity. GRHL2 enhanced the expression of ICAM-1, augmenting NK-target cell synaptogenesis and NK killing of target cells. The expression of multiple interferon response genes, including ICAM1, anti-correlated with EMT. We identified two novel GRHL2-interacting proteins, the histone methyltransferases KMT2C and KMT2D. Mesenchymal-epithelial transition, NK-sensitization and ICAM-1 expression were promoted by GRHL2-KMT2C/D interactions and by GRHL2 inhibition of p300, revealing novel and potentially targetable epigenetic mechanisms connecting the epithelial phenotype with target cell susceptibility to NK killing.

Loss of KDM6A/UTX Accelerate the Development of Multiple Myeloma

In multiple myeloma (MM), inactivating mutations and deletions affecting the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to add activation marks on histones and remove methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. Importantly, all these coregulators are found significantly mutated in MM and their function may converge into a tumor suppressive pathway. Our goal is to understand how KDM6A loss contributes to the development of MM.We modeled the loss of KDM6A in MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology. Mutant allele frequency over time post electroporation of RNP revealed a growth advantage of KDM6A mutant alleles. By 2 weeks of growth most of the cells in culture harbored KDM6A gene disruption and exhibit elimination of KDM6A protein confirming the tumor suppressive role of KDM6A in MM. We used these isogenic polyclonal edited cell lines with KDM6A wild type or mutated to identify KDM6A binding sites and enhancers affected by the loss of KDM6A. As well, we knock-in an HA tag on endogenous KDM6A to identify DNA regions occupied by KDM6A. To understand the importance of KDM6A demethylase activity in the tumor suppressive effect of KDM6A, we developed stable cell lines with a doxycycline-inducible form of KDM6A wild-type (WT) or lacking demethylase activity (JmjC-dead). We found that about 20% of the genes deregulated by re-expression of WT and jmjC-dead KDM6A overlap suggesting that demethylase activity is not essential for all KDM6A functions in MM.Importantly, we confirmed the tumor suppressive role of KDM6A in a novel mouse model of MM in which KDM6A is deleted specifically in the B cell compartment. Briefly, we isolated CD19cre-/+ (control) or CD19cre-/+ Kdm6afl/fl fetal liver cells and transduced these cells with a constitutively activated form of the IL-6 coreceptor (L-GP130) that activates the JAK/STAT pathway. Mice transplanted with CD19cre-/+ Kdm6afl/fl fetal liver cells developed MM by 7 weeks post transplantation while mice transplanted with CD19cre+/- fetal liver cells did not developed MM by20 weeks. Necroscopy and flow cytometry analysis demonstrated infiltration of CD138+ cells in bone marrow, spleen, liver and kidney of mice that developed MM. In the future we will use this model to explore how loss of KDM6A affects chromatin structure in vivo and how it changes the characteristics of MM. These studies are expected to provide new insights that lead to the development of more effective MM therapies which directly target mechanisms of chromatin structure regulation. DisclosuresLicht:Celgene: Research Funding.

Epigenetic Regulation and Therapeutic Targeting in Myeloma

Genetic alterations of epigenetic regulators that alter the repressive trimethylation of lysine 27 on histone H3 (H3K27me3) are a recurrent feature in cancer. The histone demethylase UTX/KDM6A Is mutated in ~5% of multiple myeloma (MM) at diagnosis and is commonly absent in MM cell lines, derived from patients with advanced disease. KDM6A forms a complex containing H3K4 specific methyltransferases KMT2D and KMT2C, the histone acetyltransferase CBP/p300 and SWI/SNF chromatin-remodelers. Collectively the complex adds activation marks on histones and removes the gene repression associated H3K37me mark at enhancers. Removal of KDM6A from MM cells using CRISPR and re-expression of KDM6A in deficient cells showed that KDM6A has tumor suppressor function and regulates genes involved in cell adhesion. ChIP-studies showed that loss of KDM6A lead to decreased H3K27 acetylation and increased H3K27me at specific loci, but at other loci changes in H3K27Ac are the major effect of changes in KDM6A expression. KDM6A loss shifts the balance of gene expression to repression and KDM6A null cells had heightened sensitivity to EZH2 inhibitors. H3K27me/Ac chromatin modifications are also deregulated in MM associated with t(4;14) and overexpression of the NSD2/MMSET histone methyltransferase. NSD2 overexpression leads to a ~10-fold genome wide increase in histone 3 lysine 36 dimethylation (H3K36me2), a chromatin mark associated with gene activation and similar loss of H3K27me3. As a result, many genes are aberrantly activated and NSD2 aberrant stimulates cell growth in part through activation of c-myc. However, some loci are repressed in the presence of high NSD2 due to redistribution of EZH2. These genes may also be important for pathogenesis and growth since NSD2 high cells also show increased sensitivity to EZH2 inhibitors. Nevertheless, the global loss of H3K27me3 makes the chromatin structure of NSD2 overexpressing MM cells more “open” and these cells demonstrated increased DNA damage in response to genotoxic agents. At the same time NSD2 overexpression increased increases DNA repair processes making cells relatively resistant to chemotherapy, potentially explaining the poor prognosis of such patients. A point mutation in NSD2, E1099K is found in some MM patients, the commonly utilized MM.1 model cell line and in up to 15% of cases of relapsed childhood acute lymphocytic leukemia (ALL). This mutation increases the activity of the enzyme and also globally increases H3K36me2 and decreases H3K27me3. Removal of the mutant allele from ALL cells lines reverses of histone methylation patterns, deceased growth, altered cellular adhesion and increased susceptibility to therapy. RNA-seq analysis showed that similar to the UTX mutation, NSD2 mutation activated a program of cell adhesion, motility and signaling. Furthermore in in ALL, the activity of mutant NSD2 blocks glucocorticoid induced expression of pro-apoptotic genes, promoting tumor relapse. In summary deregulation of histone methylation patterns in MM plays an important role in disease pathogenesis and progression. Rebalancing histone modifications using appropriate inhibitors represent a potential therapeutic strategy. DisclosuresLicht:Celgene: Research Funding.

CARM1 methylates MED12 to regulate its RNA-binding ability.

The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain-containing effector molecule, TDRD3. Chromatin immunoprecipitation-seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.

Dissecting KMT2D missense mutations in Kabuki syndrome patients.

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

Histone methyltransferase KMT2D sustains prostate carcinogenesis and metastasis via epigenetically activating LIFR and KLF4

Abnormalities in epigenetic modifiers are emerging as driving events in prostate cancer (PCa). The histone methyltransferase KMT2D, a frequently aberrant epigenetic modifier in various tumors, has an undefined role in PCa. Moreover, little is known regarding KMT2D’s mutation in Chinese patients or its downstream signaling pathways and targets. Here, we profiled the mutational spectrum of 32 significantly PCa-associated genes by using disease-targeted sequencing, and found that KMT2D was highly mutated (63.04%, 29/46) in Chinese patients. Moreover, high KMT2D transcription was also associated with poor prognosis in an independent cohort (n = 51). In KMT2D-knockdown PC-3 and DU145 cells, cell proliferation (P < 0.01), invasion (P < 0.001), and migration (P < 0.01) were consequently suppressed. KMT2D depletion effectively suppressed tumor growth by 92.21% in vivo. Notably, integrative analyses of RNAseq and ChIPseq characterized two crucial genes downregulated by KMT2D, leukemia inhibitory factor receptor (LIFR) and Kruppel-like factor-4 (KLF4), which are regulators in PI3K/Akt and EMT, respectively. Our present study revealed that KMT2D epigenetically activates PI3K/Akt pathway and EMT by targeting LIFR and KLF4 and thus serves as a putative epigenetic-based target for treating PCa.

Abstract 12: KMD6A/UTX loss enhances the malignant phenotype of multiple myeloma and sensitizes cells to EZH2 inhibition

Abstract In multiple myeloma (MM), inactivating mutations and deletions encompassing the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. Future sequencing studies may show an increased incidence of mutation with disease relapse. KMD6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases working as a scaffold for a multiprotein complex containing H3K4-specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300, and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to remove the gene repression associated methylation of lysine 27 on histone H3 (H3K27me), a mark placed by EZH2. Hence loss-of-function mutations of KDM6A may affect the function of this complex, which is common in B-cell malignancies. We found that KDM6A act as a tumor suppressor in MM in vivo and in vitro, but how this exactly happens and which genes are affected is incompletely understood. We modeled the loss of KDM6A in MM in vitro, using a pair of cell lines, ARP-1 (KDM6A wild type) and ARD (homozygous KDM6A deletion), derived from the same MM patient. As well, we used CRISPR-Cas9 mediated genome editing to disrupt KDM6A gene in cell lines. We examined gene expression profiles in ARP-1 vs. ARD cells, and vs. ARD cells upon reexpression of KDM6A, in a doxycycline-inducible manner, by whole transcriptome (RNA sequencing). To understand how KDM6A demethylase activity contributes to tumor suppressive role of KDM6A, we abolished the demethylase activity using directed mutagenesis targeting the JmjC domain. Disruptive mutations in functional domain of KDM6A provide a growth advantage in MM cell in vitro. Many genes induced upon KDM6A add-back are found upregulated in KDM6A wild-type ARP-1 cells. Mass spectrometry analysis of these cell lines and the add-back system showed no difference in global H3K27me3 levels, suggesting that the tumor suppressive role of KDM6A does not involve alteration of H3K27 methylation or that the changes in this histone mark following KDM6A loss are loci-specific. Reexpression of JmjC-dead KDM6A and wt KDM6A had similar effect on growth and clonogenicity, suggesting that demethylase activity is dispensable. KDM6A demethylase activity is dispensable for the tumor-suppressive effect of KDM6A in MM. Loss of KDM6A alters the transcriptome of MM cells. We therefore hypothesized that the scaffolding properties of KDM6A may be essential for the tumor-suppressive role of KDM6A. We plan to use CRISPR-Cas9 mediated genome editing to create specific mutations and flag-insertion in KDM6A gene, in both cell lines and mouse model, to further investigate the molecular mechanism of KDM6A activity. Citation Format: Daphne Dupere-Richer, Teresa Ezponda, Christine Will, Eliza Caroline Small, Nobish Varghese, Patel Tej, Xiaoxiao Huang, Zheng Yupeng, Giovanni Tonon, Neil Kelleher, Jonathan Keats, Jonathan D. Licht. KMD6A/UTX loss enhances the malignant phenotype of multiple myeloma and sensitizes cells to EZH2 inhibition [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 12.

Mutational Landscapes of Smoking-Related Cancers in Caucasians and African Americans: Precision Oncology Perspectives at Wake Forest Baptist Comprehensive Cancer Center.

Background: Cancers related to tobacco use and African-American ancestry are under-characterized by genomics. This gap in precision oncology research represents a major challenge in the health disparities in the United States.Methods: The Precision Oncology trial at the Wake Forest Baptist Comprehensive Cancer Center enrolled 431 cancer patients from March 2015 to May 2016. The composition of these patients consists of a high representation of tobacco-related cancers (e.g., lung, colorectal, and bladder) and African-American ancestry (13.5%). Tumors were sequenced to identify mutations to gain insight into genetic alterations associated with smoking and/or African-American ancestry.Results: Tobacco-related cancers exhibit a high mutational load. These tumors are characterized by high-frequency mutations in TP53, DNA damage repair genes (BRCA2 and ATM), and chromatin remodeling genes (the lysine methyltransferases KMT2D or MLL2, and KMT2C or MLL3). These tobacco-related cancers also exhibit augmented tumor heterogeneities. Smoking related genetic mutations were validated by The Cancer Genome Atlas dataset that includes 2,821 cases with known smoking status. The Wake Forest and The Cancer Genome Atlas cohorts (431 and 7,991 cases, respectively) revealed a significantly increased mutation rate in the TP53 gene in the African-American subgroup studied. Both cohorts also revealed 5 genes (e.g. CDK8) significantly amplified in the African-American population.Conclusions: These results provide strong evidence that tobacco is a major cause of genomic instability and heterogeneity in cancer. TP53 mutations and key oncogene amplifications emerge as key factors contributing to cancer outcome disparities among different racial/ethnic groups.

Reduced Expression of Histone Methyltransferases KMT2C and KMT2D Correlates with Improved Outcome in Pancreatic Ductal Adenocarcinoma.

Genes encoding the histone H3 lysine 4 methyltransferases KMT2C and KMT2D are subject to deletion and mutation in pancreatic ductal adenocarcinoma (PDAC), where these lesions identify a group of patients with a more favorable prognosis. In this study, we demonstrate that low KMT2C and KMT2D expression in biopsies also defines better outcome groups, with median survivals of 15.9 versus 9.2 months (P = 0.029) and 19.9 versus 11.8 months (P = 0.001), respectively. Experiments with eight human pancreatic cell lines showed attenuated cell proliferation when these methyltransferases were depleted, suggesting that this improved outcome may reflect a cell-cycle block with diminished progression from G0-G1 RNA-seq analysis of PDAC cell lines following KMT2C or KMT2D knockdown identified 31 and 124 differentially expressed genes, respectively, with 19 genes in common. Gene-set enrichment analysis revealed significant downregulation of genes related to cell-cycle and growth. These data were corroborated independently by examining KMT2C/D signatures extracted from the International Cancer Genome Consortium and The Cancer Genome Atlas datasets. Furthermore, these experiments highlighted a potential role for NCAPD3, a condensin II complex subunit, as an outcome predictor in PDAC using existing gene expression series. Kmt2d depletion in KC/KPC cell lines also led to an increased response to the nucleoside analogue 5-fluorouracil, suggesting that lower levels of this methyltransferase may mediate the sensitivity of PDAC to particular treatments. Therefore, it may also be therapeutically beneficial to target these methyltransferases in PDAC, especially in those patients demonstrating higher KTM2C/D expression. Cancer Res; 76(16); 4861-71. ©2016 AACR.

Abstract 112: Genetic alterations in molecular tumor subgroups of malignant pleural mesothelioma

Abstract Background: Malignant pleural mesothelioma (MPM) is a very aggressive neoplasm linked to asbestos exposure. In spite of research efforts to develop more effective therapeutic approaches, its prognosis remains poor, because of the absence of curative treatment. The identification of molecular profiles by omic analyses is a powerful approach to better define MPM subclasses and new targeted therapies. A recent transcriptomic analysis allowed to define a robust MPM classification and to identify two subgroups, C1 and C2, linked to histology and survival. The main finding was to separate epitheloid MPM, the most frequent histologic subtype, within these two subgroups. Epitheloid MPM with a worse survival prognosis were classified in the C2 subgroup. To better define the heterogeneity of MPM and establish a more precise classification, we determined genetic alterations in C1 and C2 MPM. Material and Methods: Using a gene candidate approach, we performed Next-Generation Sequencing (Miseq, Illumina) in 84 MPM including 49 MPM primary cultures and 35 frozen tumors to identify genetic alterations in specific genes with a 200X depth. We selected 22 genes including key altered genes in mesothelial carcinogenesis (NF2, CDKN2A, CDKN2B BAP1, TP53 and LATS2) previously sequenced by Sanger method, genes mutated at low frequency in MPM (KRAS, HRAS, EGFR⋯) and genes recently suggested as altered in MPM (CUL1, KMT2D, SETD2⋯). Results: Previous mutations identified by Sanger sequencing in our collection have been confirmed. The results showed an enrichment in C&amp;gt;T transitions, which was already observed in an exome sequencing study in MPM. We identified 93 variants inducing protein structure modification. Among them, 70% were deleterious to the function of the protein (deletion/insertion, splice-site mutation, substitutions nonsense and damaging missense predicted by SIFT or Polyphen). For most of the genes, the mutation frequency was consistent with literature data. The highest alteration frequency was found in CDKN2A, CDKN2B, NF2 and BAP1 genes (over 25%), lower frequencies were found in TP53, LATS2, KMT2D and SETD2 genes (5% to 20%). The alteration frequency of four genes was different between C1 and C2 tumor subgroups. BAP1 mutations were more frequent in C1 MPM, as we showed previously. We also identified new relevant mutations notably in the methyltransferases SETD2 and KMT2D that are also associated to C1 MPM. Mutations in TP53 were significantly associated with C2 MPM. Conclusion: We defined the genetic alterations signatures of C1 and C2 tumor subgroups. Mutations in enzymes involved in chromatin organization seem to be characteristic of the C1 subgroup. Interestingly, TP53 mutations, which are known to provide tumor aggressiveness, are found in the C2 subgroup gathering patients with the worse prognosis. Ongoing analysis of 83 additional MPM would allow to confirm statistically all the associations we observed. Citation Format: Lisa Quetel, Robin Tranchant, Clément Meiller, Sandrine Imbeaud, Annie Renier, Françoise Le Pimpec-Barthes, Jessica Zucman-Rossi, Marie-Claude Jaurand, Didier Jean. Genetic alterations in molecular tumor subgroups of malignant pleural mesothelioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 112.

A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes.

The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2β. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo. Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.

Abstract IA18: Functional characterization of the tumor suppressor lysine-specific methyltransferase KMT2D in lymphoma

Abstract Genomic studies have revealed surprising new insights into the genetics of human B-cell lymphomas. Of particular note the lysine-specific methyl transferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and in diffuse large B-cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations in lymphoma are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and promotes lymphoma development in vivo. Integrative analyses of KMT2D function indicate specific and focal effects on H3K4 methylation and gene expression. These include B-cell growth and differentiation signals such as the CD40, JAK/STAT, and B-cell receptor pathways. Moreover, KMT2D targets include frequently mutated tumor suppressor genes such as TNFAIP3/A20, SOCS3, and TNFRSF14/HVEM. In this manner, KMT2D deficiency disrupts the physiological process of B-cell differentiation, facilitates malignant outgrowth, and can impede therapies acting on the CD40 and B-cell receptor pathways. Citation Format: Ana Ortega-Molina, Isaac W. Boss, Andres Canela, Heng Pan, Yanwen Jiang, Chunying Zhao, Man Jiang, Deqing Hu, Ji-Eun Lee, Hua-Tang Chen, Daisuke Ennishi, David W. Scott, Christoffer Hother, Shichong Liu, Xing-Jun Cao, Wayne Tam, Rita Shaknovich, Benjamin A. Garcia, Randy D. Gascoyne, Kai Ge, Ali Shilatifard, Olivier Elemento, Andre Nussenzweig, Ari M. Melnick, Hans-Guido Wendel. Functional characterization of the tumor suppressor lysine-specific methyltransferase KMT2D in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA18.

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.

Abstract LB-064: Characterization of the tumor suppressor function of the lysine-specific methyltransferase KMT2D in follicular lymphoma

Abstract Follicular lymphoma (FL) is a common and incurable form indolent B-cell lymphoma. Genomic studies have now catalogued many recurrent mutations in FL. Epigenetic regulators have emerged as the most common targets of somatic point mutations. For example, the histone methyltransferase KMT2D (MLL4/MLL2) is the most frequently mutated gene in FL with reported mutational frequencies of up to 84%. However, the transcriptional and biological consequences of KMT2D loss of function are currently unknown. Using a well-described murine model of FL, we showed that deficiency of KMT2D promotes the initiation of indolent FL in vivo. To explore the mechanism of KMT2D-mediated tumor suppression we used cross species comparisons of gene expression, histone H3K4 methylation marks and KMT2D genomic occupancy. These analyses converge on a relatively small group of conserved target genes. Strikingly, they include bona fide tumor suppressors and regulators of B-cell proliferation (e.g. TNFAIP3/A20, SOCS3, ARID1A, and TNFRSF14). Our results indicate that KMT2D restrains FL development through simultaneous effects on multiple key regulators of B-cell behavior. Note: This abstract was not presented at the meeting. Citation Format: Ana Ortega-Molina, Isaac Boss, Heng Pan, Yanwen Jiang, Deqing Hu, Xin Gao, Rita Shaknovich, Ali Shilatifard, Ari M. Melnick, Hans-Guido Wendel. Characterization of the tumor suppressor function of the lysine-specific methyltransferase KMT2D in follicular lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-064. doi:10.1158/1538-7445.AM2015-LB-064