ALBERT

Description: During the last years, the study of molecular alterations associated with multiple myeloma (MM) has been mostly focused on the analysis of the genome, transcriptome and DNA methylome. These analyses are showing that (epi)genetic heterogeneity and extensive perturbation of the transcriptional landscape are hallmarks of MM. Our previous analysis of the whole DNA methylome of MM revealed that this epigenetic mark globally shows a poor correlation with gene expression, and therefore did not allow us to better understand gene deregulation in MM. In contrast, the chromatin structure and histone modifications are emerging as essential epigenetic layers to understand the mechanisms underlying gene expression changes in cancer, but remain widely unexplored in MM. We have now performed a deep ChIP-seq profiling of CD138+ sorted cells from bone marrow samples obtained from four MM patients and three biological replicates of normal plasma cells (NPCs) using antibodies against H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3 and H3K9me3. Different combinations of these marks allow us to segment the MM and NPC genome into functional chromatin states, including active, weak or poised promoters, active or weak enhancers, transcriptional elongation, polycomb-repressed regions and heterochromatic regions. The initial unsupervised exploration of the data showed that the chromatin landscape of MM is widely altered as compared to NPCs. A supervised analysis of chromatin states revealed that MM globally shows a more active chromatin structure than NPCs. From over 40,000 regions identified with differential chromatin structure between MM and NPCs, 88% were de novo activated in neoplastic plasma cells. Analyzing the chromatin of individual genes, we observed that there were roughly ten times more genes gaining activity upon neoplastic transformation than those acquiring repressed chromatin marks. Interestingly, the genes showing more activate chromatin were enriched with biosynthesis and metabolic processes, while genes with repressed chromatin were related to gene ontology terms related to B cell signaling. Among those genes gaining de novo activity in MM, we selected several candidates and we are currently performing functional in vitro assays to explore their implication in MM pathogenesis. Furthermore, as extensive chromatin activation is a hallmark of MM, we are currently analyzing additional 15 MM cases and NPCs by ChIP-seq for H3K27ac (marking active promoters and enhancers) and ATAC-seq (marking active regulatory regions) to validate our initial findings and explore chromatin heterogeneity in MM. Collectively, our initial exploration of histone modification profiles in MM has revealed that MM cells acquire a more active chromatin landscape, with thousands of regions gaining activation as compared to NPCs. Reversing this global activation by epigenetic drugs, such as BET inhibitors, may represent an attractive therapeutic option for MM. During the meeting, updated information will be presented, including data from all 19 MM patients studied as well as functional data from new candidate genes involved in MM pathogenesis. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Description: Deregulation of long non-coding RNAs (lncRNAs) is emerging as a common feature of different human tumors and their investigation may uncover novel biomarkers and oncogenic mechanisms. Previous studies have suggested that the alteration of some lncRNAs may play an important role in the pathogenesis of multiple myeloma (MM); however, the complete expression landscape of lncRNAs has not been elucidated. In the present work we characterized the lncRNAs transcriptome of MM and determined the potential involvement of lncRNAs in the pathogenesis and clinical behavior of MM. To characterize the MM transcriptome, we performed paired-end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients and in 3 bone marrow PCs (BMPCs) of healthy donors, as well as in distinct normal B-cell populations (Naïve, Centroblasts, Centrocytes, Memory and Tonsilar PCs). We identified 40,511 novel lncRNAs that were expressed, accounting for more than half of MM transcriptome (56%). This group of novel lncRNAs together with previously annotated lncRNAs comprised most (82%) of the MM transcriptome. We studied the transcriptional heterogeneity in MM samples and observed that lncRNAs showed a much more heterogeneous expression than coding genes, suggesting that these elements could contribute to the biological heterogeneity of the disease. Moreover, to determine differentially expressed genes, each MM patient was compared to normal BMPCs, detecting 19,886 lncRNAs deregulated (10,351 overexpressed and 9,535 downregulated) in more than 50% of patients. We then analyzed the transcriptional dynamics of MM considering the different stages of B-cell differentiation and focused on a group of 989 lncRNAs that were upregulated specifically in plasma cells from MM in comparison with the rest of B-cell stages (MM-specific lncRNAs). Next, we aimed to determine whether upregulation of MM-specific lncRNAs in MM was under epigenetic control so we analyzed the distribution of six histone modifications with non-overlapping functions (H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3, and H3K9me3) of within the lncRNAs of interest by ChIP-seq in MM cases as compared to normal B cell subtypes. We detected 89 lncRNAs with de novo epigenomic activation. These data suggest an epigenetic rewiring in MM where the loci of most MM-specific lncRNAs are in an inactive state in normal cells and become active in MM. We focused on a specific lncRNA, LINC-SMILO, de novo epigenetically active and expressed in MM cells to determine whether upregulation of this lncRNA could play a role in the pathogenesis of the disease. Knockdown of LINC-SMILO in 3 different MM cell lines (MM.1S, MM.1R and KMS-11) using two different shRNAs, resulted in reduced proliferation and induction of apoptosis of myeloma cells. Using low input RNA-seq (MARS-seq), we found that inhibition of LINC-SMILO was associated with activation of ERVs (Endogenous retroviruses) and increase in interferon (IFN) induced genes and activation of IFN pathways, essential for MM cells survival. Finally, we aimed to determine whether the use of specific lncRNAs could improve the current prognostic stratification of MM patients using the IA11 release of CoMMpass data. We analyzed the prognostic value of lncRNAs using COX regression analysis and Backward elimination of Stepwise regression analysis, obtaining that the overexpression of the lncRNA PDLIM1P4 together with 1q amplification and 17p deletion stratified MM patients in three different risk groups (Figure 1). In summary, our study shows that the lncRNA transcriptome is widely altered in MM and suggests that some of the identified lncRNAs have marked prognostic influence and can be used as potential therapeutic targets for MM. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Melnick:KDAc Therapeutics: Membership on an entity's Board of Directors or advisory committees; Constellation Pharmaceuticals: Consultancy; Epizyme: Consultancy; Janssenn: Research Funding.

Description: Chromatin regulators including methyltransferases are frequently disrupted by inactivating mutations in a wide spectrum of cancers. In contrast gain of function mutations that increase enzyme activity are much more rare and found in only two enzymes within the family of approximately 60 SET domain methyltransferases, MMSET and EZH2. Specific recurrent mutations in either MMSET or EZH2 are detected in hematological malignancies in conjunction with increased methylation of their histone substrates, H3K36 and H3K27, respectively. We and others characterized the MMSET E1099K mutant, which generates a clear molecular hallmark of increased dimethylation of H3K36 (H3K36me2). This gain of function also induces a corresponding decrease in trimethylation at H3K27 (H3K27me3). The altered balance between H3K36me2 and H3K27me3 mimics global chromatin effects that we previously reported in multiple myeloma lines that overexpress wild type MMSET due to the t(4:14) translocation. Research led by the Pediatric Cancer Genome Project has shown that MMSET E1099K is detected in ~10% of B-cell acute lymphoblastic leukemia (ALL) and particularly enriched in samples following disease relapse. Therefore, we hypothesize that this mutation is a driver of ALL progression and mediates resistance to therapy. To address this question, we have rescued or disrupted the endogenous E1099K mutation in cell lines from relapsed ALL and characterized the resulting molecular and phenotypic effects. The sequence context of the E1099K mutation allows allele-specific CRISPR targeting, which we have exploited to create isogenic lines that differ by MMSET E1099K status. We isolated subclones from three parental lines and sequencing analysis revealed distinct outcomes: indel mutations that disrupt the E1099K allele or rescue to WT by interchromosomal gene conversion. Comparing these subclones to non-targeted controls showed that loss of E1099K causes a 3-fold reduction in H3K36me2 levels and 5-fold increase in H3K27me3. Mass spectrometry-based measurement of methylation kinetics revealed that E1099K accelerates the rate constant of conversion from unmodified H3K36 to monomethylation. This corresponds to an increased ability of the mutant enzyme to turnover substrate in vitro. Because H3K36 and H3K27 methylation both contribute to transcriptional regulation, we compared expression profiles and identified a common set of genes overexpressed in cells lines harboring the mutant allele. These overexpressed genes included components of the SLIT/ROBO, WNT/Beta-Catenin, and cell adhesion pathways. Correlating with these expression changes, several phenotypic changes resulted from loss of E1099K, such as reduction in proliferation, colony-forming ability, and adhesive properties. Loss of E1099K also increased sensitivity to chemotherapeutic agents used to treat ALL, such as doxorubicin and dexamethasone. One gene consistently upregulated in the presence of E1099K was the transcription factor ETV1. In support of ETV1 being a key mediator of E1099K-driven phenotypes we found that parental lines treated with an ETV1 inhibitor displayed reduced viability and adhesion. To further our phenotypic analysis, subclones and parental lines have been tagged with luciferase or fluorescent markers to assess invasion in models of metastasis and allow in vivo monitoring of tumors. Collectively, we have developed gene-targeting reagents specific for the MMSET E1099K mutation and used these tools to show its impact on global chromatin environment and cell phenotypes. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 523 Multiple myeloma (MM) is associated with recurrent chromosomal translocations that lead to overexpression of known and putative oncogenes. The MMSET (Multiple Myeloma SET domain) protein is overexpressed in MM patients harboring the translocation t(4;14), and is believed to be the driving factor in the pathogenesis of this subtype of MM. Additionally, through unknown mechanisms, MMSET expression is enhanced in many different tumors including neuroblastoma, colorectal carcinomas and prostate cancer. Our initial studies showed that overexpression of MMSET in myeloma cells induces a global increase in H3K36 dimethylation, a mark associated with actively transcribed genes, and a concomitant genome-wide loss of H3K27 trimethylation. This effect is due to an enhanced rate of H3K36 methylation as well as an increased rate of H3K27 demethylation, and causes a physical loosening of the chromatin structure, leading to altered gene expression. Using genomic-wide chromatin precipitation/next generation sequencing (ChIP-seq), and genetically matched cells in which the overexpressed MMSET allele was knocked out (KMS11-TKO), we found that increased H3K36 methylation due to MMSET disrupts the normal genomic H3K36me2 architecture, from a mostly promoter-enriched mark to a modification spread throughout gene bodies. Additionally, many loci activated in response to MMSET showed the loss of H3K27 methylation near the transcriptional start site. Surprisingly, while MMSET-overexpressing cells lose the H3K27me3 mark globally, specific loci display enrichment of this modification and are transcriptionally repressed. This is associated with a shift in the genomic localization of the H3K27 methyltransferase, EZH2, in the presence of MMSET. Thus, in addition to gene activation through increased H3K36 methylation, overexpression of MMSET can also induce gene repression by imposing EZH2 and H3K27me3 accumulation at specific genomic loci. Using KMS11-TKO cells, we performed a structure-function study to identify important domains of MMSET. Re-introduction of the wild-type MMSET into the TKO cells induces the H3K36/H3K27 epigenetic switch, enhances proliferation and colony formation of these cells, and leads to differential gene expression. These biological activities require all PHD finger domains of the protein (putative chromatin binding regions), the second PWWP domain (a non-specific DNA binding domain) and the functional SET/histone methyltransferase domain. When MMSET constructs containing mutations or deletions in one of these domains were overexpressed in TKO cells, the resulting protein failed to methylate histones, alter growth or change gene expression. Furthermore, point mutations in the PHD domains 2 or 3, analogous to those found in the MMSET homologue NSD1 in Sotos syndrome, prevented MMSET from binding to chromatin and altering histone methylation. By contrast, an MMSET construct lacking the C-terminal PHD4 domain bound chromatin, induced methylation of H3K36, but was unable to mediate a complete loss of the H3K27me3 mark or fully stimulate growth. Together this suggests that pathogenic activity of MMSET could be interrupted by blocking its binding to chromatin, its intrinsic H3K36 methylation activity or by blocking demethylases of H3K27 that might interact with MMSET. To validate MMSET as a therapeutic target, we used KMS11 t(4;14)+ cells that express MMSET specific shRNA in the presence of doxycycline. In vitro, doxycycline-mediated knockdown of MMSET leads to loss of H3K36 methylation, gain of H3K27 methylation and decreased proliferation. KMS11 cells rapidly formed tumors when injected into immunocompromised mice. Doxycycline treatment caused a dramatic decrease in the volume of established tumors and extended survival in mice. Interestingly, removal of the doxycycline treatment restarted tumor growth in some, but not all animals. This suggests that, at least in some cases, targeting MMSET alone may be sufficient to completely eliminate the tumor burden. Together, our work elucidates some of the mechanisms used by MMSET to induce an oncogenic phenotype and identifies domains to be considered in designing novel inhibitors of MMSET function. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions, such as transcriptional aberrations, may play a role in its development. Moreover, MDS prevalence is almost exclusive to older patients, suggesting that elderly-related alterations may predispose to the development of this clinical entity. Thus, study of the transcriptional lesions occurring in the aging-MDS axis could shed some light of the molecular bases of the disease. To characterize the transcriptional profile of HSCs in aging and MDS, we isolated CD34+, CD38-, CD90+, CD45RA- cells from 11 untreated MDS patients with unilineage and multilineage dysplasia (median of 75 y/o), as well as from 16 young and 8 elderly healthy donors (median of 21 and 70 y/o, respectively), and their expression profile was analyzed using MARS-seq. Unsupervised principal component analysis demonstrated that the three groups of HSCs clustered separately, indicating that different expression profiles characterize healthy young and elderly, and MDS-associated HSCs. To better understand the gene expression deregulation of HSCs, we analyzed the transcriptional dynamisms along the aging-MDS axis, detecting groups of genes following different patterns of expression. Some gene clusters showed exclusive alteration either in aging or in the progression from elderly HSCs to MDS-HSCs, other groups of genes presented a continuous alteration along the axis, and some displayed opposite regulation in aging and in the transition to MDS (Figure 1). Genes showing specific downregulation in aging were involved in DNA damage sensing and repair, and in cell cycle regulation, whereas genes overexpressed in this process were enriched in apoptosis regulators and in cancer-associated genes, including AML-related factors. These findings indicate that transcriptional changes in aging may predispose for MDS and AML, and potentially other malignancies. Interestingly, we detected a group of genes in which the age-mediated upregulation of gene expression was reversed to that of young HSCs in MDS, indicating a "rejuvenation" profile of malignant HSCs. These genes were involved in response to inflammation, to different types of stress conditions such as hypoxia or radiation, and to cytokines. Elderly HSCs may upregulate such genes in response to the known inflammatory microenvironment of elderly bone marrow. Intriguingly, the decrease in expression detected in MDS suggests that malignant HSCs lose the ability of reacting to such stimuli, possibly favoring their survival in a hostile microenvironment. Finally, the analyses performed allowed for the identification of genes showing MDS-specific deregulation. Genes specifically overexpressed in MDS compared to normal (both young and elderly) HSCs, we enriched in transcriptional and epigenetic regulators, and among them, we detected the presence of DDIT3/CHOP, a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. To determine its potential effects on hematopoietic deregulation, DDIT3 was exogenously overexpressed in healthy HSCs. Notably, its upregulation produced an erythroid bias in an ex-vivo differentiation system, with an increase in the percentage of erythroblasts and a decrease in granulocytes and monocytes compared to HSCs transduced with the empty vector. Transcriptomic analysis of transduced HSCs not subjected to differentiation demonstrated how DDIT3 overexpression produced an erythroid-prone state of HSCs, suggesting it may act as a pioneer factor in MDS-HSCs. Furthermore, gene set enrichment analysis showed that DDIT3 overexpression produced an MDS-like transcriptional profile, suggesting this factor may be key in the acquisition of the disease. Altogether, our results demonstrate that HSCs undergo transcriptional changes in the aging-MDS axis that may alter their intrinsic functions as well as their response to the microenvironment, ultimately contributing to the acquisition of the disease. In particular, our data show that DDIT3 may be a potential driver of MDS transformation. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Díez-Campelo:Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 469 In multiple myeloma recurrent chromosomal translocations lead to the overexpression of oncogenes such as MAF, MAFB and MMSET. 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 expression of 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. MMSET gene is overexpressed in about 15% of cases of MM due to chromosomal translocation (4;14). In most cases upregulation of MMSET is accompanied by the overexpresson of FGFR3. Overexpression of FGFR3 would be expected to increase MAP kinase signaling and c-MYC expression. However, in 30% of cases of t(4;14) myeloma, only MMSET is overexpressed. Hence we hypothesized that MMSET might also stimulate c-MYC expression. We showed that MMSET is a histone methyltransferase that has promiscuous activity in vitro. In vivo, MMSET overexpression was correlated with elevated dimethylation of H3K36 and depressed trimethylation of H3K27 levels, altered chromatin structure, gene expression profiles, and cell growth. However, the exact genetic targets underlying the MMSET activity are not well understood. We found that MMSET overexpression was associated with increased c-MYC protein but not mRNA expression. Depletion of MMSET from myeloma cells was associated with decreased levels of c-MYC protein but not mRNA and no change in the half-life of the MYC proteins. This suggested that MMSET controlled MYC at the post-transcriptional level by preventing its translation, an effect reminiscent of the action of microRNAs. miRNA profiling of t(4;14) myeloma cells before and after shRNA-mediated depletion of MMSET showed that MMSET repressed the expression of many miRNAs including miR-126*, which was predicted to target c-MYC. We therefore hypothesized that by repressing miR126* expression MMSET could stimulate c-MYC protein expression. Accordingly, miR126* but not a point mutant of mir126* inhibited translation of a construct fusing the 3' untranslated region of c-MYC to luciferase. Furthermore a point mutation within the 3'UTR of c-MYC abrogated the effect of miR126* overexpression on the reporter. Overexpression of miR126* in t(4;14) myeloma cells suppressed c-MYC expression and cell growth. Furthermore, growth suppression and inhibition of c-MYC expression in myeloma cells after MMSET knock-down was partially blocked by an antagomir directed against mir126*. Importantly, 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 KAP1 a partner protein identified by affinity chromatography and mass spectroscopic analysis. KAP1 mediated repression has been associated with increased H3K27 and H3K9 methylation at promoter sites and accordingly on the miR126/126* promoter, MMSET expression was associated with MMSET and KAP1 recruitment to the promoter, increased H3K9 and H3K27 methylation and decreased histone acetylation. KAP1 depletion in myeloma cells was associated with increased expression of mir126*, decreased c-MYC levels and decreased myeloma growth. Lastly to model the effects of an MMSET inhibitor in combination with 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 but the combination of a sub-optimal dose of the HDAC inhibitor combined with MMSET depletion led to complete silencing of c-MYC expression and increased cell death. Collectively, these data suggest a new mechanism of oncogenesis by MMSET in MM, and emphasize the key role of c-MYC overexpression in this disease. Disclosures: No relevant conflicts of interest to declare.

Description: Increasing amount of evidence indicates that the deregulation of non-coding elements is a common feature of cancer and therefore, its investigation may uncover new molecular oncogenic mechanisms. In multiple myeloma (MM), the altered expression of a small number of long non-coding RNAs (lncRNAs) has been associated with progression and decreased survival, suggesting that these elements may play a more important role in this disease than previously expected. Nevertheless, an extensive high-throughput analysis that characterizes the deregulation of lncRNAs in MM has not yet been performed. To characterize the transcriptome, including all genomic types of lncRNAs, of MM we performed a paired end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients, as well as PC samples from tonsils (TPCs, n=5) and bone marrow (BMPCs, n=3) of healthy donors as controls. Principal component analysis (PCA) demonstrated that normal PC samples from tonsil and bone marrow cluster separately, suggesting that in spite of being the same cell type, their coding and non coding transcriptomes are very different. Therefore, we selected BMPCs as the normal counterparts for comparison with BM of MM samples. PCA analysis also demonstrated that the well known heterogeneity of MM patients rely not only on the coding transcriptome but also on the lncRNA expression profile. Comparison of MM to BMPCs samples showed 70 previously annotated lncRNAs that were deregulated in MM patients, with 3 lncRNAs showing higher and 67 lower expression than normal BMPCs. Moreover, we identified 40.552 novel MM-specific lncRNAs that were present in at least 3 of the 38 patients, highlighting the magnitude of the deregulation of these non coding elements in MM. To determine the functional role of altered lncRNAs in the biology of MM plasma cells we focused on the study of LINC-MSL1 (Myeloma-Specific LncRNA 1). Analysis of the expression of this lncRNA at different stages of B-cell differentiation (Naïve, Germinal Center, Memory and PC) indicated that it is not expressed at any stage, except for a modest expression in BMPCs. Interestingly, its overexpression was detected in 40% of MM specimens when compared to normal BMPCs which was validated by qPCR in an independent cohort of MM patients. To determine whether the expression of this lncRNA is regulated by epigenetic mechanisms, we studied the DNA methylation state of this gene. DNA methylation analysis in MM demonstrated that the CpGs located upstream of LINC-MSL1 were differentially methylated in comparison with normal counterpart BMPC. These CpGs showed 70% DNA methylation in control samples, about 40% in MGUS, whereas the average of MM was about 20%, showing a remarkable hypomethylation. We validated these results by pyrosequencing, which showed a significantly lower DNA methylation at the promoter region in comparison with B cell populations from tonsil, normal BMPCs and cell lines that do not overexpress LINC-MSL1. We also have observed a gain of active chromatin states analyzed by ChiP-seq in the promoter region of LINC-MSL1 in MM patient samples. These data suggest that epigenetic mechanisms, namely the progressive hypomethylation and the gain of active histone modifications, are the cause of the overexpression of LINC-MSL1 in MM. To analyze the role of the overexpression of LINC-MSL1 in MM, we engineered two MM cell lines that show high levels of LINC-MSL1, MM.1S and MM.1R, to express shRNAs against this lncRNA. Knockdown of LINC-MSL1 by two different shRNAs resulted in a reduced proliferation of the cell lines over time. This effect was not associated with a cell cycle arrest but with a marked increased in the percentage of Annexin V-positive apoptotic cells, indicating that the overexpression of LINC-MSL1 is necessary for the survival of MM cells. All together, these data demonstrate that the alteration of lncRNAs is an important an unexplored feature that contributes to MM pathogenesis. The overexpression of LINC-MSL1 is essential for MM survival and is very specific of MM BMPCs, suggesting it could be a relevant therapeutic target. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Melnick:Janssen: Research Funding.

Description: MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, and is believed to be the driving factor in the pathogenesis of this subtype of MM. Overexpression of MMSET also occurs in solid cancers, including neuroblastoma, colon and prostate. MMSET overexpression in MM and prostate cells leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3). This altered epigenetic landscape is accompanied by changes in proliferation, gene expression, and chromatin accessibility. Prior work linked methylation of histones, including H3K36, 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 might play a role in DNA damage repair and response. To investigate the role of MMSET in DNA damage repair, we transfected U2OS cells with a linearized vector expressing a neomycin-resistant gene. In the presence of G418, only cells that are able to integrate this plasmid through non-homologous end joining (NHEJ) can survive. siRNA knockdown of MMSET led to a decrease in cell survival, suggesting that MMSET is necessary for efficient DNA repair. We also used U2OS cells engineered to express the AsiSI enzyme fused to an estrogen receptor hormone-binding domain. Upon tamoxifen treatment, double strand breaks (DSBs) are induced at multiple AsiSI recognition sites, accompanied by an increase in γH2AX foci. The extent of repair after AsiSI-induced damage was ascertained by the ability of a DNA fragment that spans a specific cut site to be PCR amplified. With MMSET knockdown, there was a 〉10 fold increase in unrepaired DNA. ChIP analysis showed that with the depletion of MMSET, γH2AX persisted at the cut site. ChIP for specific effectors of DNA damage showed a marked decrease of recruitment of CtIP and RAD51 to the DSB. However, immunoblot analysis showed that CtIP and RAD51 levels were drastically decreased with MMSET depletion, thus explaining the loss of their recruitment to DSBs. In contrast, XRCC4 levels were maintained with MMSET siRNA, but its recruitment to the DSB decreased. CtIP is important for both NHEJ and homologous recombination (HR), RAD51 is critical for HR, and XRCC4 is necessary for NHEJ, suggesting that MMSET is important in multiple pathways of DNA repair. To study the effect of MMSET in MM, we used the t(4;14)+ KMS11 cell line, NTKO, and genetically matched TKO cells in which the overexpressed MMSET allele was knocked out. NTKO cells have elevated levels of DNA damage at baseline, as measured by a comet assay and by the presence of elevated numbers of 53BP1-positive foci. Upon addition of the DNA damaging agent melphalan, NTKO cells showed increased damage as measured by an increase in the tail moment by the comet assay. Paradoxically, upon treatment of these cells with the DNA damaging agents, NTKO cells survived better than TKO cells. NTKO repaired DNA damage at an enhanced rate and continued to proliferate after a significant DNA damage insult, whereas TKO cells accumulated DNA damage and entered cell cycle arrest. We repleted TKO cells with constructs expressing either wild-type MMSET or an HMT-dead (Y1118A) isoform. Upon treatment, cells expressing the wild-type MMSET have showed enhanced DNA repair and continued proliferation after DNA damage, whereas cells expressing the HMT-dead protein repaired DNA damage more slowly and entered cell cycle arrest. The HMT activity of MMSET was critical for the induction of expression of genes required for multiple DNA repair pathways including CHEK2, DDB2, DDIT3, RAD51, and MRE11, again suggesting that MMSET modulates DNA repair by affecting expression of critical components of the repair machinery. The clinical relevance of these finds becomes more apparent in vivo. Luciferase-tagged KMS11 cells harboring doxycycline-inducible MMSET shRNA were injected into nude mice. After one week, mice were treated with doxycycline and injected with melphalan or saline. Knockdown of MMSET or melphalan treatment alone decreased tumor growth but eventually all mice had progressive disease. Only when MMSET was knocked down and chemotherapy given were the mice rendered tumor free. These findings indicate a new mechanism for the ability of MMSET to enhance DNA repair and identify the protein as a potential therapeutic target in MM and other cancers. Disclosures: No relevant conflicts of interest to declare.