ALBERT

Description: Multiple myeloma (MM) is a hematological malignancy characterized by various genetic abnormalities including translocations involving the IgH gene at 14q32. Amongst these, t(11;14) is one of the most common translocations. Recent clinical data suggests a significant impact of Venetoclax, a small molecule inhibitor of BCL2, in this subgroup of MM patients, representing the first example of personalized medicine in MM and opening a wide range of research aiming at elucidating its mechanism of action. However, despite the initial positive response to the drug, a significant proportion of patients eventually develop resistance and relapse. To delineate the mechanisms that contribute to the development of an acquired drug-tolerant/resistance phenotype, we modeled the response to Venetoclax in 2 MM cell lines (KMS27 and KMS-12PE with IC50 of 35.47nM and 3.64nM, respectively). Whereas the vast majority of cells plated into 96-well plates were killed within a few days of exposure to a high dose of drug concentration, we detected a small fraction of viable, largely quiescent cells, which were expanded by culturing them in high doses of Venetoclax. We successfully generated 4 independent clones from each cell line, that were single cell-cloned with continued growth in the presence of high doses of Venetoclax. These clones labelled as drug-tolerant expanded persisters (DTEP) were investigated for the mechanisms driving drug tolerance and resistance against Venetoclax. First, we observed that altered expression of apoptotic regulators were associated with Venetoclax resistance in DTEP cells. We indeed observe a significant increase in the anti-apoptotic proteins MCL1 and BCL-XL in DTEP clones, which translated in our observation of improved sensitivity to MCL1 and BCL-xL inhibitors (S63845 and A-1155463 respectively). We performed both whole genome sequencing (WGS) and RNA-seq to evaluate if DTEP cells undergo transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprograming during development of acquired drug resistance. While, WGS analysis didn't show any significant differences between parental and resistant clones, transcriptomic analysis showed both shared and unique transcriptome signatures in the DTEP clones. Gene set enrichment analysis (GSEA) of the common significantly modulated genes in the resistant clones revealed that the genes belonging to the PKA-ERK-CREB pathway were significantly upregulated in resistant clones, while apoptotic genes were downregulated compared to parental cells. Western blot analysis confirmed activation of ERK and the downstream target cAMP response element-binding (CREB) gene in resistant clones; and importantly treatment with the ERK inhibitor U0126 rescued the resistance to Venetoclax, providing a synergistic activity in resistant clones but not in parental cells, with decreased cell viability and increased apoptotic cell death. To evaluate if the ERK pathway was also associated with intrinsic resistance to Venetoclax, we assessed a panel of 24 MM cell lines and then calculated Pearson correlation coefficients between the measured drug activity and individual gene expression levels (by RNA-seq) across all cell lines and subjected the resulting rank-ordered gene list to GSEA. This analysis showed that mechanisms driving the DTEP phenotype are different from those associated with the intrinsic resistance to Venetoclax. RNA processing and splicing pathways were strongly enriched, with high expression of these genes correlating with increased sensitivity. Moreover, among the genes correlated with a resistant phenotype, we observed that the gene G0S2 was significantly downregulated in the resistant cell lines. G0S2 is a tumor suppressor gene that binds and inhibits BCL2. Interestingly, we observed that while G0S2 is downregulated in MM compared to normal plasma cells, t(11:14) patients have a higher expression. We are now in the process of validating G0S2 in MM and its contribution to Venetoclax sensitivity in MM. In conclusion, we here provide evidences of molecular mechanisms of acquired resistance to Venetoclax with activation of the ERK pathway as one of the prime targets. Combining Venetoclax with ERK inhibitor may therefore prevent or overcome the acquired resistance to Venetoclax observed in MM patients. Disclosures Fulciniti: NIH: Research Funding. Munshi:C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Description: Multiple myeloma (MM) is a complex hematological malignancy characterized by gene pathway deregulations. Initial sequencing approaches have failed to identify any single frequent (〉25%) mutation in the coding genome. We, therefore performed a deep (average coverage 〉 80X) whole genome sequencing (WGS) on 260 MM samples (208 newly diagnosed and 52 first relapse after uniform treatment) to comprehensively identify recurrent somatic alterations in non-coding regions. We have identified the most frequently involved genes affected by perturbation in neighboring non-coding region and integrate their expression using our matching deep RNA-seq data from the same patients. One of the most prominent examples is mutations in the 5' untranslated region and intron 1 of the BCL7A gene in 76% of myeloma patients. Integration of WGS with RNA-seq data confirmed significant downregulation of its expression (p values 〈 1e-5) in the MM cells as compared to normal plasma cells (PC). This led us to investigate the consequences of BCL-7A loss in MM. To evaluate the role of BCL7A in MM, using gain of- (GOF) and loss-of-function (LOF) approaches, we have utilized a large panel of MM cell lines with differential expression of BCL7A at the RNA and protein levels. Ectopic expression of BCL7A in a panel of 3 MM cell lines with low basal levels of BCL7a significantly reduced cell viability and colony formation over time. Inhibition of cell viability was associated with induction of apoptotic cell death in the BCL7A overexpressing cells compared to control cells. LOF studies in 3 MM cell lines with relatively higher expression of BCL7a using 3 BCL7A-specific shRNA constructs showed a more proliferative phenotype, with increased growth and viability and enhanced colony formation. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7a-KO cells had higher proliferative rate compared to WT cells and add back of lentiviral BCL7a plasmid reversed this effect. BCL7A is part of the SWI/SNF chromatin remodeling complex. Mutations in the genes encoding m-SWI/SNF subunits are found in more than 20% of human cancers, with subunit- and complex-specific functions. We confirmed that when expressed, BCL7A interacts with BCL11A into the SWI/SNF complex in MM cells. Comparative, mass spectrometry analysis in fact revealed SMARCC2 (BAF170), an integral subunit of SWI/SNF complex, to bind with BCL7A-BCL11A complex. However, BCL7A loss causes decreased SMARCC2 incorporation into SWI/SNF, thus suggesting that presence of BCL7A is crucial in the formation of SWI/SNF complex in MM cells and might play an important role in chromatin remodeling. Interestingly, oncogenes DEK (DNA binding oncogene) and TPD52 (tumor protein D52) involved in cancer cell proliferation and chromatin remodeling formed complex with BCL11A in BCL7A KO MM cells. Additionally, several anti-apoptotic proteins such as ANXA-1 and BCL2 are in complex with BCL11A when BCL7A is lost, suggesting the formation of an anti-apoptotic complex with consequences on MM cell survival. Currently ongoing studies are investigating the molecular mechanism of non-coding mutations impacting BCL7A expression and pathways affected by its downregulation with impact on MM cell growth and survival. In conclusion, we report biological consequences of a frequent (〉75% patients) non-coding mutation in MM with cellular and molecular effects of BCL7A loss in which implicates a functional role of the m-SWI/SNF complex in driving a MM cell proliferative phenotype. Disclosures Anderson: Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder . Munshi:Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

Description: Uncontrolled proliferation is a hallmark of tumorigenesis and is associated with perturbed transcriptional profiles. The proliferative program in Multiple myeloma (MM), a complex disease with heterogeneous genetic changes, is controlled by transcription factors (TFs) and chromatin-associated factors. The dependency on these transcriptional regulators, leading to the dysregulated proliferation, is not predicted by genetic changes, making the tumor cells more sensitive to inhibition of these regulators than normal cells.The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs is not well-defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. Our genome-wide epigenomic profile in myeloma has identified the existence of two non-overlapping regulatory axes controlled by promoter and enhancer-driven processes, governing distinct biological functions. We have utilized E2F1 as promoter proximal transcription factor, and evaluated its transcriptional and functional interrelationship with enhancer-associated factors, such as BET bromodomain transcriptional co-activators. We identified that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in MM cells. Global chromatin analysis revealed two distinct regulatory axes, with E2F and MYC predominantly localized to active gene promoters of growth/proliferation genes and CDK9 and BETs disproportionately at enhancer-regulated tissue-specific genes. This divergent BRD4 enhancer and E2F promoter axes is also observed in diffuse large B-cell lymphoma, suggesting a more broader transcriptome control process. Dual inhibition of E2F and BETs displays a superior activity against MM cell growth and viability, both in vitro and in vivo, compared to single perturbation alone providing an important molecular mechanism for combination therapy. Moreover, at low doses of BRD4 inhibitor JQ1, the addition of E2F1 depletion down-regulates the promoter controlled proliferation gene expression axis. As for many TFs, direct pharmacologic inhibition of E2F remains a difficult challenge in drug discovery. However, E2F is not entirely "undruggable" as inhibitors of upstream regulators of the pRB-E2F axis are available. For example, a number of Cyclin dependent kinases (CDK) 4/6 inhibitors, including Palbociclib are now being investigated in clinical trials in in fact approved by the FDA in select malignancies. CDKs are serine threonine kinases that modulate cell cycle progression. CDK4 and CDK6 together with D-type cyclins and cyclin E/CDK2 complexes control the commitment to cell cycle entry from quiescence and the G1 phase. These kinase complexes can phosphorylate RB, releasing E2F to modulate the expression of E2F target genes that are required for S phase entry. We investigated combination of low doses of JQ1 and Palbociclib and observed a profound effect on E2F promoter driven transcriptional activity, and was highly synergistic with JQ1 in a large panel of MM cell lines and primary MM cells from newly diagnosed and relapsed patients. Cell cycle analysis revealed complete G1 arrest after treatment. Importantly, the combination regimen was not effective in healthy donor PBMCs activated with PHA, suggesting a favorable therapeutic index. Transcriptomic changes to assess the impact on promoter and SE-driven processes are ongoing and will be presented. In conclusion, these data implicates the existence of a sequestered cellular functional control that may be perturbed in cancer to maintain the tumor cell state. Simultaneous targeting of non-overlapping promoter and enhancer vulnerabilities impairs the myeloma proliferative program, with potential for development of a promising therapeutic strategy in MM and other malignancies. Disclosures Young: Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder. Munshi:OncoPep: Other: Board of director.

Description: Multiple Myeloma (MM) is a complex disease with distinct molecular and clinical characteristics. Recent large collaborative efforts have identified number of driver genes. However over 95% of all somatic alterations occur in non-coding regions and very little is known about how they affect the disease. We performed a deep (average coverage 〉 80X) whole genome sequencing (WGS) on 260 MM samples (208 newly diagnosed and 52 first relapse after uniform treatment) to comprehensively analyze recurrent somatic alterations in non-coding regions. We detected median 11,852 (Range 4,802-87,396) mutations and indels per sample with overall more than 3.9M total somatic mutations. Introns (3.6 mutations/per Mb) and intergenic regions (4.06 mutations/per Mb) had significantly higher number of mutations per megabase compared to Exons (2.7 mutations/per Mb) (p 〈 1e-5). Mutations in coding regions in our data was similar to published whole exome sequencing studies. We observed 46 [range 7 - 219] structural variants (SVs) per sample with 98% involving non-coding regions. We found that number of SVs significantly correlated with overall survival (p value = 1.7e-5). We detected chromothripsis (〉=7 oscillating copy number change and significant clustered SVs and/or clustered translocations) in 24% of newly diagnosed samples; and kataegis hotspots on chromosome 3q27-3q28 (24%), 11q13 (5.8%) and 12q24 (5.3%). By clustering SV breakpoints across the genome we have identified 3 SV hotspots on chromosome 17q21, 7q34, and 11q13. We next interrogated the non-coding regions to identify genomic loci with higher than expected mutation count compared to background mutation rate. We have identified 456 loci that are significantly enriched in non-coding regions (5' UTR, 3'UTR, promoter, intergenic, intronic, and distal regulatory regions) [adjusted p value 〈 1e-5 and observed in 〉=10% newly diagnosed MM]. These loci are then assigned to genes or gene neighborhoods to evaluate their potential impact. We have identified the most frequently involved genes affected by perturbation in neighboring non-coding region and integrate their expression using our matching deep RNA-seq data from the same patients. Of these the most prominent examples are 1.) 3'UTR mutations are enriched in CD93 gene, which plays critical role in B cell development with loss of expression in CD138+ MM cells compared to normal plasma cells (p value 〈 1e-5); 2.) Promoter region - we have identified 635 mutations in 2kb region in BCL6 coming from 76% of all newly diagnosed samples. BCL6 (p value 〈 1e-5) has significantly downregulated expression in MM. Interestingly, but not surprisingly this hypermutated region showed high intensity of H3K27Ac activity in normal cells; 3.) 5'UTR - BCL7A (27.9%) and LPP (11.7%) were top two 5' UTR mutated target genes and RNA-seq data confirmed significant downregulation of their expression (p values 〈 1e-5 and 0.0048 respectively) in the MM cells. Additionally, BCL7A (48%) also showed significant enrichment of intronic mutations. A similar mutational hotspots were observed within the vicinity of additional functionally important genes in myeloma including ROBO1/2, ILF2, IRF8 and BCL2A1. Our data also showed that these frequent mutations have higher cancer cell fraction (CCF) [median CCF 〉 0.75] suggesting their occurrence earlier in the disease development. To validate the function of these mutations, we have started to carry out gain/loss of function studies. Our analysis with BCL7A shows that BCL7A knockdown increases the cell viability while its overexpression decreased growth, colony formation and increased apoptosis. This tumor suppressor function of BCL7A is being further analyzed in light of our mutational data in the nearby non-coding region. In conclusion, this large deep whole genome sequencing data from newly-diagnosed MM patients identifies a vast majority of non-coding mutations with potentially significant functional and biological role in MM. Our integrative approach using both WGS and RNA-seq data from the patients now provides us important tools to further characterize the impact of these mutations and develop opportunities for targeted therapeutics. Disclosures Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Moreau:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thakurta:Celgene Corporation: Employment, Equity Ownership. Anderson:Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Avet-Loiseau:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.

Description: High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Description: Unrepaired DNA double-strand breaks (DSBs) pose a serious threat to genomic stability, potentially leading to the formation of oncogenic mutations, as well as translocations, deletions and amplifications. Almost all multiple myeloma (MM) patients are characterized by such copy number alterations and structural variations. Therefore, we evaluated and report that MM cells have increased ongoing spontaneous DNA damage which could be exploited to create synthetic lethality in combination with inhibitors of homologous recombination (HR). We evaluated various types of DNA damage in peripheral blood mononuclear cells (PBMCs)from 15healthy controls, 4 MM cell lines and CD138+ bone marrow plasma cells (BMPCs) from 16 patients with monoclonal gammopathy of undetermined significance, 18 with smoldering myeloma (SMM) and 15 with MM. Relative to normal PBMCs, MGUS and MM cells showed significantly higher levels of endogenous DNA breaks (measured as olive tail moments with single-cell gel electrophoresis), abasic sites (Elisa-based detection) as well as DNA break repair efficiency (all P

Description: Identification of mechanisms underlying genomic instability is necessary to understand disease progression, including development of drug resistance. Our previous data demonstrates that dysregulation of DNA repair and maintenance/modification activities (including homologous recombination (HR), apurinic/apyrimidinic nuclease and APOBEC) significantly contribute to genomic instability in multiple myeloma (MM). However, how these and other pathways involved in genomic instability are dysregulated, remains to be explored. Since kinases play a critical role in the regulation of the maintenance of genomic integrity, we have performed a genome-wide kinome profiling to identify those involved in genomic instability in cancer. First, we analyzed genomic database for ten human cancers (including MM) from TCGA with both tumor cell gene expression and SNP/CGH array-based copy number information for each patient.We assessed genomic instability in each patient based on the total number of amplification and deletion events. We next interrogated all 550 kinases expressed in humans and identified those whose expression correlated with copy number alteration (based on FDR ≤ 0.05) in all tumor types. We identified six kinases whose elevated expression correlated with increased genomic instability defined by genomic amplification/deletion events in all ten cancers, including MM. To demonstrate functional relevance of these kinases, we conducted a CRISPR-based loss of function screen (using 3 guides per gene) in MM cells and evaluated the impact of each gene-knockout on micronuclei, a marker of ongoing genomic rearrangements and instability. For all six kinases, at least one guide resulted in ≥ 65% inhibition of micronuclei formation. Moreover, for five out of the six kinases, at least two guides showed ≥ 60% inhibition of micronuclei. All together, these data establishes a strong relevance of these kinases with genomic instability in MM. PDZ Binding Kinase (PBK) was among top kinases impacting genome stability in this data set with 2 out of 3 guides causing 〉 88% and 3rdguide causing 35% inhibition of micronuclei formation. We further report that inhibition of PBK, by knockdown or small molecule, inhibits DNA breaks, RAD51 recombinase expression and homologous recombination in MM cells. We further investigated molecular mechanisms involved in PBK-mediated genomic instability in MM. Expression profiling using RNA sequencing of MM cells treated with a specific PBK inhibitor showed that top ten pathways downregulated by treatment were mostly DNA repair/recombination followed by replication and G2/M checkpoint. Interestingly, we identified a notable overlap between PBK-regulated genes with FOXM1 target genes. FOXM1 is a major transcriptional regulator of genes involved in DNA repair, G2/M regulation and chromosomal stability. We, therefore, investigated PBK/FOXM1 interaction and show that PBK interacts with FOXM1 in MM cells. Moreover, the inhibition of PBK, by knockdown or small molecule, inhibits phosphorylation of FOXM1 as well as downregulates FOXM1-regulated HR and cell cycle genes RAD51, EXO1 and CDC25A. These results suggest that PBK-dependent phosphorylation of FOXM1 activity controls transcriptional networks involved in genomic instability in MM. Ongoing work is investigating role of PBK and other kinases in progression of MGUS/SMM to active MM and their impact on ongoing genomic changes with influence on multiple DNA repair pathways including HR. In conclusion, we describe a kinase panel that may have significant role in maintaining genome stability, and their perturbation may allow to improve genome stability in MM. Disclosures Munshi: Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy.

Description: Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.