Young, Richard A.

Therapeutic targeting of CDK7 has proven beneficial in pre-clinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in multiple myeloma (MM) patient cells; and its selective targeting counteracts E2F activity via perturbation of the CDKs/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression increasing survival in several MM mouse models including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

Abstract Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models. Gene expression analysis after CDK7 inhibition in several MM cell lines 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, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs. To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented. In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. Disclosures Shirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: 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; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

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.

Abstract Abstract 1409 MYC is among the most prolific oncogenes in cancer, yet pharmacologic strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have undertaken to target c-Myc transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain-containing proteins as transcriptional regulatory factors for c-Myc. BET inhibition with JQ1 rapidly downregulates c-Myc transcription, followed by depletion of chromatin-bound c-Myc and genome-wide downregulation of Myc-dependent target genes. In translational model systems of multiple myeloma and Burkitt's lymphoma, both canonical MYC -dependent hematologic malignancies, JQ1 treatment produces a potent antiproliferative effect associated with cell cycle arrest. In multiple myeloma, adhesion to bone marrow stroma is associated with upregulation of BRD4, a BET bromomdomain coactivator protein. Inhibition of Myc function with JQ1 leads to impaired adhesion to stroma and cellular senescence, a classical Myc-specific phenotype. Mechanistically, JQ1 treatment depletes BRD4 from IgH enhancers, leading to prompt and robust downregulation of MYC transcription. In vivo efficacy of JQ1 in two disseminated models of multiple myeloma and in a Burkitt's lymphoma human xenograft establishes the therapeutic rationale for BET bromodomain inhibition in these diseases. Together, these studies identify a mechanistic rationale for targeting c-Myc in human cancer, and potentially other undruggable oncogenes driven by immunoglobulin rearrangement. Note: G.C.I. and J.E.D. have made equal contributions to this research; C.S.M. and J.E.B. are jointly senior authors Disclosures: Richardson: Millennium: Advisory Board; Celgene: Advisory Board; Johnson & Johnson: Advisory Board; Novartis: Advisory Board; Bristol Myers Squibb: Advisory Board. Ghobrial:Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celgene:; Novartis:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar:; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding. Bradner:Acetylon Pharmaceuticals: Scientific Founder; SHAPE Pharmaceuticals: Scientific Founder; Tensha Therapeutics: Scientific Founder.

Abstract The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the MYC gene locus. The clinical outcome for patients with high levels of MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Beisi Xu, Shizhen Zhu, Xiaoling Zhang, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. MYC activation through enhancer hijacking or focal enhancer amplification drives a subset of high-risk pediatric neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA19.

Abstract The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that c-MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic c-MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the c-MYC gene locus. The clinical outcome for patients with high levels of c-MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish c-MYC as a bona fide oncogene in a clinically significant group of high risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Xu Beisi, Shizhen Zhu, Xiaoling Zhang, Zhaodong Li, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. c-MYC drives a subset of high-risk pediatric neuroblastoma and is activated through mechanisms including enhancer hijacking and focal enhancer amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4135.

Abstract The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here, we show that the family member MYC is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (∼10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic MYC activation include focally amplified distal enhancers and translocation of highly active enhancers from other genes to within topologically associating domains containing the MYC gene locus. The clinical outcome for patients with high levels of MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Significance: Amplification of the MYCN oncogene is a recognized hallmark of high-risk pediatric neuroblastoma. Here, we demonstrate that MYC is also activated as a potent oncogene in a distinct subset of neuroblastoma cases through either focal amplification of distal enhancers or enhancer hijacking mediated by chromosomal translocation. Cancer Discov; 8(3); 320–35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 253