ALBERT

Description: High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.

Description: In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while 〉80% and 〉75% of these genes have detectable transcript levels (RPKM〉1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Description: Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases (e.g. CRBN and VHL) to induce neopmorphic ubiquitination and proteasomal degradation of target oncoproteins, with potent preclinical activity against diverse neoplasias. Despite intense recent efforts to develop pharmacological "degraders" against many different oncoproteins, the mechanisms regulating tumor cell sensitivity to different classes of these "degraders" remain incompletely understood. To address this question in an unbiased manner, we performed genome-scale CRISPR/Cas9-based gene editing loss-of-function (LOF) studies in MM.1S multiple myeloma (MM) cells treated with CRBN-mediated degraders of BET bromodomain proteins (dBET6) or CDK9 (Thal-SNS-032); or with VHL-mediated degraders of BET bromodomain proteins (ARV-771 or MZ-1). We observed that MM cell resistance to any of these "degraders" does not involve genes with recurrent LOF in MM patients and association with high-risk MM (e.g. for TP53, PTEN, negative regulators of cell cycle, et.c.), suggesting that these degraders may exhibit activity against tumor cells with prognostically adverse genetic features. In tumor cells resistant to the CRBN-mediated degraders dBET6 and Thal-SNS-032, we observed significant enrichment of sgRNAs targeting CRBN itself or (to a lesser extent) other components or regulators of its cullin RING ligase (CRLCUL4A) complex, including members of the COP9 signalosome (COPS7A, COPS7B, COPS2, COPS3, COPS8, GPS1, etc.), DDB1, or the E2 ubiquitin conjugating enzyme UBE2G1. In tumor cells resistant to the VHL-mediated degraders MZ-1 and ARV-771, we observed pronounced enrichment of sgRNAs for CUL2, VHL itself, other members (e.g. RBX1, elongin B/C [TCEB1, TCEB2] of the CUL2 complex with VHL), as well as COP9 signalosome genes (COPS7B, COPS8) and UBE2R2. We also validated, using individual sgRNAs for several of these candidate genes that their CRISPR knockout can decrease tumor cell response to dBET6 and Thal-SNS-032 treatment (e.g. for CRBN, COPS7B, COPS2, or COPS8) or MZ-1 and ARV-771 (e.g. for VHL, COP7B and COPS8). Notably, the sgRNAs against COP9 signalosome genes conferred less pronounced decrease in sensitivity to VHL-, than CRBN-based, degraders, suggesting that COP9 signalosome loss has differential roles in the function of CUL4ACRBN vs. CUL2VHL and potentially other CRL complexes. Tumor cells isolated from our CRISPR knockout screens with confirmed resistance to a given degrader were then treated with other degraders operating through the same or different E3 ligase; and against the same or different oncoprotein: we observed cross-resistance between degraders operating through the same E3 ligase against different oncoproteins, but not for degraders targeting the same protein via different E3 ligase/CRLs: this result is consistent with our observation for substantial gene-level differences (despite pathway-level similarities) for resistance mechanisms for CRBN- vs. VHL-based degraders. In conclusion, our study systematically defined at genome-scale the resistance mechanisms of tumor cells against degraders which leverage the same E3 ligase against different targets; or target the same oncoprotein through different E3 ligases/CRL complexes. We observed that for multiple types of degraders, tumor cell resistance is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein. The observed pathway-level similarities and major individual gene-level differences in resistance mechanisms for CRBN- and VHL-mediated degraders likely reflects the different composition and regulation of the respective CRL complexes mediating the action of these classes of degraders Our observations suggest that preventing or delaying resistance to pharmacological degradation of oncoproteins may require concurrent or sequential/alternating use of degraders operating through different E3 ligases and ideally, different CRL complexes; while synthetic lethal strategies to prevent COP9 signalosome LOF may also be contemplated to counteract a common, but quantitatively less pronounced, potential mechanism of resistance for several different classes of degraders. Collectively, our study highlights important new directions in the development of new pharmacological degraders for blood cancers and other neoplasias. Disclosures Richardson: Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Oncopeptides: 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; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Licht:Celgene: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria. Gray:C4 Therapeutics: Consultancy. Mitsiades:TEVA: Research Funding; Janssen/ Johnson & Johnson: Research Funding; EMD Serono: Research Funding; Takeda: Other: employment of a relative; Abbvie: Research Funding.

Description: The discovery that thalidomide derivatives recruit the E3 ligase CRBN to induce neomorphic degradation of proteins critical for multiple myeloma (MM) cells stimulated the research into proteolysis-targeting chimeric compounds (PROTACs), led to development of several CRBN- or VHL-based PROTACs against various oncoproteins and put a new spotlight on the biology and therapeutic targeting of E3 ligases in human neoplasias. However, so far only a few of the ~600 known/presumed E3 ligases have been leveraged for generation of PROTACs. The mechanisms regulating the function of most E3 ligases have not been systematically examined. Because the function of an E3 ligase is considered essential for anti-tumor activity of its respective PROTACs, we applied CRISPR knock-out (KO) systems to identify candidate regulators of E3 ligase function, via characterizing the the network of genes which modulate MM cell responses to PROTACs. We thus performed genome-scale CRISPR-based gene editing (for loss-of-function, LOF) studies in MM.1S cells treated with PROTACs targeting BET bromodomain proteins through MDM2 (A1874), CRBN (dBET6) or VHL (ARV-771 or MZ-1) or targeting CDK9 through CRBN (Thal-SNS-032); and validated key hits with individual sgRNAs in different MM cell lines. The top individual LOF events conferring resistance to PROTACs did not involve a compensatory mechanism or "work-around" the loss of the respective oncoprotein, but were predominantly associated with LOF of the respective E3 ligase; or with LOF for genes with known or plausible role in regulating the respective E3 ligases. For instance, sgRNAs against members of the COP9 signalosome complex decreased MM cell responses to CRBN- and (to a lesser extent) VHL-, but not MDM2-based PROTACs. PROTACs leveraging different E3 ligases were regulated by different cullin ring ligase (CRL) complex members (e.g. CUL2, RBX1, TCEB1, TCEB2 for VHL- vs. DDB1 for CRBN- vs. no CRL member for MDM2-based PROTACs) or E2 conjugating enzymes (UBE2R2 vs. UBE2G1 for VHL- vs. CRBN-based PROTACs). Collectively, these results suggest that MDM2 regulation is largely CRL- and COP9-signalosome independent; while VHL regulation is less COP9 signalosome-dependent compared to CRBN. These mechanistic differences suggest that PROTACs targeting the same oncoprotein through different E3 ligases should not be associated with cross-resistance, a result which we validated in experiments involving sequential administration of different PROTACs against BRD4/3/2. In turn, this observation implied that developing PROTACs that leverage a more extended spectrum of E3 ligases may facilitate sequential uses of existing and these new PROTACs to delay or prevent treatment resistance. Building on results of our genome-scale CRISPR essentiality screens, we examined the dependency landscape of known E3 ligases of MM (n=20 cell lines) and 500+ non-MM cell lines. CRBN is redundant for nearly all MM or non-MM cell lines tested, while most other E3 ligases leveraged for PROTACs (e.g. MDM2, BIRC2, DCAF15, DCAF16, RNF114) are essential for only modest or small subsets of human cancer cell lines, suggesting that resistance to respective PROTACs may readily emerge through LOF of these E3 ligases without major fitness cost to tumor cells. We thus sought to identify E3 ligases which are highly expressed in subsets of human tumor cell lines (at levels well above the large majority of normal tissues) and are major dependencies for these "high expressor" cell lines: we identified MDM2 as a major dependency for p53-wild-type cell lines (consistent with MDM2 role as E3 ligase for p53) and we validated this result by documenting the preferential activity of a MDM2-based PROTAC for BRD4/3/2 against p53 wild-type cells. We also identified other E3 ligases genes with well-known roles in tumor cell biology (e.g. members of anaphase promoting complex/cyclosome); as well as E3 ligases (e.g. KCMF1, RNF4) which, to our knowledge, have not been leveraged for design of PROTACs, but warrant consideration given their patterns of essentiality in "high expressor" tumor cells. Our study provides insights on differential regulation and distinct patterns of essentiality for different E3 ligases and informs the design of new PROTACs which leverage different E3 ligases to help delay/overcome treatment resistance in MM and beyond. Disclosures Schlossman: Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: 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; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

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.