Abstract 2834: Genomewide CRISPR studies of sequential treatment with CRBN-based degronimids: Insights into the molecular evolution of treatment resistance in myeloma and beyond

Degronimids are bifunctional agents in which a thalidomide-like moiety brings the E3 ligase CRBN close to proteins which bind to the second functionality of these compounds, leading to ubiquitination and proteasomal degradation of these latter proteins. Our groups and others have examined BET bromodomain (BRD)-containing proteins BRD2,3, and 4 (dBET6) or CDK9 (ZZ1-33, referred to herein as dCDK9). We documented that both dBET and dCDK9 compounds trigger rapid induction of multiple myeloma (MM) cell death in vitro. To dissect the mechanisms regulating the response vs. resistance of MM cells to degronimids, we performed genome-wide CRISPR/Cas9-based gene editing screens using lentiviral particles of a pooled sgRNA library (Brunello; ~70,000 sgRNAs against ~20,000 genes; 4 sgRNAs/gene; plus additional non-targeting control sgRNAs). In screens with both CRBN-mediated "degraders", we observed consistent and pronounced enrichment of sgRNAs for CRBN itself, and also other upstream components or regulators of the complex that regulates CRBN function (e.g. DDB1, CAND1), E2 ubiquitin conjugating enzymes (e.g. UBE2G1) and multiple members of the signalosome complex (COPS7A, COPS7B, GPS1, et.c.). These results indicate that a primary mechanism of degronimid-resistance relates not to adaptation to the degradation of their respective targets, but to via loss of function (LOF) of CRBN or its positive regulators, to prevent degradation of the degronimid target(s). Also, we observed that dCDK9-resistant MM.1S cells which established from our CRISPR screen, upon more extensive treatment with either dCDK9 or dBET led to further enrichment of the CRBN sgRNA-containing tumor cells, with significant decrease in the fraction of the population with sgRNAs for other hits. These observations imply that LOF for positive regulators of CRBN activity may provide protection in short-term, but the role of several of these genes for MM cell proliferation/survival may limit the longer-term fitness of these populations, allowing cells with CRBN LOF to outcompete other potential forms of resistance. This finding may also indirectly explain why clinical resistance to thalidomide or its derivatives, CRBN-mediated "degraders of IKZF1/IKF3, are reported to involve LOF of CRBN, rather than most of its regulators of CRBN. Our CRISPR studies document the concordance in mechanisms of resistance to "degronimids" targeting different proteins for degradation; and how time-course analyses in these CRISPR screens can reveal the dynamics between sub-populations of tumor cells harboring sgRNA-mediated LOF for different candidate genes. Citation Format: Ryosuke Shirasaki, Sara Gandolfi, Ricardo De Matos Simoes, Geoffrey Matthews, Sondra Downey, Olga Dashevsky, Tang Huihui, Michal Sheffer, Eugen Dhimolea, Megan Bariteau, Jeffrey Sorrel, Nick Kwiatkowski, Thinghu Zhang, Nathanael Gray, Constantine Mitsiades. Genomewide CRISPR studies of sequential treatment with CRBN-based degronimids: Insights into the molecular evolution of treatment resistance in myeloma and beyond [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 2834.