Integrated Functional Genomic and Pharmacological Characterization of the Kinome of Myeloma Cells

Abstract

Small molecule kinase Inhibitors represent the main therapeutic modality for targeting aberrant kinase signaling in cancer. The recent emergence of CRISPR/Cas9 based functional genomic tools facilitates the genome-wide assessment of cancer cell dependencies, and evaluation of the relative dependence of tumor cells on diverse types of kinases inhibited by small molecule agents with different specificities, as well as other kinases for which selective inhibitors may not be currently available. In this study, we integrated the phenotypic information from the response of multiple myeloma cell lines to small molecule kinase inhibitors (16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure; targeting a total of 43 known primary oncogenic targets) vs. CRISPR-based functional data on the relative dependency of MM lines on the respective targets of these inhibitors. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors, consistent with the high proliferative rates of MM cell lines in vitro and the role of the corresponding genes as broad-spectrum dependencies across not only MM, but also a collection of 300+ cell lines from other neoplasias. We observed virtually no anti-MM in vitro activity for selective inhibitors of c-met, ALK, EGFR, c-kit, PDGFR, VEGFR, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases):, consistent with these results, the corresponding kinases genes were not essential for in vitro survival and proliferation of MM cell lines in our CRISPR studies. We observed modest to minimal response to selective pharmacological inhibitors for PDK1, PI3K (excluding those that also inhibit mTOR), and Akt. Interestingly, at the functional genomic level, PI3KCA was a prominent dependency (and more pronounced than other PI3K isoforms) for several MM cell lines, while individual isoforms of AKT were typically not essential genes for most or all of the MM cell lines tested, suggesting potential redundancy between these isoforms or feedback loops as bypass mechanisms attenuating the effect of pharmacological inhibitors. Prominent and selective MM kinase dependencies at the functional genomic level include PIM2 (providing indirect explanation for the clinical activity observed in Phase 1 trials in MM of a pan-PIM inhibitor) and IKBKB (consistent with the role of the NFKB pathway in MM). Consistent with prior studies on small inhibitors against IGF1R, its gene is a dependency in most MM lines tested and one of the most recurrent dependencies among receptor tyrosine kinases. Both pharmacological and CRISPR-based results suggest heterogeneous responses of MM cells to loss-of-function of MEK1/2 and, more broadly to diverse members of the MAPK family. Comparison of pharmacological results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our results provide novel insights into the relative role of different classes of kinases in MM; the similarities and potential differences in the impact of pharmacological inhibition vs. genomic perturbation of these kinases. These integrated functional genomic and pharmacological studies of the kinome provide a framework for the potential development of inhibitors against previously understudied or underappreciated kinases; and the development of molecular or functional genomic assays that may potentially guide future efforts towards individualized administration of kinase inhibitors in MM. DisclosuresMitsiades:TEVA: Research Funding; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; Ono: Research Funding; Abbvie: Research Funding; Takeda: Other: Employment of family member.