Functional mapping of multiple myeloma kinome with library of small molecule inhibitors

The functional role of individual kinases in multiple myeloma (MM) cells has been primarily interrogated using RNAi approaches, which are mechanistically dissimilar to the pharmacological action of small molecule inhibitors used in the clinic; and in stroma-free cultures, which don’t account for stroma-induced signaling events. To address these dissimilarities, we performed functional mapping of the kinome in 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. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors (consistent with high proliferative rates of MM cell lines in vitro); modest to minimal for selective PDK1, PI3K (excluding those that also inhibit mTOR), and Akt inhibitors; virtually none for selective inhibitors of c-met, ALK, EGFR, HER2, c-kit, PDGFR, VEGFR, Flt3, FAK, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases). We also observed modest activity for FGFR3 inhibitors against FGFR3expressing cell lines; and none for BRAF inhibitors in our BRAF wild-type cell lines (some of which had increased proliferation upon treatment; consistent with known adaptive activation of signaling through C-RAF). Testing 8 different MEK1/2 inhibitors identified lines with low, intermediate vs. high response; the latter being KRAS-mutant and BRAF-wild-type cells with high baseline p-ERK levels (e.g. due to MEK2-activating mutation in one cell line). Importantly, comparison of 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 studies demonstrate the feasibility of functional annotations of the kinome in MM using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of cell lines. Our results provide insight into the relative role of different classes of kinases in MM, and how the genetic (e.g. MEK2 or BRAF mutation status) and microenvironmental context can influence the role of these kinases and possible future individualized administration of inhibitors against these kinases in MM. BP-032