Functional Mapping of Multiple Myeloma Kinome Using a Small Molecule Inhibitor Library

In multiple myeloma (MM) and other neoplasias, several kinases have been extensively evaluated as potential therapeutic targets using RNAi-based approaches or pharmacological inhibitors. Attempts to map the functional dependence of MM cells on individual kinases have primarily utilized RNAi, a mechanistic approach inherently dissimilar to small molecule inhibitors that are applied in the clinic. For many of these oncogenic kinases, large numbers of such inhibitors have been designed: these inhibitors often exhibit very similar effect on their primary designated target(s), but also perturb other secondary kinases, which may vary for different inhibitors within the same class. Using large sets of such inhibitors can enable comparative analyses to reveal the functional roles of both the respective primary target(s), as well as non-overlapping secondary targets. We therefore pursued the functional mapping of the kinome dependencies of 16 MM cell lines, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure), which target a total of 43 known primary oncogenic targets. In this study, we observed universally potent activity of Aurora (n=18 compounds), PLK (n=5), and mTORC1/2 (n=20) inhibitors; this observation is consistent with the high proliferative rate of MM cell lines in vitro. In contrast, we observed modest to minimal cell-autonomous susceptibility of MM cells to selective inhibitors of PDK1, PI3K (excluding those that also inhibit mTOR), and Akt: this suggests that PDK1- and Akt-independent mechanisms mediate the effect of PI3K signaling on the survival of most of these cell lines. In addition, we observed lack of response in virtually all tested cell lines to inhibitors of c-met (n=17 inhibitors), ALK (n=2), EGFR superfamily members (EGFR, HER2; n=25 inhibitors), c-kit (n=3), PDGFR(n=5), VEGFR (n=21), Flt3 (n=7), FAK (n=2), Syk (n=5), Src (n=5) and BTK: this result was observed even in those cell lines with detectable transcript against the respective kinases. Notable exceptions to this pattern were inhibitors that, in addition to their primary target, also possess activity to other kinases with known roles in MM (e.g. potent activity of FAK or ALK inhibitors that also target IGF1R, such as TAE226 and GSK1838705A, respectively). Consistent with prior experience, several FGFR3 inhibitors showed modest activity against FGFR3- expressing cell lines (e.g. KMS11, KMS18, OPM2, KMS34). Our screen also revealed several previously underappreciated classes of inhibitors with "non-consensus", heterogeneous, activity across the tested MM cell lines. For instance, we identified 3 clusters of cell lines with high (e.g. AMO1, Karpas-620); intermediate (e.g. KMS20, MM1S), and low responsiveness, to 8 different MEK1/2 inhibitors. Notably, both Karpas-620 and AMO1 cells are KRAS-mutant, BRAF-wild-type and have inherently high levels of p-ERK; while AMO1 cells also harbor a MEK2-Q60P mutation, previously reported to positively regulate the kinase domain activity of MEK2 and induce resistance of BRAF-V600E mutant melanoma cells to MEK1/2 inhibitors. These results raise the possibility that the response to MEK1/2 inhibitors and the role of specific mutations, such as MEK2-Q60P, are tumor-type dependent and/or influenced by concurrent BRAF mutation status. Notably, BRAF inhibitors (n=7) were inactive as cytoreductive agents against our cell line panel of BRAF wild-type cells; while several MM cell lines exhibited significantly increased proliferation upon treatment with these inhibitors. This stimulation has been previously noted in melanoma and has been attributed to activation and signaling through C-RAF; it also suggests that treatment of MM patients harboring both V600E-BRAF mutant and wild-type clones with BRAF inhibitor may decrease the burden of the former clone(s), but select for outgrowth of the latter. In summary, our studies establish the value of using large libraries of small-molecule kinase inhibitors in phenotypic assays against panels of tumor cell lines, as an approach to functionally annotate the kinome dependencies across a given neoplasia, such as MM. Furthermore, our studies provide insight into the possible clinical implications that specific molecular lesions (e.g. mutation status of MEK2 or BRAF) can have on the individualized administration of kinase inhibitors targeting the respective pathway.