Constitutive Vs. Stroma-Induced Kinase Dependencies in Myeloma Cells: Functional Mapping Using Small Molecule Inhibitors As Chemical Probes
Authors: Dhimolea Eugen , King Emily , Sheffer Michal , Hu Yiguo , Awate Pallavi P. , Sarkar Subhashis , Matthews Geoffrey M. , Murnane Megan , Bariteau Megan A. , Aftab Blake T. , Mitsiades Constantine S 
Published: October 2019

Abstract
The functional characterization of kinases in multiple myeloma (MM) cells has mainly involved the use of RNAi, a mechanistic approach inherently dissimilar to small molecule inhibitors that are applied in the clinic. Furthermore, in the bone marrow (BM) microenvironment, where MM cells primarily reside, BM stromal cells (BMSCs) and other nonmalignant cell populations can function as "accessory" cells and alter the vulnerability of MM cells to diverse targeted therapies, including kinase inhibitors. In order to characterize how the kinase dependencies of MM cells differ in the presence vs. absence of BMSCs, we evaluated 16 MM cell lines and their response, in monoculture vs. co-culture conditions, to a panel of 273 kinase inhibitors (100nM, 24-72 h exposure), which target a total of 43 known primary oncogenic targets. We observed that virtually all tested cell lines showed no response, in either monocultures or co-cultures, to inhibitors of c-met, ALK, Abl, EGFR superfamily members (EGFR, HER2), c-kit, PDGFR, Flt3, FAK, VEGFR and Syk, including cell lines with detectable transcript levels for the respective kinases at baseline or stroma-induced increase in co-cultures. These results suggest that the respective kinases do not represent major dependencies for MM cell lines in either the cell-autonomous state or in the context of interaction with stromal cells. Dual PI3K/mTOR inhibitors exhibited similarly potent anti-tumor activity in both monocultures and stromal co-cultures of essentially all MM cell lines tested; while mTOR inhibitors devoid of PI3K activity had examples (e.g. in U266 and KMM2 cells) of stroma-induced resistance, likely reflecting a role of PI3K signaling in mediating, at least in some MM cells, stroma-induced decrease in dependence to mTOR function. Inhibitors of IKK or JAK (e.g. ruxolitinib, baricitinib) exhibited higher activity against several MM cell lines in the presence compared to absence of BMSCs. This stroma-induced sensitization to these kinase inhibitors is consistent with the role of BMSC-derived cytokines or cell adhesion molecules in inducing in MM cells NF-kappaB transcriptional activation or JAK/STAT3 signaling. Interestingly, several other classes of kinase inhibitors, e.g. against Aurora, PLK, MEK, Akt, exhibited more heterogeneous stroma-induced effects on their activity (decrease against some cell lines vs. sensitization against others; without an obvious common pattern of these effects for each of these kinase inhibitors across different cell lines). These results suggest that the dependence of MM cells on the corresponding kinases and the signaling networks they regulate are not only subject to stroma-induced changes, but also exhibit a higher degree of cell type-dependent plasticity than previously appreciated. In further support of the complexity of these stoma-induced events, we also observed that BRAF inhibitors (e.g. vemurafenib, dabrafenib, PLX-4720, AZ-628, et.c.) induced at least modest increase in proliferation of several BRAF-wild-type MM cell lines cultured in the absence of stromal cells, but co-culture with BMSC blunted this effect in some cell lines (JJN3, AMO1, OPM2), and modestly enhanced it in others (e.g. KMS34). BRAF inhibitor-induced proliferation of BRAF-wild-type cell lines has been previously reported in different tumor types and has been attributed to activation and signaling through C-RAF: our current observations suggest that, in MM patients harboring both V600E-BRAF mutant and wild-type clones, the impact of treatment with BRAF inhibitor on decreasing the burden of the former clone(s) vs. selecting for outgrowth of the latter may actually be subject to complex, genotype-dependent, influence of the BMSCs of the local microenvironment. In summary, our studies demonstrate the feasibility of functionally annotating the kinase dependencies in MM and potentially other neoplasias by using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of tumor cell lines. Furthermore, our studies suggest that future efforts to individualize the administration of kinase inhibitors in MM should take into account not only the genotype of MM cells in the respective patient, but also the heterogeneous impact that nonmalignant "accessory" cells such as BMSCs can have on the MM cell dependence on each respective kinase and its downstream targets.

Disclosures
Aftab: Cleave Biosciences, Inc.: Research Funding; Onyx Pharmaceuticals, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Omniox, Inc.: Research Funding. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding.