In Vitro Anti-Myeloma Activity of the Multitargeted Kinase Inhibitor Midostaurin in the Context of Heterotypic Cocultures of Myeloma Cells with Nonmalignant Microenvironmental Accessory Cells
Authors: Lawasut Panisinee , Jacobs Hannah M. , Delmore Jake E , Negri Joseph , McMillin Douglas W. , Weisberg Ellen L , Griffin James D. , Richardson Paul G. , Anderson Kenneth C , Mitsiades Constantine S. 
Published: October 2019

Abstract
Abstract 2923
Midostaurin (PKC412; Novartis Pharmaceuticals) is a multi-targeted kinase inhibitor currently being evaluated in clinical trials in acute myelogenous leukemia (AML), because of its potent activity in cells expressing mutant FLT3. Prior preclinical studies from our groups have shown that PKC412 has FLT3-independent anti-MM activity, and the effects on AML cells is suppressed by the presence of conditioned media from bone marrow stromal cells (BMSCs), such as the immortalized BMSC line HS-5 (Weisberg et al. Mol Cancer Ther 2007). In this study, we evaluated whether the microenvironment-dependent drug resistance to PKC412 applies to not only AML cells, but also to cells from MM and other FLT3-negative malignant cells. We tested a panel of cells from MM (n=8), FLT-ITDneg AML (n=1), CML (n=2) and breast cancer (n=2) for their response to PKC412 in the presence or absence of BMSCs and other non-malignant accessory cells using tumor cell compartment-specific bioluminescence imaging (CS-BLI), as in our antecedent studies (McMillin et al. Nat Medicine 2010). We also compared the PKC412 response of the aforementioned neoplastic cells when cultured in vitro in the presence or absence of conditioned media (CM) from different types of BMSCs known to confer PKC412 resistance in FLT3-mutant AML cells. Consistent with our previous studies of PKC412 treatment in conventional cultures of MM cells in isolation, we observed that PKC412 exhibits an anti-proliferative effect within the first 24 hrs of treatment, with major reduction of the numbers of viable cells at 48 and 72 hrs. At sub-micromolar doses that did not significantly affect the viability of non-malignant accessory cells tested, PKC412 had similar (or for some MM cell lines had more pronounced) activity against the MM cells, both in the presence and absence of the non-malignant accessory cells tested (HS-5, HS-27a, NIH-3T3 cells with or without transfection with human CD40L, etc.). In contrast, under the same experimental conditions, coculture with either BMSCs or exposure to their conditional media, decreased the response of MM cells to dexamethasone. These results suggested in contrast to the impact on FLT3mut AML, that the anti-MM activity of PKC412 is preserved (and in some cases slightly enhanced) when the MM cells interact with microenvironmental accessory cells and/or their secreted growth/survival factors. To obtain insight on possible mechanistic foundations of these observations, we examined the pattern of kinases inhibited by PKC412 at sub-μM concentrations (using FLT3 and FGFR3, known targets of PKC412 as positive controls). The results of in vitro kinase activity assays showed that PKC412 potently suppresses the aforementioned positive controls, but also exerts >50% inhibitory effect on the in vitro activity of additional kinases such as Akt2, Pim1, GSK3a, PDK1, p70S6K, SRC and Aurora A. Many of these kinases are known to participate in proliferative/anti-apoptotic signaling cascades downstream of cytokine/growth factor receptors or cell adhesion-mediated events triggered during MM–stromal interactions. We therefore conclude that the influence of the tumor microenvironment on the anti-neoplastic effects of PKC412 may be tumor-type dependent. The anti-MM activity of PKC412 is not subject to drug resistance triggered by non-malignant accessory cells, and conversely is occasionally moderately enhanced by these MM-stromal interactions. Mechanistically, this observation may be attributed in part to the multi-targeted nature of this inhibitor and, in particular, its aggregate impact on several kinases known to mediate stroma-induced proliferative and antipoptotic signaling in MM.

Disclosures:
Griffin: Novartis Pharmaceuticals: Consultancy, Research Funding. Richardson:Millennium: ; Celgene: ; Johnson & Johnson: ; Novartis: ; Bristol Myers Squibb:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Mitsiades:EMD Serono: Research Funding; AVEO Pharma: Research Funding; Amgen: Research Funding; OSI Pharmaceuticals: Research Funding; PharmaMar: licensing royalties; Amnis Therapeutics: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.