Microenvironment-Dependent Synthetic Lethality: Implications for Tumor Pathophysiology and Anti-Cancer Drug Discovery.
Authors: McMillin Douglas , Delmore Jake , Weisberg Ellen , Negri Joseph M. , Klippel Steffen , Mitsiades Nicholas , Munshi Nikhil C. , Griffin James D , Richardson Paul G. , Anderson Kenneth C. , Kung Andrew L. , Mitsiades Constantine S. 
Published: October 2019

Abstract 1722

Poster Board I-748

Conventional anti-cancer drug screening in vitro has traditionally been performed in the absence of accessory cells of the tumor microenvironment. These normal cells of the bone marrow milieu can profoundly alter anti-tumor drug activity. To address this major limitation of traditional in vitro models, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. In this platform, tumor cells (e.g. myeloma, leukemia and solid tumors) stably expressing bioluminescent reporters are co-cultured with non-malignant accessory cells (e.g. stromal cells) to selectively quantify tumor cell viability to treatments in presence vs. absence of accessory cells. We applied CS-BLI to test various chemical libraries and showed that this platform is high-throughput scalable. We also identified stroma-induced chemoresistance in diverse malignancies, including imatinib-resistance in leukemic cells, as well as MM cell resistance to certain investigational agents. The majority of compounds screened in our studies were less active against tumor cells in the presence of stromal cells compared to their absence. Most interestingly, however, we identified a fraction of compounds which were more active against tumor cells in the presence of stromal cells. For example, we identified reversine, a compound exhibiting this stroma-dependent synthetic lethality in vitro, which we further confirmed in vivo, as it is active in an orthotopic model of diffuse MM bone lesions, but not in conventional subcutaneous xenografts. Mechanistically, in vitro kinase activity assays showed that reversine exhibits a distinct pattern of inhibition against targets such as Auroras, JAK2, and SRC, but not against other important kinases for MM survival, such as AKT1, 2, 3, FGFR3, or GSK3. These observations are compatible with the role of SRC and JAK kinases as downstream regulators of IL-6/IL-6R signaling, a key cascade triggered by tumor-stromal interactions in MM. Further mechanistic evaluation of this interaction at the transcriptional level showed that a stromal-induced gene expression signature in MM tumor cells correlates with inferior overall survival in patients with advanced MM (APEX dataset) and includes enhanced amplitude of signatures for activated Akt, Ras, NF-κB, HIF-1á, myc, hTERT, and IRF4; as well as signatures for biological aggressiveness and stem cell self-renewal. This suggests that selective inhibitors which block the activity of these pathways may exhibit tumor specific stromal-dependent synthetic lethality. Historically, synthetic lethality has focused on how tumor cells harboring specific constitutive oncogenetic lesions are responsive to certain agents, but not in absence of these genetic events. Our study introduces the notion that a synthetic lethal phenotype, rather than being exclusively genotype-dependent, can also be driven by the extrinsic influences of the tumor microenvironment. Importantly, the CS-BLI system can probe both genetically- and microenvironment-related synthetic lethality in a high-throughput scalable manner. This allows the testing of a large number of permutations, including multiple candidate therapeutics, cell lines, and non-malignant accessory cells, thus enabling the previously intractable large-scale evaluation of how genetics and microenvironment play a role in modulating cancer cell response to treatment. Importantly, unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells when interacting with stroma. These agents, in the past, may have been discarded from further preclinical or clinical development. We now provide a system with which to evaluate the role of the tumor microenvironment and identify novel agents capable of overcoming its protective effects.

Disclosures

Munshi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richardson:Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.