Abstract B02: Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer

Lung cancer is the foremost cause of cancer-related deaths in the U.S. KRAS mutations are one of the most frequent oncogenic aberrations in lung adenocarcinoma patients. Despite harboring identical mutations, the response of distinct tumor cells to therapeutic agents varies due to epigenetic reprogramming of the tumor cells. Epithelial-mesenchymal transition (EMT) contributes to phenotypic heterogeneity and determines the response of tumor cells to therapeutic agents. Kras-mutant NSCLC cells have been shown to acquire EMT as a mechanism to evade therapeutic targeting. We have previously observed that murine and human epithelial lung cancer cells are highly sensitive to MEK inhibition whereas mesenchymal tumor cells remain largely resistant. We aim to identify the mechanisms conferring survival advantage and an intrinsic resistance to MEK inhibition to mesenchymal cells. Using a loss-of-function shRNA dropout screen for targets with FDA-approved drugs, we identified cyclin dependent kinase 4 (CDK4) as an important driver of growth in mesenchymal cells both in vitro and in vivo. Murine NSCLC cells are resistant to cell cycle arrest induced by serum starvation, which corresponds to higher protein levels of CDK4 and cyclin D1 allowing cells to transition from G1 to S phase. After release of cells from arrested state, the mesenchymal cells transition into S phase more rapidly than the epithelial cells, suggesting a higher dependency on CDK4-cyclin D1 complex of mesenchymal tumor cells. An Ex Vivo Tumor (EVT) system to culture lung tumors in a 3D matrix environment that retains tumor cell heterogeneity was utilized to test the therapeutic sensitivity of distinct tumor cell subpopulations. In response to treatment with a CDK4 inhibitor, we observed a depletion of mesenchymal tumor cells within heterogeneous EVTs. In combination with MEK inhibitor treatment, there was a net decrease in size and viability of EVTs. Single-agent treatment with CDK4 inhibitor upregulated pErk in mesenchymal tumor cells, increasing their sensitivity to MEK inhibition and achieving an additive response with combination treatment. Mechanistically, the effects are mediated by FOXM1, a transcriptional regulator of cell cycle. FOXM1 positively regulates CDK4-cyclin D1 activity and is negatively regulated by the miR-200 family, which is preferentially expressed in epithelial cells. Upon induction of miR-200 expression, there was a decrease in FOXM1. CDK4, cyclin D1 and pRB levels follow a similar trend, suggesting the phenotype observed in mesenchymal cells is mediated by FOXM1. Since EMT is modulated by the miR-200/Zeb1 axis, higher levels of the transcriptional repressor Zeb1 and consequently FOXM1 in mesenchymal tumor cells confer a survival advantage and an intrinsic resistance to MEK inhibition. Combinatorial treatment approaches with MEK and CDK4 inhibitors targeting different subpopulations within a tumor are an effective strategy to combat resistant outgrowth of epigenetic subsets in a heterogeneous Kras mutant tumor. Citation Format: Aparna Padhye, B. Leticia Rodriguez, Jared J. Fradette, Christin Ungewiss, Don L. Gibbons. Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B02.