A Pooled Shrna Screen In 3d Cultures Of Primary Tumor Propagating Cells Identifies Regulators Of Innate Chemoresistance In Kras-Driven Nsclc

Despite advances developing targeted therapies for non-small cell lung cancer (NSCLC), patients carrying activating KRAS mutations still have no effective targeted therapeutics and respond poorly to chemotherapy. Discovering drug targets that work in combination with existing chemotherapeutics to inhibit innate chemoresistance and prevent re-initiation of tumor growth could reduce the likelihood of relapse and extend patient survival. Heterogeneous tumor propagating cell (TPC) populations in NSCLC have been shown to have increased chemoresistance, an enhanced ability to re-seed tumors in immunocompromised mice, and express genes associated with poor prognosis. To target chemoresistance in TPCs, we have developed a 3D culture model for functional genomic screens using primary tumor cells from a mouse model of NSCLC driven by activation of KRAS and p53 loss. Using pooled shRNA libraries, we have performed a focused screen in 3D TPC cultures of over 3,000 shRNAs targeting 600 genes associated with chemotherapeutic response in TPCs, TPC gene expression, DNA damage response, or bulk tumor resistance. We selected targets for validation, characterization, and in vivo studies if knockdown of a gene selectively sensitized TPCs to platinum-based chemotherapy or inhibited growth of TPCs. Multiple DNA damage response pathways were implicated in chemoresistance including known regulators, such as ATR and the translesion synthesis pathway. Many genes expressed selectively by TPCs alone or in response to chemotherapy were required for chemoresistance, including regulators of mitochondrial transport, RNA export, and DNA replication. A parallel screen using the KRAS-mutant LKR10 cell line was used as a control for shRNA representation and to determine which targets are required for TPCs to survive in 3D but not in 2D cultures. 3D-specific vulnerabilities include genes that are expressed highly in TPCs or in chemoresistant tumors and represent potential KRAS vulnerabilities that exist in the TPC population. Most chemoresistance targets and KRAS vulnerabilities are not shared by LKR10 cells grown in 2D suggesting growth in a multidimensional matrix exposes therapeutic opportunities that are distinct from cells grown as monolayers. Although functional genomic screens using primary tumor cells in 3D have limited throughput, focused screens utilizing primary tumor cells have the potential to discover therapeutic opportunities that exist in vivo that have not been uncovered in screens utilizing cell lines in 2D. Citation Format: David Richard Simpson, Dedeepya Vaka, Yanyan Zheng, John Tamaresis, Alejandro Sweet-Cordero. A pooled shRNA screen in 3D cultures of primary tumor propagating cells identifies regulators of innate chemoresistance in KRAS-driven NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4393.