Abstract A03: Genome-wide siRNA synthetic lethal screen for sensitizers of pancreatic cancer cells to gemcitabine

Abstract

Abstract Pancreatic cancer is estimated to claim 37,390 lives in the United States in 2012 (NCI). One year survival rate for patients who are diagnosed with pancreatic cancer is 24%, and overall 5-year survival is only 5%. The difficulty of early stage diagnosis of the disease undoubtedly contributes to the low survival statistics. At the time of diagnosis, over 52% of the patients have distant disease and 26% have regional spread (ACS). Therefore, identifying biomarkers to improve early detection of the disease is an important aspect in improving treatment efficacies. Despite an accrued wealth of knowledge, including the deep understanding of the RAS pathway, this aggressive disease still remains one of the deadliest of cancers. Pancreatic cancers are notoriously insensitive to chemo- and radiation therapy, all of which target processes essential for genome maintenance. Gemcitabine, a nucleoside analog that blocks DNA replication, remains a major therapy for patients with advanced pancreatic cancer. However, gemcitabine only extends survival by several months over other treatments. There is an urgent need to identify novel molecular targets that sensitize pancreatic cancer cells to chemotherapeutic agents. Identifying mechanisms of chemoresistance of pancreatic cancer will provide new targets that enhance cell killing by cytotoxic agents such as gemcitabine. We performed a synthetic lethal whole-genome siRNA screen to identify genes that promote survival of pancreatic cancer cells to gemcitabine. 27 genes were validated to promote survival of pancreatic cancer cells to gemcitabine. Because our screen cannot take into account the microenvironment of the pancreatic cancer cell, we identified survival/resistance pathways that were intrinsic to the cell. We have focused our efforts on the Vitamin D receptor (VDR), a transcription factor that is best known for regulating genes critical for bone morphogenesis. VDR however is expressed ubiquitously and regulates diverse biological pathways in different tissues. In the skin, where exposure to UV promotes the synthesis of vitamin D, VDR is thought to play a role in UV protection. Our finding that VDR in pancreatic cancer cells is a critical determinant of gemcitabine survival maybe an adaptation of its UV protective role in skin. We have isolated a BxPC3 pancreatic adenocarcinoma cell line that is stably depleted of VDR by shRNA and used it to validate specificity. The sensitization effect of VDR depletion can be alleviated by adding back siRNA resistant Wt-VDR, but not ligand or transactivation mutants. Pharmacological inhibition of VDR with TEI9647 antagonist also sensitized cells to gemcitabine. We show that depletion of VDR by RNAi enhances killing of gemcitabine by reducing the LC50 x-fold. This degree of sensitization is comparable to that achieved with inhibitors of the DNA damage checkpoint. The mechanism of killing is independent of checkpoint override, a known pathway that promotes drug survival to DNA damaging drugs. Timelapse movies show that death occurs while gemcitabine treated cells are arrested in the cell cycle, as opposed to premature entry into mitosis. Our screen therefore succeeded in identifying a pharmacologically sensitive pathway for gemcitabine survival of pancreatic cancer cells. We suggest that VDR activates a transcription protram that promotes survival of cancer cells to both extrinsic and intrinsic genotoxic stresses. Inhibiting VDR may enhance killing of tumor cells by gemcitabine but may also prevent cells from surviving intrinsic genotoxic stresses. Citation Format: Vikram Bhattacharjee, Timothy J. Yen. Genome-wide siRNA synthetic lethal screen for sensitizers of pancreatic cancer cells to gemcitabine. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A03.