Abstract B20: KrasG12D effector dependencies in the maintenance of pancreatic ductal adenocarcinoma

Abstract

More than 90% of pancreatic ductal adenocarcinoma (PDAC) tumors in humans feature activating mutations in the proto-oncogene KRAS. In mice, KrasG12D mutation alone is sufficient for PanIN formation; when coupled with mutational inactivation of TP53 (Tp53R172H), PanIN formation is accelerated and these neoplastic lesions progress to invasive and metastatic disease at high frequency. Several groups have demonstrated that inactivation of mutant Kras in established PanIN and PDAC leads to redifferentiation and apoptosis of the tumor, respectively. More recently, several combinatorial genetic and pharmacologic strategies have been employed that highlight RAF kinases as key oncoeffectors for KRAS addiction, but that RAF inhibition alone demonstrates little efficiency against KRAS mutant tumors. Additionally, previous studies suggest a requirement for both C-raf and EGFR blockade or the combination of RAF-MEK or RAF-PI3K inhibitors to inhibit to tumor growth. However, a systematic evaluation of dependencies of Kras effectors in tumor maintenance has not been explicitly performed. To investigate effector dependencies in the KrasG12D setting, we have developed an organoid platform to generate isogenic lines bearing doxycycline-inducible small hairpin (sh)RNAs coupled to GFP inserted into mouse pancreatic tumor organoids derived from KPC mice. These shRNAs are directed against each of the major Kras effector molecules, including B-raf, C-raf, Pi3k, RalA, RalB, and Rac1, with Kras shRNAs and Renilla shRNA used as controls. We identified 2 effective shRNAs per target, with strong and durable inhibition of each protein observed after doxycycline treatment. To investigate the hierarchy of Kras effectors, we employed a competition assay of the shRNA organoids, and observed the drop-out of Kras, C-raf, and Rac1 shRNA-bearing tumor organoid lines. Cell number and volumetric analyses confirmed strong dependencies on Kras, C-raf, and Rac1. Chromatin accessibility and transcriptomic analyses of Kras- , C-raf- , and Rac1-depleted organoids revealed consensus features of abrogated tumor maintenance and key molecular nodes in tumor maintenance. Together, we have employed a mouse organoid platform to create a rigorous map of the requirements for the key effectors of mutant Kras, and to dissect the biologic mechanisms shared by effectors disruptive of the mutant Kras epigenetic and transcriptional landscape. In turn, we anticipate these findings can inform the subsequent development of novel therapies to address these vulnerabilities. Citation Format: Adrien Grimont, Zhen Zhao, Steven D. Leach, Rohit Chandwani. KrasG12D effector dependencies in the maintenance of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B20.