Abstract 3: In vivo CRISPR activation screening identifies tissue-specific oncogene selection

Abstract

Abstract Background: While the genetic landscapes of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) are relatively well described, the contribution of individual genes and transcriptomic programs to progression and metastasis remain incompletely understood. To quantify the tissue-specific selective advantage of transcriptomic drivers of tumor progression and metastasis, we have developed a platform for in vivo CRISPR activation (CRISPRa) oncogene screening. Material and Methods: Our platform consists of an autochthonous CRISPRa-competent mouse tumor model (PPKS; P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM), coupled with in vivo delivery of Cre/sgRNA lentiviral libraries. To evaluate tissue-specific differences in oncogene selection, we designed a CRISPRa library targeting 450 of the most frequently altered genes across human adenocarcinoma. We next generated lung and pancreas tumors through administration of controlled doses of Cre/sgRNA library in vivo. Tumors and metastatic lesions were isolated and guide enrichment was determined using NGS. We also performed CRISPRa screening on proto-oncogenic (PPKS) pancreatic organoids orthotopically transplanted into syngeneic or immunodeficient mice (NU/J). The results from our CRISPRa screening were confirmed using single-gene activation in vivo and in pancreatic organoids. Results: We found that pancreatic tumors displayed dramatically higher levels of guide selection relative to lung tumors. While, on average, 99.7% of guides were represented in primary lung tumors at survival endpoint, only 40.4% of guides were detected in pancreatic tumors. We identified very strong selection for Egfr-activation in primary lung tumors, followed by MafB, Sox2 and Myc. In contrast, we found near-complete dominance of Myc-activation in pancreatic tumors. Since EGFR and MYC represent two of the most frequently altered genes in NSCLC and PDAC, respectively, this indicates our ability to recapitulate clinically observed oncogene selection. To determine if the selection for Myc-activation in the pancreas was driven by immune response, we performed screening in pancreatic organoids transplanted into immune competent and incompetent mice and found that Myc was uniformly the most highly enriched target. To confirm our findings, we generated Myc-activated proto-oncogenic organoids and autochthonous tumors, and both displayed accelerated tumor formation relative to non-targeted controls. Conclusions: We present one of the first implementations of autochthonous in vivo CRISPR activation screening, allowing for direct observation and quantification of oncogene selection and competition in vivo. Our model faithfully recapitulated oncogene selection observed in patient tumors by identifying EGFR and MYC as dominating oncogenes in the context of KRAS mutant lung and pancreas tumors, respectively. Citation Format: Fredrik Ivar Thege, Sonja M. Woermann, Anirban Maitra. In vivo CRISPR activation screening identifies tissue-specific oncogene selection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3.