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Conventional genetically engineered mouse models (GEMMs) are time consuming, laborious and offer limited spatio-temporal control. Here, we describe the development of a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Unlike conventional GEMMs, our model system allows for flexible, sustained and timed activation of one or more target genes using single or pooled lentiviral guides. Using Myc and Yap1 as model oncogenes, we demonstrate gene activation in primary pancreatic organoid cultures in vitro and enhanced tumorigenic potential in Myc-activated organoids when transplanted orthotopically. By implementing our model as an autochthonous lung cancer model, we show that transduction-mediated Myc activation leads to accelerated tumor progression and significantly reduced overall survival relative to non-targeted tumor controls. Furthermore, we found that Myc-activation led to the acquisition of an immune suppressive “cold” tumor microenvironment. Through cross-species validation of our results using publicly available RNA/DNA-seq data sets, we were able to link MYC to a previously described, immunosuppressive transcriptomic subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC/immune-targeted therapies. Overall, our work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening.

Conventional genetically engineered mouse models (GEMMs) are time consuming, laborious and offer limited spatio-temporal control. We have developed a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Our model system allows for flexible, sustained and timed activation of one or more target genes, in vitro or in vivo, using single or pooled lentiviral guides. Using Myc and Yap1 as model oncogenes, we implemented this platform to study the effect of oncogene activation on the tumorigenic potential of primary pancreatic organoids, as well as in an autochthonous model of lung adenocarcinoma. We found that Myc-activation in pancreatic organoids increased their tumorigenic potential and resulted in significantly shorter survival relative to controls when transplanted orthotopically. In vivo Myc activation in the lung accelerated tumor progression and resulted in significantly shorter overall survival relative to non-targeted tumor controls. Furthermore, we found that Myc-activation drives the acquisition of an immune suppressive “cold” tumor microenvironment. Through cross-species validation of our results, we were able to link MYC to a previously described, immunosuppressive transcriptomic subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC/immune-targeted therapies. Our work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening. Citation Format: Fredrik Ivar Thege, Dhwani N. Rupani, Bhargavi B. Barathi, Anirban Maitra, Andrew D. Rhim, Sonja M. Wörmann. Development of a platform for programmable in vivo oncogene activation and screening using CRISPRa technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 918.

Introduction: Pancreatic cancer is characterized by early metastasis and resistance to chemotherapy. To elucidate the transcriptional reprogramming and drug resistance that emerges during the metastatic cascade, we set out to characterize the transcriptomic profiles and drug response of matched sets of cancer cell organoids derived from multiple sites (primary tumor, metastatic lesion, and circulating tumor cells) in tumor-bearing PKCY (p53L/wt, KrasLSL-G12D/wt, ptf1acre/wt, Rosa26LSL-YFP/LSL-YFP) mice. Methods: To establish primary and metastatic organoids, tumor lesions are excised, enzymatically digested, and plated in Matrigel. Previously described organoid cell culture media (containing R-spondin1, Wnt3A, Noggin, FGF10, and EGF) was used to maintain organoids in culture. To allow for the culture and analysis of circulating tumor cell (CTC) organoids, we developed a CTC isolation and culture protocol. Briefly, whole blood is collected from tumor-bearing mice using a terminal cardiac puncture into the right ventricle, followed by RBC lysis and plating in Matrigel. To allow for high-throughput drug screening, we developed a single organoid per 384-well approach where organoid growth and response to drug treatment can be quantified using automated fluorescent imaging. Results: We established organoid cultures from primary tumors and metastatic lesions (liver or lung) from 30 tumor-bearing PKCY mice, with a 100% success rate. In addition, we successfully established CTC organoid lines from 10 metastatic PKCY mice, with an overall success rate of 40% (10/25). The success rate was higher (60%) in mice harboring both macroscopic liver and lung metastasis, whereas mice harboring only liver or lung metastasis displayed a lower success rate (33% and 40%, respectively). We have established 5 matched sets of primary, metastatic (liver and/or lung), and CTC-derived organoids as wells as normal pancreatic ductal organoid lines from CY (ptf1acre/wt, Rosa26LSL-YFP/LSL-YFP) mice, serving as noncancer controls. Pancreatic origin of all organoids was confirmed with the YFP lineage-label. Preliminary drug testing of an 8-drug panel (gemcitabine, paclitaxel, 5FU, irinotecan/SN38, oxaliplatin, 5-azacytidin, SAHA, and EPZ6438) revealed several cases of increased drug resistance in organoids derived from metastatic lesions and CTCs relative to matched primary tumor organoids. To expand on this analysis, we are implementing a high-throughput drug screen evaluating a large library of small compound drugs and inhibitors and correlating the results to transcriptomic analysis. Conclusion: We developed an approach for mapping the pharmacotranscriptomic landscape of pancreatic cancer organoids derived from primary tumor, metastatic lesions, and CTCs. We are exploring our initial findings further using high-throughput drug screening, transcriptomic analysis, and orthotopic transplantations into syngeneic mice. Citation Format: Fredrik I. Thege, Bhargavi B. Barathi, Dhwani N. Rupani, Sonja M. Woermann, Andrew D. Rhim. Mapping the pharmacotranscriptomic landscape of pancreatic circulating tumor cell organoids [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 C55.