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Conventional genetically engineered mouse models (GEMM) are time-consuming, laborious, and offer limited spatiotemporal control. Here, we describe the development of a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Unlike conventional GEMMs, this model system allows for flexible, sustained, and timed activation of one or more target genes using single or pooled lentiviral guides. Myc and Yap1 were used as model oncogenes to demonstrate gene activation in primary pancreatic organoid cultures in vitro and enhanced tumorigenic potential in Myc-activated organoids when transplanted orthotopically in vivo. Implementation of this model as an autochthonous lung cancer model showed that transduction-mediated activation of Myc led to accelerated tumor progression and significantly reduced overall survival relative to nontargeted tumor controls. Furthermore, Myc activation led to the acquisition of an immune suppressive, “cold” tumor microenvironment. Cross-species validation of these results using publicly available RNA/DNA-seq datasets linked MYC to a previously described immunosuppressive molecular subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC- and immune-targeted therapies. Overall, this 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. Significance: A streamlined platform for programmable CRISPR gene activation enables rapid evaluation and functional validation of putative oncogenes in vivo.

Conventional genetically engineered mouse models (GEMM) are time-consuming, laborious, and offer limited spatiotemporal control. Here, we describe the development of a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Unlike conventional GEMMs, this model system allows for flexible, sustained, and timed activation of one or more target genes using single or pooled lentiviral guides. Myc and Yap1 were used as model oncogenes to demonstrate gene activation in primary pancreatic organoid cultures in vitro and enhanced tumorigenic potential in Myc-activated organoids when transplanted orthotopically in vivo. Implementation of this model as an autochthonous lung cancer model showed that transduction-mediated activation of Myc led to accelerated tumor progression and significantly reduced overall survival relative to nontargeted tumor controls. Furthermore, Myc activation led to the acquisition of an immune suppressive, “cold” tumor microenvironment. Cross-species validation of these results using publicly available RNA/DNA-seq datasets linked MYC to a previously described immunosuppressive molecular subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC- and immune-targeted therapies. Overall, this 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.Significance:A streamlined platform for programmable CRISPR gene activation enables rapid evaluation and functional validation of putative oncogenes in vivo.

Supplementary Figure from A Programmable In Vivo CRISPR Activation Model Elucidates the Oncogenic and Immunosuppressive Functions of MYC in Lung Adenocarcinoma

Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-binding protein that deaminates adenosine (A) to inosine (I). A-to-I editing alters post-transcriptional RNA processing, making ADAR1 a crucial regulator of gene expression. Consequently, Adar1 has been implicated in organogenesis. To determine the role of Adar1 in pancreatic development and homeostasis, we conditionally deleted Adar1 from the murine pancreas (Ptf1aCre/+; Adar1Fl/Fl). The resulting mice had stunted growth, likely due to malabsorption associated with exocrine pancreatic insufficiency. Analyses of pancreata revealed ductal cell expansion, heightened interferon-stimulated gene expression and an increased influx of immune cells. Concurrent deletion of Adar1 and Mavs, a signaling protein implicated in the innate immune pathway, rescued the degenerative phenotype and resulted in normal pancreatic development. Taken together, our work suggests that the primary function of Adar1 in the pancreas is to prevent aberrant activation of the Mavs-mediated innate immune pathway, thereby maintaining pancreatic homeostasis.

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.

Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-binding protein that deaminates adenosine(A) to inosine(I). A-to-I editing alters post-transcriptional RNA processing making ADAR1 a critical regulator of gene expression. Consequently, Adar1 has been implicated in organogenesis. To determine the role of Adar1 in pancreatic development and homeostasis, we specifically deleted Adar1 from the murine pancreas (Ptf1aCre/+; Adar1Fl/Fl). The resulting mice had stunted growth likely due to malabsorption associated with exocrine pancreas insufficiency. Analyses of pancreases revealed ductal expansion, heightened interferon-stimulated gene expression and an increased influx of immune cells. In addition, we observed an increased prevalence of CD4+ T and natural killer cells in their splenic tissue. These results indicate an association between loss of pancreatic Adar1 with dysregulation of systemic immunity. Concurrent deletion of Adar1 and Mavs, a signaling protein implicated in the innate immune pathway rescued the degenerative phenotype and resulted in normal pancreatic development. Taken together, our work suggests that the primary function of Adar1 in the pancreas is to prevent aberrant activation of the Mavs-mediated innate immune pathway, thereby maintaining pancreatic homeostasis.Summary statementThis work defines the role of Adar1 in pancreatic development and homeostasis.

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.

While genetic mutations have been shown to drive pancreatic ductal adenocarcinoma (PDAC), epigenomic alterations in cancer cells may also contribute to tumor initiation and progression. Adenosine-to-inosine (A-to-I) editing is the most common type of RNA editing in the cell and is mediated by the adenosine deaminase acting on RNA (ADAR) enzyme. ADAR has been shown to be upregulated in various tumor types, it improves tumor cell viability and loss of Adar1 in melanoma cells increases efficacy of checkpoint blockade therapy in vivo. However, its function in PDAC is currently not well understood. In order to investigate the role of Adar1 in PDAC we developed a mouse model with a conditional knockout of Adar1 in the pancreas using the pancreas specific ptf1a Cre. However, the CYA (ptf1a [cre/wt], Rosa26 [LSL-YFP/LSL-YFP], Adar [F/F]) mice are born runted compared to littermate controls and die preweaning. We find that a conditional loss of Adar1 in the pancreas induces acinar cell death and the pancreatic tissue degenerates over a period of fifteen days. Adar1 inhibits interferon (IFN) responses that are activated by cellular double-stranded RNA (dsRNA) sensors through mitochondrial antiviral signaling protein (Mavs). To rescue the apoptotic and inflammatory phenotype of the CYA mice, we developed a CYAM mouse (ptf1a [cre/wt], Rosa26 [LSL-YFP/LSL-YFP], Adar [F/F], Mavs [-/-]). We find that these mice develop normally and their pancreas is devoid of apoptotic cells. Furthermore, to understand the function of Adar1 in pancreas homeostasis of adult mice, we developed an inducible model wherein in adult CiYA (ptf1aCre-ERTM [cre/wt], Rosa26 [LSL-YFP/LSL-YFP], Adar [F/F]) Adar1 loss was induced with intraperitoneal tamoxifen injections. We find that loss of Adar1 in the CiYA adult pancreas induces cell death and inflammation and the mice decline rapidly. We tested expression of a panel of 5 ISGs (Mx1, Isg15, IfnB1, Ifit1, Ifit2) by RT-qPCR and show that there is an elevated expression of ISGs in the pancreas of CiYA mice compared to WT controls. In conclusion, we show that pancreatic acinar cells are extremely sensitive to Adar1 loss both during developmental stages and in adult mice. Current in vivo models to study the function of Adar1 in development and cancer are limited by the lethal phenotype caused by upregulation of interferon stimulated genes (ISGs) and cell death in tissues where Adar1 is knocked out. Our work establishes a mouse model wherein pancreas-specific role of Adar1 in development and cancer can be studied without activation of Mavs-mediated inflammation. We are currently exploring how the CYAM mouse model can be utilized to advance our understanding of Adar1’s role in pancreas development and cancer. Citation Format: Dhwani N. Rupani, Robert W. Cowan, Andrew D. Rhim. Loss of Adar1 in pancreatic acinar cells leads to cell apoptosis and inflammation [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 A45.

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.

The transferrin receptor is the primary means of iron importation for most mammalian cells and understanding its regulatory mechanisms is relevant to hematologic, oncologic, and other disorders in which iron homeostasis is perturbed. The 3′ UTR of the transferrin receptor mRNA contains an instability element that is protected from degradation during iron depletion through interactions of iron regulatory proteins (IRPs) with five iron-responsive elements (IREs). The structural features required for degradation and the site of IRP binding required for in situ protection remain unclear. An RNA-CLIP strategy is described here that identifies the predominant site of IRP-1 interaction within a nontransformed primary cell line. This approach avoided complications associated with the use of elevated concentrations of protein and/or mRNA and detected interactions within the native environment of the mRNA. A compensatory mutagenesis strategy indicates that the instability element at minimum consists of three non-IRE stem–loops that can function additively, suggesting that they are not forming one highly interdependent structure. Although the IREs are not essential for instability, they enhance instability when IRP interactions are inhibited. These results are supportive of a mechanism for a graded response to the intracellular iron resulting from a progressive loss of IRP protection.