Abstract 4342: Direct genome editing of patient-derived xenografts for rapid in vivo functional genomics (2019)

Abstract Patient-derived xenografts (PDXs) are high fidelity in vivo tumor models that have become valuable tools for cancer research, particularly in the study of emerging cancer therapeutics. However, PDXs are rarely used in mechanistic and functional genomic studies, due in part to the technical barriers imposed by continuous passaging in vivo. We sought to develop a CRISPR-Cas9 platform that would leverage the robust nature of PDXs and enable in vivo functional genomic studies in these cancer models. To this end, we created pSpCTRE-CD4, an all-in-one doxycycline-inducible Cas9 lentiviral vector, which features a novel truncated CD4 selection marker that enables selection of transduced PDX cells by flow cytometry, bypassing the need to culture these cells ex vivo. The inducible TRE3GS promoter controlling Cas9 expression is in the reverse orientation, which reduces potential leaky expression of Cas9 and also places this promoter in close proximity to the constitutive EFS promoter controlling CD4 and rtTA expression. Interestingly, this promoter orientation causes a dramatic ~100-fold increase in CD4 expression over basal levels in the presence of doxycycline and thus provides an indicator for cells that induce Cas9. This property of pSpCTRE-CD4 allows for the study of heterogeneous tumor cell populations without the need to select for single cell clones, which is a common practice in inducible cell line systems. In vitro functional studies of pSpCTRE-CD4 showed that this vector is tightly regulated by doxycycline. We transduced 27 lung cancer PDXs with pSpCTRE-CD4 lentivirus and observed transduction efficiencies ranging from 0-30%. Of these, we successfully enriched 8 Cas9 PDX models derived from multiple lung cancer subtypes. We functionally validated these Cas9 PDXs by introducing an sgRNA targeting the essential gene RPA1 in an in vivo clonal competition assay. For each of the Cas9 PDXs tested, we observed a significant decrease in the sgRPA1:sgNon-targeting ratio for two independent RPA1 sgRNAs in mice treated with doxycycline, indicating a loss of fitness in these PDXs when RPA1 is depleted. Similarly, in JHU-LX55a-Cas9, a KRAS mutant lung adenocarcinoma PDX, we observed a significant decrease in the fitness of cells with an sgRNA targeting KRAS. We are using this system to investigate genetic dependencies within these PDXs and have designed a small sgRNA library targeting the druggable genome to perform CRISPR screens in these models. Additionally, we have designed a recombinant AAV vector that expresses a single sgRNA as well as template to introduce point mutations via homology-directed repair and we are using this system to interrogate variants of unknown significance identified in large-scale tumor sequencing studies. The CRISPR-Cas9 platform we have developed will greatly expand the use of PDXs and enable functional genomic studies to be performed in this robust in vivo model system. Citation Format: Christopher H. Hulton, Emily A. Costa, Charles M. Rudin, John T. Poirier. Direct genome editing of patient-derived xenografts for rapid in vivo functional genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4342.