Abstract PR-003: In vivo genome-wide CRISPR/Cas9 screens conducted in an immunocompetent mouse model of glioblastoma identify novel in vivo tumor liabilities and potential mechanisms of resistance to chimeric antigen receptor T-cell (CAR-T) therapy
Authors: Koch Catherine E. , Whittaker Charles A. , Hemann Michael T. 
Published: March 2024

Abstract
Glioblastoma (GBM) is the most common and deadly adult brain cancer with a post-diagnosis survival of 8-15 months even with treatment, highlighting a desperate need for new therapeutic strategies. One promising strategy involves the use of chimeric antigen receptor T-cell (CAR-T) therapy, created by engineering patient T-cells to express a CAR that recognizes a tumor-specific surface antigen thus allowing for directed tumor cell killing. While CAR-T therapy has experienced unprecedented success in treating hematologic malignancies, its application in solid tumors has proven challenging. However, early clinical studies in GBM have shown promise including a case in which a patient with disseminated GBM experienced a complete response. Unfortunately, the patient relapsed highlighting both potential therapeutic efficacy and our limited knowledge of resistance and relapse mechanisms. Established resistance mechanisms include tumor target antigen loss or intrinsic CAR-T failure, but there are certainly others that have yet to be identified. Furthermore, solid tumors present unique challenges and may possess distinctive resistance mechanisms related to hostile, immunosuppressive tumor microenvironments and vast inter-tumoral heterogeneity. Ultimately, improved understanding could lead to increased therapeutic efficacy through CAR-T combination therapies or direct CAR-T cell modifications. Beyond the promise of CAR-T however, there still remains a scarcity of effective treatment options for GBM, underscoring a critical need to gain a better fundamental understanding in order to create additional novel therapeutic strategies. In order to both understand mechanisms of resistance to CAR-T therapy in GBM and uncover novel tumor dependencies for therapeutic exploitation, I took an unbiased, functional genomics approach. I conducted a series of in vivo genome-wide screens in an orthotopic, immunocompetent mouse model of GBM using a novel, modular, small-pooled CRISPR/Cas9 single guide (sgRNA) library. Cas9-expressing tumor cells with library sgRNAs were intracranially injected into mice that subsequently received no treatment or direct intertumoral injection of non-targeting control or targeting CAR-T cells. Top depletion and enrichment hits were used to generate 2 separate validation libraries one containing CAR-T sensitization or resistance hits and the other containing hits essential for in vivo tumor growth and therefore potentially exploitable as novel therapeutic targets. Validation libraries were screened in the same manner as the original genome-wide screen and top hits are actively being validated with planned in-depth mechanistic follow up. Importantly, to date, these are the first in vivo genome-wide CRISPR/Cas9 screens conducted in an orthotopic, immunocompetent mouse model of GBM. Ultimately, these results could aid not only in the identification of novel therapeutic strategies for GBM and potential improvements in CAR-T clinical efficacy, but also in a broader understanding of the fundamental biology that underlies treatment resistance in GBM.
Citation Format: Catherine E. Koch, Charles A. Whittaker, Michael T. Hemann. In vivo genome-wide CRISPR/Cas9 screens conducted in an immunocompetent mouse model of glioblastoma identify novel in vivo tumor liabilities and potential mechanisms of resistance to chimeric antigen receptor T-cell (CAR-T) therapy [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-003.