KS01.7.A IN VIVO CRISPR SCREENING UNCOVERS VULNERABILITIES FOR CANCER IMMUNOTHERAPY AGAINST GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma remains a formidable challenge due to its complex intra- and intertumoral heterogeneities and immunosuppressive microenvironment, which have contributed to the limited progress in treatment over the past decades. A comprehensive approach, involving the strategic combination of therapies, is essential to effectively address the challenges associated with glioma treatment. L19-TNF, a tumor-targeted cytokine-based therapy that triggers anti-tumor immunity, is currently being tested in clinical trials for newly diagnosed and recurrent glioblastoma patients. To enhance its efficacy, we sought to identify synergistic or additive drug targets using unbiased in vivo screens in immunocompetent glioma mouse models. MATERIAL AND METHODS To achieve this, we employed a novel genome-wide in vivo CRISPR screening approach to uncover druggable vulnerabilities in glioma under the selective pressure of L19-TNF immunotherapy in the CT-2A orthotopic murine glioblastoma model. This platform overcomes challenges, such as the bottleneck during cell engraftment, by enabling timed sgRNA activation and incorporating an in vivo tumor-internal reference library. This strategy mitigates off-target effects and enhances the precision of genetic perturbations. Subsequently, we refined our findings with a second, targeted in vivo CRISPR screen to validate the most promising therapeutic candidates. RESULTS Using this approach, we discovered previously unrecognized vulnerabilities of glioblastoma that converge to ferroptosis. To validate its therapeutic potential, we tested small molecules triggering ferroptosis in combination with L19-TNF immunotherapy in preclinical glioblastoma mouse models. This dual therapy significantly prolonged survival in mice. We also confirmed target expression in glioblastoma patient samples by RNA sequencing, supporting its clinical relevance. Additionally, we validated the hits using orthogonal ex vivo drug-screens in freshly obtained patient-derived glioblastoma tissue samples. CONCLUSION Our research marks a considerable advancement in systematically uncovering gene candidates using in vivo CRISPR screening. These findings lay the foundation for the rational design of synergistic approaches, offering exciting prospects for more effective combination therapies for glioblastoma.