EXTH-16. UNBIASED DISCOVERY OF SYNERGISTIC VULNERABILITIES AGAINST GLIOBLASTOMA: AN INNOVATIVE<i>IN VIVO</i> CRISPR PROFILING PLATFORM TO UNLEASH THE POWER OF TUMOR-TARGETED CYTOKINE THERAPY

Abstract BACKGROUND Glioblastoma poses a formidable challenge due to its intricate intra- and intertumoral heterogeneities and its associated immunosuppressive milieu. The standards of care have limited activity, and no major advances have been made in decades. One promising avenue is tumor-targeted cytokine-based therapies, particularly L19-TNF, which is undergoing clinical trials in Europe and the US for patients with newly diagnosed and recurrent glioblastoma. However, there is still an opportunity to find safer and more tolerable combination partners to improve the efficacy of L19-TNF immunotherapy. To address this challenge, we employed a cutting-edge in vivo CRISPR screening approach to identify synergistic vulnerabilities with a focus on druggable targets. METHODS We systematically investigated the dependencies of tumor growth and key signaling pathways under the pressure of L19-TNF immunotherapy in the CT-2A orthotopic murine glioma model. We used an innovative in vivo CRISPR screening approach that enables timed sgRNA activation to mitigate off-target effects and overcome orthotopic tumor cell implantation challenges, such as the bottleneck of cell engraftment. RESULTS We first conducted a genome-wide in vivo screen and identified 400 top-scoring candidate genes, which we subsequently tested again in a targeted in vivo screen to refine our drug selection process. Through this, we uncovered novel and previously unexplored druggable targets for glioblastoma. Out of these, we validated four promising target-specific drugs in preclinical glioblastoma models in downstream in vivo experiments. In addition, we investigated modes of action and resistance. CONCLUSION Our research marks a significant advancement in pursuing novel treatment strategies for glioblastoma. We have unveiled a range of synergistic combinations and paved the way for the rational design of combinatory treatment approaches to maximize the therapeutic efficacy of L19-TNF immunotherapy, laying the groundwork for future clinical translation.