JS04.7.A IDENTIFICATION OF BCL FAMILY MEMBERS AS MODULATORS OF REGORAFENIB EFFICACY IN EXPERIMENTAL GLIOMA

Abstract BACKGROUND Glioblastoma are aggressive primary tumors of the central nervous system with limited systemic treatment options. Regorafenib, a multi-tyrosine kinase inhibitor, demonstrated improved median overall survival in the phase 2 REGOMA trial for progressive glioblastoma, compared to the control arm with Lomustin. However, these results were not confirmed in the GBM AGILE adaptive clinical trial platform. To discover potential modifiers of the therapeutic efficacy of regorafenib, we investigated functionally guided combination therapy strategies in experimental glioma models. MATERIAL AND METHODS We employed a genome-wide CRISPR-Cas9 functional genomics approach, including both activation and knockout screens, followed by genetic, pharmacological, and functional validation. Regorafenib-induced molecular changes were analyzed at the RNA and protein levels. Selected functionally guided combination therapies were evaluated in vivo using several well-established orthotopic glioma mouse models, including the syngeneic, immunocompetent SMA560/VM/Dk model. Post-treatment brain tumor tissue was further analysed via immunohistochemistry. RESULTS We discovered potential regulators of regorafenib efficacy, including ARAF, BCL2, BCL2L1, FOXC1, ITGB3, PLCE1 and SERAC1. Combining regorafenib with the Bcl-2/Bcl-xL inhibitor navitoclax significantly improved therapeutic efficacy compared to monotherapy in vitro, ex vivo, and in vivo. RNA sequencing revealed regorafenib-dependent modulation of the Bcl-2 downstream target chemokine receptor 1 (CCR1). We further characterized the role of CCR1 as a functional mediator of the observed synergy with Bcl-2 family inhibition. Additionally, we detected regorafenib-induced changes in the myeloid compartment of the glioma-associated microenvironment in post-treatment murine tissue. CONCLUSION We utilized a functional genomics-based target discovery approach with subsequent multi-level validation to identify modulators of regorafenib efficacy. Our findings reveal actionable targets and support the rationale for combinatorial treatment strategies, particularly co-inhibition of Bcl-2 family proteins. These results provide a strong biological rationale for clinical evaluation of regorafenib in combination with navitoclax.