Glioblastoma (GBM) is a highly aggressive brain cancer and is usually fatal within a year of diagnosis. While immune checkpoint blockade therapies, such as Programmed Cell Death Ligand 1 (PD-L1) inhibitors, have shown success in various cancers, in recurrent GBM only 8% of patients revealed objective responses. Non- responsive GBMs display an immunosuppressive profile and higher levels of tumor-infiltrating myeloid cells (TIMs), consisting of brain microglia and peripheral-derived cells. Tackling the immunosuppressive microenvironment is the major challenge in GBM immunotherapy. The goal of the study is to identify mechanisms of GBM-mediated immune suppression and develop effective immunotherapy. In human GBM specimens and mouse models, we previously identified elevated activation of Proline-Rich Tyrosine Kinase 2 (Pyk2) and Focal Adhesion Kinase (FAK) signaling, associated with an immunosuppressive environment compared to healthy brain tissue. Pyk2 knock-out tumors exhibited increased infiltration of inflammatory myeloid cells and antigen-specific CD8+ lymphocytes. Additionally, an inverse relationship was found between Pyk2/FAK activation and Neurofibromatosis 1 (NF1) expression in human GBM samples, while NF1- cells demonstrated higher expression of PD-L1, which was reduced by knocking down Pyk2 and FAK. Based on these findings we hypothesize that Pyk2 and FAK signaling in GBM cells coordinate the development of an immunosuppressive tumor environment by regulating the release of cytokines and expression of PD-L1 in GBM cells. Targeting Pyk2/FAK enhances the sensitivity of GBM to anti-PD-L1 therapy. The NF1 loss exhibit increased sensitivity to Pyk2/FAK inhibitors, suggesting a potential therapeutic strategy for NF1- GBMs. The combination of Pyk2/FAK inhibitor defactinib and PD-L1 inhibitor pembrolizumab is anticipated to enhance anti- tumor immune responses and improve therapeutic outcomes in PD-L1 inhibitor non-responsive patients. In this study we will utilize NF1+ and NF1- mouse GBM models and primary human GBM cell cultures, coupled with cell biology approaches, to dissect the underlying mechanisms of Pyk2 and FAK signaling in regulating the activation state of TIMs. Additionally, the effectiveness of Pyk2/FAK inhibition as a sensitization strategy for the PD-L1 inhibitor pembrolizumab will be assessed in mouse GBM models, with a focus on NF1- tumors. These findings have the potential to enhance treatment outcomes in GBM and guide personalized treatment decisions based on the NF1 status of the tumor. To test our hypothesis, we propose the following specific aims: Specific Aim 1. To identify the role of Pyk2 and FAK signaling on the immunological microenvironment in NF1- and NF1+ GBM tumors. Specific Aim2. To identify the role of Pyk2 and FAK signaling in PD-L1 expression in GBM cells. Specific Aim3. To assess the effectiveness of combined Pyk2/FAK and PD-L1 pharmacological inhibition in reducing GBM tumor growth.

This project aims to improve immunotherapeutic outcomes for glioblastoma (GBM) patients by investigating the role of Pyk2 and FAK signaling in regulating PD-L1 expression in GBM and their impact on the tumor immune microenvironment. The study will explore the potential of Pyk2/FAK inhibitors as sensitizers for anti-PD-L1 therapy in GBM, considering the importance of incorporating the NF1 status of the tumor in treatment decisions. The findings will contribute to personalized therapeutic approaches and treatment choices, with the goal of enhancing outcomes for GBM patients.