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.
