Abstract B046: Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma
Authors: Ramirez Neisha M. 
Published: November 2024

Glioblastoma (GBM) is often fatal within a year despite treatment, with immune checkpoint blockade therapies benefiting only about 10% of patients. Non-responsive GBMs exhibit an immunosuppressive profile and higher levels of tumor-associated myeloid cells (TAMs). Addressing this immunosuppressive microenvironment is a major challenge in GBM immunotherapy. Our recent studies have identified a positive correlation between Proline-Rich Tyrosine Kinase 2 (Pyk2) activation in GBM tumor cells and the cytokine expression profile of TAMs. This identified Pyk2 as a potential prognostic marker for the immune state in GBM tumor microenvironment. We hypothesize that Pyk2 and MEK/Erk signaling in GBM cells regulate the release of chemokines and cytokines through extracellular vesicles (EVs) by modulating the actin cytoskeleton, thereby impacting TAM activation. The study aimed to investigate the role of Pyk2/MEK/Erk signaling in EV release in GBM cells, potentially revealing new therapeutic targets. Two humans primary GBM cell lines, with and without Pyk2 CRISPR/Cas9 knockout (Pyk2KO), were used. Flow cytometric analysis of EVs, enriched from cell-conditioned medium, identified a shift towards larger in diameter EVs populations in Pyk2 KO cells, compared to wild-type (WT) cells. Analysis using Integrin as a plasma membrane marker showed that 84.70% of Integrin+ and 15.30% of Integrin- EVs were derived from WT cells, whereas Pyk2KO cells produced 89.07% Integrin+ and 10.93% Integrin- EVs. Treatment of WT cells with the Erk/MEK inhibitor Avutometinib (1µM) altered the EV population’s ratio to 79.11% Integrin+ and 20.89% Integrin-. Similarly, Avutometinib treatment of Pyk2KO cells resulted in 78.83% Integrin+ and 21.17% Integrin- EVs, similar to the ratio observed in Avutometinib-treated WT cells. Western blot and PCR analysis of EVs content revealed a significant reduction in CCL2, CCL5, tumor necrosis factor (TNF), and vascular endothelial growth factors (VEGF) in EVs from Pyk2KO GBM cells, compared to WT. In vivo studies using GL261/C57Bl/6 mouse glioma implantation model and flow cytometric analysis of TAMs from tumors generated with WT and Pyk2KO GL261 cells demonstrated increased infiltration of TNF+/IFNg+ CD8+ lymphocytes and Ly6C+/CD206- myeloid cells, alongside a reduction in CD45+/CD11b+/CD33+/MHCII- myeloid-derived suppressor cells. PCR analysis of TAM isolated from Pyk2KO tumors revealed lower gene expression of TNF, CCL5, CCL2, VEGFa, and epidermal growth factor (EGF) compared to WT tumors. The study highlights that Pyk2/MEK/Erk signaling regulates the immune microenvironment in GBM by influencing EV release, which impacts TAM cell activation. Pyk2 primarily regulates the release of Integrin- EVs, while MEK/Erk predominantly regulates EVs shed from the plasma membrane. The findings underscore the interplay between Pyk2 and MEK/Erk signaling in regulating EV biogenesis and composition.

Citation Format: Neisha M. Ramirez. Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B046.