TMIC-12. Pyk2 and MEK/Erk signaling regulate extracellular vesicle biogenesis and cargo in glioblastoma

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor with a median survival of less than one year. Immunotherapy is effective in only 10% of cases, partly due to an immunosuppressive tumor microenvironment shaped by tumor-derived extracellular vesicles (EVs), which influence immune responses by activating tumor-associated myeloid cells (TAMs) and promoting tumor growth. We hypothesize that Pyk2(Proline Tyrosine Kinase 2) and MEK/Erk signaling in GBM cells regulate EV biogenesis via actin cytoskeletal remodeling, impacting TAM activation. Primary human GBM cell lines with and without CRISPR/Cas9-mediated Pyk2 knockout (Pyk2KO) were used to investigate this. EVs were analyzed by flow cytometry, western blotting, and PCR, and MEK/Erk signaling was inhibited using Avutometinib (1 µM). Pyk2KO shifted the EV profile toward larger-diameter particles. Using Integrin as a plasma membrane marker, 84.7% of EVs from wild-type cells were Integrin+, compared to 89.1% in Pyk2KO cells. MEK/Erk inhibition alone reduced Integrin+ EVs to 79.1%, and the combination with Pyk2KO further decreased this to 78.8%. Additionally, Pyk2KO significantly reduced EV cargo levels of CCL2, CCL5, TNF, and VEGF, key modulators of TAM activation and angiogenesis. These findings suggest that Pyk2 regulates EVs originating from the endosomal compartment, whereas MEK/Erk signaling enhances the shedding of plasma membrane-derived EVs. These pathways influence EV size, membrane origin, and immune-regulatory cargo. This study reveals the role of Pyk2 and MEK/Erk in EV biogenesis in GBM.