CSIG-10. MARCKS EFFECTOR DOMAIN PHOSPHORYLATION STATE AS A REGULATOR OF TUNNELING NANOTUBES IN GLIOBLASTOMA AND NORMAL HUMAN ASTROCYTE BIOLOGY

Abstract BACKGROUND Glioblastoma (GBM) is well known to interact with surrounding tumor and stromal cells in a variety of ways to maintain its pathogenicity. Emerging evidence has identified tunneling nanotubes (TNTs), dynamic actin-rich structures that mediate direct intercellular communication, as critical facilitators of GBM progression and therapeutic resistance, yet mediators of TNT formation remain poorly understood. Myristorylated Alanine-Rich C Kinase Substrate (MARCKS), an actin-binding protein regulated by PKC, may influence TNT dynamics. We hypothesized that MARCKS effector domain (ED) phosphorylation via PKC regulates TNT formation and mitochondrial transfer between GBM cells and astrocytes, contributing to chemoresistance. METHODS We employed serum-free TNT-promoting co-culture models of PTEN-null GBM cells (patient-derived xenograft JX14 cells or U87) and normal human astrocytes (NHAs), using immunofluorescence confocal microscopy to detect MARCKS localization, and quantitative assays to measure TNT number, length, and mitochondria transfer. Pharmacological modulation of PKC with phorbol ester agonist (PMA) or inhibitor (Enzastaurin), and treatment with a MARCKS ED peptide (MED2), assessed effects on TNTs. Doxycycline-inducible U87 MARCKS ED mutants (WT, pseudo-phosphorylated [PP], non-phosphorylatable [NP]) were used to dissect MARCKS phosphorylation roles. CGGA database analysis examined correlations between MARCKS and TNT-related genes in primary classical GBM. RESULTS JX14 co-cultured with NHAs exhibited increased temozolomide resistance. MARCKS localized to heterotypic TNTs, and MED2 reduced TNT number, length, and mitochondrial transfer. PKC activation increased TNT formation and transfer, while inhibition produced opposite effects. U87-PP cells displayed abundant TNTs and transfer similar to PKC activation, whereas U87-NP cells showed reduced TNTs. MARCKS positively correlated with MyosinX, ACTB, ACTG1, and RHOT1 in GBM patient data. CONCLUSIONS MARCKS ED phosphorylation via PKC promotes functional TNT formation and mitochondrial trafficking between GBM cells and astrocytes, contributing to chemoresistance. Targeting MARCKS or PKC may represent a novel strategy to disrupt TNT-mediated resistance mechanisms in GBM.