DNAR-01. Post-translational arginine methylation of Sam68 regulates DNA repair and radiation resistance in glioblastoma

Abstract OBJECTIVE Glioblastoma (GBM) radiation (RT) resistance is governed by numerous levels of biology including post-translational modifications. Protein arginine methylation is altered in many GBMs and can be targeted in the clinic, but how it relates to DNA repair is uncertain. We sought to understand how protein arginine methylation mediates DNA repair and RT resistance in GBM. METHODS We assessed RT-induced protein arginine methylation marks by both Western blotting and methylation-specific immunoprecipitation coupled with mass spectrometry (IP-MS). To test functional impact, we generated knockdown cells targeting the identified protein using shRNA and re-expressed either wild-type (WT) or methylation-deficient mutant proteins. DNA damage repair kinetics were analyzed through γH2AX foci immunofluorescence staining, and radiosensitivity was measured via clonogenic survival assays. Protein interactions were examined using co-immunoprecipitation (co-IP). RESULTS Western blotting showed a global increase in monomethyl arginine (MMA) levels in GBM cells following RT, especially around 50-70 kDa. Gel extraction of this band of interest followed by MMA-specific IP-MS identified Src-Associated substrate during Mitosis of 68 kDa (Sam68), an RNA-binding protein implicated in cancer, as a candidate protein that undergoes arginine methylation following RT. RT enhanced MMA at residues R331 and R340 within the Arg-Gly-rich (RGG) domain of Sam68. We confirmed an RT-induced increase in MMA level of Sam68 by IP-western-blotting. Knockdown of Sam68 slowed RT-induced double-strand break repair, evidenced by persistent γH2AX foci at 6- and 24-hours post-RT. This defect was rescued by re-expressing WT Sam68 but not the methylation-deficient mutant(R331K/R340K). Sam68-knockdown and R331K/R340K mutation conveyed enhanced radiosensitivity to GBM cells in clonogenic survival assays. Mechanistically, RT enhanced interaction between Sam68 and poly(ADP-ribose) polymerase 1 (PARP1), a key enzyme in DNA repair, in an MMA-dependent manner. CONCLUSION Arginine methylation of Sam68 is critical for efficient DNA repair and RT resistance in GBM, potentially mediated through its interaction with PARP1.