CNSC-40. GTP SIGNALING PATHWAY PROMOTES NHEJ REPAIR BY INDUCING NUCLEAR ACTIN POLYMERIZATION IN GLIOBLASTOMA

Abstract OBJECTIVES We recently identified that GTP promotes non-homologous end joining (NHEJ) by triggering a GTP-Rac1-PP5-Abi-1/S323 cascade and we sought to define the molecular mechanisms. METHODS Crispr/Cas9, CDNA overexpression and immunoblot were used to modulate and confirm protein levels. Cellular fractionation and immunofluorescence (IF) were used to determine the localization of proteins. Laser micro-irradiation was used to induce DNA damage and monitor the recruitment of DNA repair proteins. RESULTS We found that GTP-activated protein Rac1 and Abi-1, the two key components of the newly identified pathway, shuttled into the nucleus post-RT in glioblastoma (GBM) cells. These effects were further enhanced by the GTP precursor guanosine but blocked by GTP depletion. Furthermore, Abi-1 and Rac1 accumulated in the nucleus of the cells expressing constitutively active Rac1 but not dominant negative Rac1 after RT, which suggests that Rac1/Abi-1 nuclear translocation is Rac1 activity dependent. We previously discovered that GTP-Rac1 promotes DNA repair by facilitating the dephosphorylation of Abi-1 (S323). Here, we discovered that dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D) shuttled into nucleus after RT. Because Rac1 canonically binds to Abi-1 and promotes actin polymerization in the cytosol, we next wanted to determine if RT-induced nuclear Rac1/Abi-1/S323A could enhance nuclear actin polymerization, which could enhance NHEJ repair. We found that Abi-1/S323A but not Abi-1/S323D increases nuclear filament actin formation post-RT and activating the GTP-Rac1-Abi-1/S323A axis (through guanosine supplementation, expression of constitutively active Rac1 or expression of dephosphomimetic Abi-1/S323A) enhanced the recruitment and stability of KU80 at sites of DNA damage, which could be blocked by actin polymerization inhibitor cytochalasin D. Furthermore, inhibiting actin polymerization overcame radiation resistance in GBM cells with an active GTP-Rac1-Abi-1/S323A axis. CONCLUSIONS GTP-Rac1-PP5-Abi-1/S323 cascade promotes NHEJ repair by inducing the nuclear assembly of filament actin and disrupting this regulation could improve treatment responses in GBMs.