RM-04 * RETINOBLASTOMA BINDING PROTEIN 4 (RBBP4) MODULATES TEMOZOLOMIDE RESPONSE THROUGH REGULATION OF MGMT EXPRESSION IN GLIOBLASTOMA
Through shRNA library screen we identified RBBP4 as a modulator of TMZ response in glioblastoma (GBM). Consequently, we investigated the mechanisms whereby RBBP4 modulates TMZ response using shRNA to silence this gene in MGMT-expressing T98G and U138 GBM cells. The cytotoxicity was evaluated using fluorescence-based CYQUANT proliferation assay. A total of 4 shRNA constructs significantly suppressed RBBP4 in both T98G and U138. Cells expressing non-specific targeting shRNA (NT-shRNA) were used as control. RBBP4 knockdown significantly sensitized TMZ both in T98G and U138 cells; the relative fluorescence for the TMZ-treated (100 µM) control T98NT-shRNA cells was 1.17 ± 0.15, whereas for T98RBBP4-shRNA clones were 0.54 ± 0.02, 0.29 ± 0.03, 0.36 ± 0.05, and 0.34 ± 0.03, respectively (p < 0.001). Similar sensitization was observed in U138 cells; relative fluorescence for the TMZ-treated (300 µM) control U138NT-shRNA cells was 0.70 ± 0.05 and for U138RBBP4-shRNA clones were 0.42 ± 0.06, 0.27 ± 0.01, 0.28 ± 0.02, and 0.30 ± 0.01, respectively (p < 0.001). Interestingly, knockdown of RBBP4 in T98G was accompanied with a synthetic lethality to PARP inhibition and increased response to TMZ-induced DNA damage, as evidenced by increased phosphorylation of KAP1, CHK1 and CHK2. Moreover, phosphorylation of H2AX in response to TMZ treatment was significantly higher in T98RBBP4-shRNA clones. Consistent with deficient homologous recombination (HR), T98RBBP4-shRNA clones significantly expressed less RAD51 compared with the control T98NT-shRNA cells. Even more interesting, RBBP4 knockdown silenced MGMT expression in both T98G and U138, which was accompanied by decreased recruitment of acetylated H3K9 coupled with increased recruitment of tri-methylated H3K9. Moreover, RBBP4 knockdown was coupled with loss of p300 recruitment to bind MGMT promoter region. Collectively, these findings suggest that RBBP4 modulates TMZ response in GBM cells through epigenetic regulation of MGMT, and perhaps through RAD51-mediated HR repair of DNA damage in selected GBM cells.