Abstract 2910: Pamiparib-induced replication stress augments the efficacy of temozolomide in GBM PDX models

Temozolomide (TMZ) is the main chemotherapy for GBM, but resistance develops in most patients, highlighting the need for sensitizing strategies. PARP inhibitors (PARPi), which disrupt DNA repair, are being investigated as TMZ-sensitizing agents. Here we evaluated the efficacy and explored molecular mechanisms of pamiparib-mediated TMZ sensitization. In vitro, low concentrations of pamiparib (0.1-1.0 μM) synergized with TMZ and enhanced DNA damage, particularly during the S- and G2/M phases. In vivo, pamiparib monotherapy (0.75 or 3 mg/kg, twice daily) was ineffective, however, TMZ afforded survival benefit in orthotopic patient-derived xenograft (PDX) models (GBM12, GBM22, and GBM84) of MGMT-methylated primary GBM. Pamiparib combined with TMZ (25 or 50 mg/kg daily) prolonged survival in GBM12 [median survival 272 vs. 114 days with TMZ (P=0.02), 30 days with placebo (P<0.001)] and GBM22 (median survival 88 vs. 60 days with TMZ (P=0.05), 26 days with placebo, (P<0.001)] but not in GBM84 [median survival 355 vs. 404 days with TMZ (P=0.44), 37 days with placebo (P<0.001)]. These findings align with prior studies indicating that PARPi can potentiate TMZ efficacy in a subset of primary GBM models sensitive to TMZ. TMZ induces cytotoxic DNA adducts, N7MeG, N3MeA, and O6MeG. While N7MeG and N3MeA are repaired by base-excision repair (BER), MGMT removes O6MeG. Suppressed MGMT expression, due to promoter methylation, drives TMZ sensitivity in ∼40% of primary GBM cases. In MGMT-methylated tumors, O6MeG-thymine mispairs trigger futile mismatch repair, causing replication stress. Although BER is PARP-dependent, BER inhibition did not replicate the TMZ-sensitizing effects of pamiparib. Beyond DNA repair, PARP regulates replication stress by recruiting translocases like SMARCAL1 and controlling endonucleases such as MRE11 and EXO1, protecting stressed forks and nascent DNA. We targeted these components using siRNA and small-molecule inhibitors to elucidate their roles in TMZ sensitization. Unexpectedly, silencing SMARCAL1 or MRE11, or inhibiting MRE11 with mirin, did not affect pamiparib-mediated sensitization in U251 cells. While EXO1 silencing attenuated DNA damage signaling, it had minimal impact on TMZ sensitivity or pamiparib-mediated sensitization. These results suggest that fork protection mechanisms may be epistatic to PARP but redundant for TMZ sensitivity. Alternatively, replication stress may be mitigated by lesion bypass via translesion synthesis (TLS), where replicative polymerases are replaced with low-fidelity polymerases. Consistent with this, silencing RAD18, involved in TLS, enhanced sensitivity to both TMZ and pamiparib, with the combination being more effective than either drug alone. In conclusion, pamiparib enhances TMZ efficacy in a subset of primary GBM. While further in vivo validation is needed, TLS deficiency may be a key determinant of pamiparib-mediated sensitization. Citation Format: Shiv K. Gupta, Ann C. Mladek, Sonia Jain, Katrina K. Bakken, Zeng HU, Brett L. Carlson, Danielle M. Burgenske, Jann N. Sarkaria. Pamiparib-induced replication stress augments the efficacy of temozolomide in GBM PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2910.