Replication Associated Base Excision Repair Regulates Replication Stress Induced PARP1/PARP2 Activation and Impacts PARP and PARG Inhibitor Resistance
Date of Award
Basic Medical Sciences
Robert W. Sobol, Ph.D.
Endogenous and exogenous genotoxins pose a significant challenge to the DNA replication machinery, contributing to replication stress, a phenotype common in precancerous and cancerous cells but not common in normal cells. Replication stress is enhanced in cancer cells due to oncogene activation, inactivation of tumor suppressor genes, unexpected DNA structures, DNA base lesions, DNA nicks and ssDNA gaps, incomplete maturation of Okazaki fragments, an insufficient nucleotide pool and/or a lack of replication machinery proteins, among multiple other reasons. Replication stress is a target for cancer chemotherapy development. Replication stress causes a tremendous amount of DNA damage at the replication fork. Despite these excessive levels of DNA damage, cancer cells continue replication. I hypothesized that cancer cells use replication associated BER to resolve these DNA lesions and develop therapy resistance. Therefore, we propose that targeting the replication associated BER factors XRCC1, POLB, APTX and LIG3 will overcome cancer therapy resistance, including upon treatment with PARP or PARG inhibitors. PARP1 and PARP2 get activated in response to the damage from replication stress and then recruit XRCC1. I found (i) using two independent cell synchronization methods that PARP activation (PARylation) is a spontaneous cancer cell signal in xvii response to replication stress, (ii) using CRISPR/Cas9, both PARP1 and PARP2 are critical for PARylation in response to replication stress, (iii) using Split-TurboID, we found that the PARP1-XRCC1 activated complex recruits the BER factors POLB, APTX, LIG3 and the replication markers RPA1, RPA2, RPA3 during replication, (iv) using a PAR assay, loss of the BER proteins XRCC1, POLB, APTX, LIG3 enhances replication dependent PARylation and, (v) using confocal microscopy, the BER factors XRCC1, POLB, APTX, LIG3 colocalize with the replication marker RPA2, presumably at the replication fork. These findings collectively confirm that replication dependent PARP1/2 activation recruits BER factors to suppress PARylation from replication stress. PARG inhibition causes PAR accumulation and slows fork progression by activating CHK1. I found that PARG inhibitor resistant cells use replication associated BER factors (by increasing their recruitment upon PARGi) to resolve the PAR signal and develop resistance. I successfully targeted these BER factors (XRCC1, POLB, APTX and LIG3) to overcome PARGi resistance in ovarian and glioblastoma cancer cells. I found that in the absence of these BER factors, PARGi initially hyperactivates the ATR-CHK1 protective pathway to slow down fork progression and increase BER protein (XRCC1, POLB, APTX and LIG3 and replication factors) recruitment to suppress PARylation. However, prolonged PARG inhibition increases Caspase3/7 activity and causes cell death when BER proteins are absent. I further demonstrated that the loss of these BER proteins reverses PARPi resistance in ovarian cancer cells. The PARP1-XRCC1 activation complex recruits BER factors concurrently with the DSB repair pathway proteins BRCA1 and BRCA2. BRCA1/2 deficient cells use these BER pathway proteins most probably as a “backup xviii pathway” to develop resistance against PARP inhibitor treatment, similar to that seen upon restoration of BRCA1/2 expression. I also found that deletion of the BER genes XRCC1, POLB, APTX and LIG3 is synthetically lethal with BRCA2 deficient cells, further supporting our finding of replication associated BER as a “backup pathway” to develop resistance. Overall, these findings have defined replication associate BER as an essential pathway to regulate PAR accumulation in response to replication stress that may offer new targets to overcome PARP or PARG inhibitor resistance.
Ibrahim, Md, "Replication Associated Base Excision Repair Regulates Replication Stress Induced PARP1/PARP2 Activation and Impacts PARP and PARG Inhibitor Resistance" (2022). Theses and Dissertations. 116.
Available for download on Saturday, December 16, 2023
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