The Role of Apurinic/apyrimidinic Endonuclease 1 (APE1) in Temozolomide Resistance

Abstract

Temozolomide (TMZ) is a DNA-methylating chemotherapeutic agent used as a first-line treatment for glioblastoma. However, the development of TMZ resistance urgently requires new strategies to improve TMZ efficacy. We found that the most effective way to re-sensitize TMZ-resistant cells is by inactivating the apurinic/apyrimidinic site endonuclease (APE1). Different DNA repair pathways cause TMZ resistance in mismatch repair-proficient (MMR+) TMZ-sensitive versus MMR-deficient (MMR-) TMZ-resistant cells. In MMR+ cells, O6-methylguanine (O6-meG) triggers a “futile cycle” of MMR, likely causing replication forks to stall at MMR-induced ssDNA gaps. In MMR- cells, another TMZ-induced base adduct, 3- methyladenine (N3-meA), causes DNA replication blocks and is mainly responsible for cell death. 3-meA is repaired by the base excision repair (BER) pathway. Without APE1, BER is initiated by the MPG DNA glycosylase and stalls at the abasic site step, leading to the accumulation of ssDNA breaks, which results in cell death. We validated that novel, highly specific APE1 inhibitors (XPose Therapeutics) re-sensitize MMR- cells to TMZ and may help prevent the build-up of TMZ resistance in patients. Additional inactivation of translesion synthesis (TLS) further increases sensitivity of APE1-/- cells to TMZ. A double knockout of REV7 (a regulatory subunit of Pol ζ) and APE1 is probably lethal, indicating an essential role for Pol ζ in abasic site bypass. Conversely, Y-family polymerases (Pol η, κ, and ι) are functionally redundant if Pol ζ persists. We found that APE1 knockout is highly mutagenic, showing a BRCA-like mutational signature. Disrupting TLS significantly reduced the mutation counts observed in APE1-deficient cells, demonstrating that error-prone TLS bypass of abasic sites is a major driver of mutagenesis. Using optimized alkaline CometChip conditions, we quantified ssDNA breaks and established a direct correlation between DNA break accumulation and TMZ sensitivity. Furthermore, PRIMPOL deficiency significantly reduced ssDNA gap formation in TLS-deficient cells, confirming that PRIMPOL-mediated repriming generates post-replicative gaps that are normally filled by TLS polymerases but persist as DNA breaks when TLS fails. Our research identified APE1 as a critical vulnerability in TMZ-resistant glioblastoma and provided mechanistic insights into how BER and TLS pathways interact to determine TMZ sensitivity. Contributions The plasmid used for knocking out APE1 was created by Ratih Khoirunnisa at the IBS Center for Genomic Integrity. The clonogenic survival assays on some cell lines treated with temozolomide and APE1 inhibitors were carried out with assistance from Sandar Kyaw, Ratih Khoirunnisa, and David Manoharan at the IBS Center. Computational analysis of the WGS data was conducted by Hyeyeon Won, supervised by Professor Semin Lee at the Computational Biology Lab, Department of Biological Engineering, UNIST. Most of comet assays at pH13 were performed by Hajoong Park. Asema Maratova and Adilet Turtemir advised us how to optimize the CometChip assay conditions for TK6 cells. We are especially grateful to Professor Orlando for his help and advice in this regard.