Resistance to genotoxic therapies remains a major contributor to tumor recurrence and treatment failure, yet the mechanisms by which cancer cells escape these therapies through DNA damage response (DDR) activation are not fully understood. Here, we identify a DDR regulatory pathway in which glycogen synthase kinase 3 β (GSK3B), a multifunctional serine/threonine kinase, governs DNA double-strand break (DSB) repair pathway choice by phosphorylating 53BP1 at threonine 334 (T334) - a site distinct from canonical ATM targets. This phosphorylation event disrupts 53BP1's interaction with nonhomologous end joining (NHEJ) effectors PTIP and RIF1, promoting their dissociation from DSBs and inhibiting 53BP1-driven NHEJ. Simultaneously, T334 phosphorylation facilitates the recruitment of CtIP and RPA32 for DNA end resection and promotes homologous recombination (HR) by enabling BRCA1 and RAD51 loading. Notably, the phospho-deficient T334A mutant of 53BP1, unlike 53BP1 loss, accumulates aberrantly at DSBs along with PTIP/RIF1, impairs end resection, and suppresses HR activity. Importantly, both genetic and pharmacologic disruption of the GSK3B-53BP1 axis sensitizes tumors to PARP inhibitors (PARPi) independently of BRCA1 status. Together, these findings reveal a GSK3B-dependent mechanism that regulates DSB repair pathway choice and provide a rationale for targeting this axis to enhance PARPi efficacy in solid tumors regardless of BRCA1 status.
Keywords: Cell biology; DNA repair; Oncology.