Novel Role of Glycogen Synthase Kinase-3β in Determining Cancer Cell Response to Genotoxic Stress through Regulation of 53BP1 Function

Abstract

Glycogen synthase kinase-3β (GSK3β) is a multifunctional serine/threonine kinase involved in various cellular processes and signaling pathways. Accumulating evidence suggests GSK3β plays a role in cancer treatment with effects on tumorigenesis and treatment response. We have previously shown that inhibition of GSK3β protects neurons from ionizing radiation-induced apoptosis through upregulation of non-homologous end joining (NHEJ) mediated repair of double strand breaks (DSBs). Here, we investigate the molecular mechanism underlying GSK3β regulation of NHEJ. Using biochemical, molecular, and genetic approaches, we investigated the physical and functional interaction between GSK3β and 53BP1. Cultured human tumor cells were utilized as model system to further characterize how GSK3β controls 53BP1 function in DNA DSB recognition and repair. Finally, we employed a cell biology approach to genetically and pharmacologically manipulate GSK3β activity and test how the GSK3β-53BP1 axis impacts tumor cytotoxic response to PARP inhibitor (PARPi) and radiation therapy. We illustrate that GSK3β directly interacts with 53BP1 and phosphorylates 53BP1 at threonine 334 amino acid (T334) within a region heavily phosphorylated by several stress kinases. Phosphorylation at T334 inhibits 53BP1's function in recruitment to DNA DSB sites as well as canonical NHEJ. Furthermore, our results identify GSK3β regulation of 53BP1 function in NHEJ is achieved through suppression of downstream mediators, RIF1 and PTIP, and their function. In contrast, GSK3β enhances single strand DNA resection and promotes homologous recombination (HR) repair. Most importantly, genetic and pharmacologic inhibition of GSK3β-53BP1 signaling axis dramatically enhances the cytotoxic response of BRCA1-deficit cancer cells to PARPi. The assessment of the effect of GSK3β-53BP1 axis on tumor cell response to radiation treatment is underway. This study establishes the connection between GSK3β and DSB repair through its phosphorylation and regulation of 53BP1 pathways. Moreover, it demonstrates that GSK3β kinase activity uniquely results in inhibition of 53BP1 as opposed to other kinases that enhance 53BP1 function. Importantly, this novel signaling axis provides a strategy for targeting cancer cell resistance to PARPi.