Phosphoglycerate Mutase 1 Is Required For Homologous Recombination Repair Via Sustaining The Stability Of CtIP

Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet the molecular mechanisms remain poorly understood. Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway (PPP) flux and serine biosynthesis via catalyzing the conversion of 3-phosphoglycerate (3-PG) into 2-phosphoglycerate (2-PG), thus is an attractive target for cancer therapy.To gain insights into the global cellular processes that PGAMl is potentially involved, we conducted a mass spectrometry based proteomic study. This effort identifies multiple cellular processes that are potentially affected by PGAM1 inhibition, among which we were particularly interested in DNA damage response.The stuy starteted with an assay to test cell sensitivity change to different DNA damaging agents, known to generate different forms of DNA lesions. PGAM1 knockdown cells unanimously exhibited hypersensitivity to camptothecin (CPT) or cisplatin (CDDP). CPT and CDDP are known to selectively kill proliferating cancer cells via causing replication-dependent DNA double-stand breaks (DSBs). Using yH2AX as a surrogate marker, we compared the occurrence of drug-induced DSBs in PGAMl depleted cells and scramble control cells, and observed different kinetics in yH2AX level following drug withdrawal, indicating a defect in DSBs repair caused by PGAMl deletion. HR reporter assay enabled us to discover that PGAM 1 knockdown remarkably decreased homologous recombination (HR) efficiency, whereas PGAM1 knockdown barely affected non-homologous end-joining (NHEJ) efficiency.We then asked whether the enzymatic activity of PGAM1 was required for its role in supporting HR repair. Reconstitution of PGAM1 enzymatically inactive mutants failed to rescue the deficient repair following CPT treatment. Further introduced PGMI-004A, an enzymatic inhibitor of PGAM1, and methyl-2-PG, a cell permeable 2-PG derivative that was converted to 2-PG in cells, we found that the enzymatic activity of PGAM1 was required for HR repair. Using HR reporter assay and supplement of 6PGD product deoxyribonucleotide triphosphate (dNTP), we discovered epistatic relationship between PGAM1 and 6PGD in HR repair.We then focused in the procedure of HR repair, and found PGAM1 knockdown cells showed reduction in both CPT-induced ssDNA generation and RPA foci formation. Further analysis revealed PGAM1 facilitated DSBs processing via impairing CtIP stability in an dNTP dependent manner. Immunoblotting and RT-qPCR analysis revealed that imblanced dNTPs led to elevated p21 levels, which could premature E3 ubiquitin ligase of CtIP. Further ChIP-qPCR assay allowed us to detect the increased recruitment of p53/p73 in p21 gene promoter region, suggested a model that p53 and p73 play a key role in coping with the imbalance of dNTP level in cancer cells via upregulating p21.Our results above revealed a previously unappreciated role of PGAM1 in regulating HR repair that required its enzymatic activity. HR deficient cancer, particularly BRCA1/2 deficient breast cancer, is exquisitely sensitive to the newly approved PARP inhibitor Olaparib. Our findings may suggest broader benefit of Olaparib expanded by PGAM1 enzymatic inhibition. To address this question, we chose triple-negative breast cancer MDA-MB-231 cells with proficient BRCA1/2, and proved this therapeutic potential using xenograft mice models. While Olaparib alone failed to show any therapeutic effect, its combination with PGMI-004A largely reduced intratumoral 2-PG level, decreased CtIP protein level, and increased intratumoral level of DSB lesions, suggesting Olaparib and PGMI-004A as a combination regimen for BRCA proficient breast cancer.In summary, this study discovered a previously unappreciated role of PGAM1 in regulating HR repair that required its enzymatic activity. Inactivation of PGAM1 catalytic activity resulted in decreased dNTP pool, which accounted for decreased CtIP stability and thereby translated the impact of PGAM1 to DSBs end processing, homologous recombination repair and genomic integrity. These mechanistic insights also lead us to exploit the PARP inhibitors in BRCA1/2 proficient cancer such as triple-negative breast cancer through combination with PGAM1 enzymatic inhibition.