Exploring novel roles of metabolic enzymes MTHFD2 and PFKFB3 in cancer genome stability and their potential as anticancer therapeutic targets

Sammanfattning: Altered tumor metabolism has been described as early as the 1920s, but it was only in recent decades that proteomic and metabolomic studies revealed that the ways in which tumors rewire their nutrient and energy pathways are more diverse and have more implications for treatment outcome than previously thought. There is now a great interest in characterizing promising metabolic targets and identifying novel ways by which to exploit them for cancer treatment. This thesis work is part of an ongoing effort to elucidate the molecular mechanisms behind metabolic cancer targets specifically at the interface of genome stability, their role in the pathogenesis of different tumor types and genetic contexts, and their suitability as drug targets for clinically relevant treatment strategies. In Paper I, we present a new role for the glycolysis enzyme 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase 3 (PFKFB3) in homologous recombination (HR). We used gene silencing and pharmacological inhibitors to investigate the role of PFKFB3 in the response to DNA damage induced by ionizing radiation (IR). We found that PFKFB3 promotes the recruitment of DNA repair factors and supplies nucleotides for DNA synthesis through its interaction with ribonucleotide reductase (RNR). We also validated the antitumor preclinical potential of PFKFB3 inhibitor KAN0438757 and showed it specifically sensitized cancer cells to IR. In Paper II, we solve the first crystal structure of human one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) in complex with its cofactors and a weak inhibitor, LY345899. We developed biochemical activity and target engagement assays to evaluate the binding and inhibition of MTHFD2 by LY345899 in cancer cell models. With the newfound structural insights to determine key residues important for substrate and cofactor binding, we were able to undertake a structure-based drug discovery program targeting MTHFD2 detailed in Paper III. Paper III expands on the groundwork laid out in Paper II to develop first-in-class, highly potent and cell active inhibitors of MTHFD2 (MTHFD2i). Again, using gene silencing techniques, we identified a novel role for MTHFD2 in genome maintenance, which we confirmed with our small molecule inhibitors. We show that MTHFD2i induce replication stress and apoptosis selectively in transformed cells as a result of impaired de novo thymidylate synthesis and genomic uracil misincorporation. We established an in vivo model of acute myeloid leukemia (AML) and showed that MTHFD2i significantly prolonged survival and outperformed the standard of care compound cytarabine (AraC), providing proof-of-concept for the translational potential of MTHFD2i as anticancer drugs. In Paper IV, we further elaborate on the role of MTHFD2 in genome maintenance in response to DNA damage. We found that MTHFD2 accumulates and associates to chromatin upon DNA double strand breaks (DSBs) and promotes DNA repair through HR. Loss of MTHFD2 significantly impairs HR activity, with MTHFD2i specifically sensitizing cancer cells to PARP inhibitors in vitro and delaying tumor growth when combined with a PARP inhibitor in vivo. Taken together, these studies showcase these two metabolic enzymes, PFKFB3 and MTHFD2, in a new light as novel DNA damage response (DDR) targets. Our findings provide compelling evidence to propose the intersection of cancer metabolism and genome stability as an untapped source of novel anticancer targets warranting more mechanistic and drug development efforts.

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