Targeting MTHFD1 and MTHFD2 as cancer treatment

Abstract: One-carbon (1C) metabolism provides building blocks for nucleotide synthesis and therefore plays a central role in DNA replication and repair. To sustain rapid proliferation, cancer cells often upregulate their 1C metabolism, including the enzymes MTHFD1 and MTHFD2, as a part of their metabolic rewiring. Previously, MTHFD2 in particular has been indicated as a potential drug target, mainly due to its cancer-enriched expression profile. Interestingly, both MTHFD1 and MTHFD2 have also emerging nuclear functions besides their canonical metabolic activities in the 1C pathway. However, the nuclear localization of MTHFD2 and its role in the DNA damage response are not well understood. Moreover, evaluation of the therapeutic potential of targeting MTHFD1 and MTHFD2 in cancer is hampered by the lack of potent inhibitors of these enzymes. In this thesis, we aimed to develop small-molecule MTHFD1/2 inhibitors and characterize their mechanism of action, as well as study the nuclear role of MTHFD2 in DNA repair. In Paper I, we develop a series of small-molecule MTHFD1/2 inhibitors, including TH9619. We study the mechanism of action of these inhibitors and show that they cause thymidylate depletion, followed by excessive misincorporation of uracil into DNA, induction of replication stress and cell death in acute myeloid leukemia cells. These new inhibitors selectively induced apoptosis in leukemia cells while largely sparing nontumorigenic cells and displayed efficacy in a mouse xenograft model of acute myeloid leukemia. In Paper II, we further investigate the mechanism of action of MTHFD1/2 inhibitors, focusing on TH9619. We reveal that TH9619 engages with nuclear MTHFD2 but does not disrupt formate overflow from mitochondria since it cannot target mitochondrial MTHFD2. Mechanistically, TH9619 caused accumulation of 10-formyl-tetrahydrofolate downstream of mitochondrial formate release due to its inhibition of MTHFD1. Trapping of 10-formyl-tetrahydrofolate ultimately led to thymidylate depletion and cell death in MTHFD2-expressing colorectal cancer cells. Lastly, in Paper III, we identify a nuclear role of MTHFD2 in the early steps of DNA double-strand break repair in cancer cells. We found that MTHFD2 rapidly accumulated in the nucleus following ionizing radiation, which was mediated by the ATM and DNA-PK kinases, and co-localized with DNA damage sites. Depletion of MTHFD2 led to impaired phosphorylation of BRCA1, defective DNA end resection and decreased HR and NHEJ repair activity. Moreover, inhibition of MTHFD2 with TH9619 exacerbated DNA damage after irradiation in repair-proficient cancer cells and synergized with PARP inhibitors. In conclusion, this thesis details the complex mechanism of action of MTHFD1/2 inhibitors and highlights their therapeutic potential in cancer. Our work also demonstrates a critical role of MTHFD2 in facilitating double-strand break repair.