Abstract B109: MTHFD1/2 inhibitor TH9619 targets the DNA damage response and causes cancer-specific folate trapping with an unprecedented therapeutic window
Authors: Martilla Petra , Green Alanna C , Kiweler Nicole , Chalkiadaki Christina , Wiita Elisée , Cookson Victoria , Lesur Antoine , Eiden Kim , Bernhardin François , Long Maeve , Jemth Ann-Sofie , Mortusewicz Oliver , Homan Evert , Bonagas Nadilly , Ramos Louise , Sanjiv Kumar , Mancina Emilio Guillén , Pham Therese , Slipicevic Ana , Henriksson Martin , Meiser Johannes , Helleday Thomas 
Published: December 2023

Abstract
The MTHFD2 protein is consistently the most overexpressed enzyme when comparing cancer and normal cells and tissue. The protein regulates formate release from mitochondria and generation of nucleotides required for rapid cell growth. An additional role of MTHFD2 in DNA repair and genomic stability in cancer cells is also emerging. The MTHFD2 has been validated genetically as an anti-cancer target by numerous laboratories, but the enzyme has been notoriously difficult to target. Here, we describe first-in-class nanomolar MTHFD2 inhibitors, with protein co-crystal structures demonstrating binding in the active site of MTHFD2 and engaging with the target in cells and tumours. Because of the structural resemblance between MTHFD1, all MTHFD2 inhibitors were also found to inhibit MTHFD1, but no other folate-binding protein, kinases or other proteins. Here, we report that MTHFD1/2 inhibitors (MTHFD1/2i) reduce replication fork speed and induce replication stress, followed by S phase arrest, apoptosis and efficient killing of cancer cells in vitro and in vivo. Mechanistically, we find MTHFD1/2i prevent thymidine production leading to the misincorporation of uracil into DNA and replication stress. We show that TH9619 targets the nuclear MTHFD2 but not mitochondrial MTHFD2. Because of this, MTHFD1/2i lead to a unique folate trap, accumulation of 10-formyl-tetrahydrofolate, which depletes cells of tetrahydrofolate leading to thymidylate depletion and selective killing of MTHFD2-expressing cancer cells. Importantly, the folate trap is not generated in non-transformed cells that do not express MTHFD2. We also show that MTHFD1/2i target the DNA damage response (DDR), and the combined effect of targeting the DDR and generating a cancer-specific folate trap explains the unique &gt; 1000-fold therapeutic index generated by these inhibitors. Furthermore, we show efficient haematological and solid cancer targeting in vitro and in vivo. Excitingly, we find potent anti-cancer efficacy and complete responses at low doses in vivo while demonstrating tolerability at 1000x fold higher doses, altogether underscoring the novelty of our approach. In conclusion, MTHFD1/2 inhibitors will offer a major new DDR inhibitor attack on cancer soon to be tested in clinical trials
Citation Format: Petra Martilla, Alanna C Green, Nicole Kiweler, Christina Chalkiadaki, Elisée Wiita, Victoria Cookson, Antoine Lesur, Kim Eiden, François Bernhardin, Maeve Long, Ann-Sofie Jemth, Oliver Mortusewicz, Evert Homan, Nadilly Bonagas, Louise Ramos, Kumar Sanjiv, Emilio Guillén Mancina, Therese Pham, Ana Slipicevic, Martin Henriksson, Johannes Meiser, Thomas Helleday. MTHFD1/2 inhibitor TH9619 targets the DNA damage response and causes cancer-specific folate trapping with an unprecedented therapeutic window [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B109.