Pyrimethamine overcomes resistance to hypomethylating agents by reducing de novo pyrimidine synthesis

The DNA hypomethylating agents (HMAs) 5-azacitidine and decitabine are the backbone of disease modifying therapy in myelodysplastic syndromes (MDS). Resistance to therapy often develops by upregulation of de-novo pyrimidine synthesis which competes directly with the DNMT1-depleting nucleotide Aza-dCTP by building natural cytidines and deoxycytidines from amino acid building blocks. Inhibition of de-novo pyrimidine synthesis can restore sensitivity of leukemic stem cells to therapy. Here we demonstrate that Pyrimethamine (PYR), an FDA approved antiparasitic, antifolate agent causes apoptosis in multiple leukemic cell lines, has synergy with venetoclax (Ven) and additive effect with HMA's in HMA and Ven resistant leukemic cell lines as well as murine models. It increases differentiation of stem and progenitor populations in primary MDS patient samples and is able to directly inhibit de novo pyrimidine synthesis thereby overcoming one of the primary mechanisms of HMA resistance. In combination with Ven, it also downregulates multiple enzymes implicated in pyrimidine synthesis. Teaser Abstract In myelodysplastic syndromes, one of the mechanisms of resistance to hypomethylating agents arises through activation of de novo pyrimidine synthesis, which competes with Aza-dCTP nucleotide. We show that pyrimethamine, an FDA-approved antifolate, induces apoptosis across leukemic models, synergizes with venetoclax, and restores sensitivity in HMA- and venetoclax-resistant leukemia. Pyrimethamine promotes differentiation of stem and progenitor cells in primary MDS samples and in combination with venetoclax directly inhibits pyrimidine synthesis, revealing a clinically actionable strategy to overcome a key mechanism of therapeutic resistance.