ENT1 blockade by CNX-774 overcomes resistance to DHODH inhibition in pancreatic cancer

Inhibitors of dihydroorotate dehydrogenase (DHODH), a key enzyme for de novo synthesis of pyrimidine nu-cleotides, have failed in clinical trials for various cancers despite robust efficacy in preclinical animal models. To probe for druggable mediators of DHODH inhibitor resistance, we performed a combination screen with a small molecule library against pancreatic cancer cell lines that are highly resistant to the DHODH inhibitor brequinar (BQ). The screen revealed that CNX-774, a preclinical Bruton tyrosine kinase (BTK) inhibitor, sensitizes resistant cell lines to BQ. Mechanistic studies showed that this effect is independent of BTK and instead results from in-hibition of equilibrative nucleoside transporter 1 (ENT1) by CNX-774. We show that ENT1 mediates BQ resis-tance by taking up extracellular uridine, which is salvaged to generate pyrimidine nucleotides in a DHODH-independent manner. In BQ-resistant cell lines, BQ monotherapy slowed proliferation and caused modest py-rimidine nucleotide depletion, whereas combination treatment with BQ and CNX-774 led to profound cell viability loss and pyrimidine starvation. We also identify N-acetylneuraminic acid accumulation as a potential marker of the therapeutic efficacy of DHODH inhibitors. In an aggressive, immunocompetent pancreatic cancer mouse model, combined targeting of DHODH and ENT1 dramatically suppressed tumor growth and prolonged mouse survival. Overall, our study defines CNX-774 as a previously uncharacterized ENT1 inhibitor and provides strong proof of concept support for dual targeting of DHODH and ENT1 in pancreatic cancer.

Automated summary

In this study, we identified CNX-774 as a previously uncharacterized ENT1 inhibitor that could sensitize pancreatic cancer cell lines to the DHODH inhibitor brequinar (BQ). Our results also suggest that N-acetylneuraminic acid accumulation could serve as a potential marker for the therapeutic efficacy of DHODH inhibitors.