Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.

We previously showed that elevated TYMS exhibits oncogenic properties and promotes tumorigenesis after a long latency, suggesting cooperation with sequential somatic mutations. Here we report the cooperation of ectopic expression of human TYMS with loss of Ink4a/Arf , one of the most commonly mutated somatic events in human cancer. Using an hTS/Ink4a/Arf −/− genetically engineered mouse model we showed that deregulated TYMS expression in Ink4a/Arf null background accelerates tumorigenesis and metastasis. In addition, tumors from TYMS-expressing mice were associated with a phenotype of genomic instability including enhanced double strand DNA damage, aneuploidy and loss of G1/S checkpoint. Downregulation of TYMS in vitro decreased cell proliferation and sensitized tumor cells to antimetabolite chemotherapy. In addition, depletion of TYMS in vivo by TYMS shRNA reduced tumor incidence, delayed tumor progression and prolonged survival in hTS/Ink4a/Arf −/− mice. Our data shows that activation of TYMS in Ink4a/Arf null background enhances uncontrolled cell proliferation and tumor growth, supporting the development of new agents and strategies targeting TYMS to delay tumorigenesis and prolong survival.

Thymidylate synthase (TS) inhibitors are an integral component of chemotherapy regimens for difficult to treat cancer subtypes. Despite initial therapeutic benefit, current inhibitors induce TS overexpression or alter folate transport metabolism feedback pathways that tumor cells exploit for drug resistance. Here we report a small molecule TS inhibitor that exhibits i) enhanced antitumor activity as compared to current fluoropyrimidines and antifolates without inducing TS overexpression, ii) is structurally distinct from classical antifolates, iii) extends survival in a pancreatic tumor mouse model, iv) and is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we confirm the compound is a multifunctional non classical antifolate and through a series of analogues identify structural features allowing direct TS inhibition while also maintaining the ability to inhibit dihydrofolate reductase (DHFR). Collectively, this work identifies new non classical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile highlighting potential for enhanced cancer therapy. Citation Format: Maria V. Guijarro, Patrick C. Kellish, Peter E. Dib, Nicholas G. Paciaroni, Akbar Nawab, Jacob Andring, Lidia Kulemina, Nicholas V. Borrero, Carlos Modenutti, Richard L. Bennett, Daniil Shabashvili, Jonathan D. Licht, Robert McKenna, Adrian Roitberg, Robert W. Huigens, Frederic J. Kaye, Maria Zajac-Kaye. First in class multifunctional non classical antifolates inhibits thymidylate synthase and extends survival in pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4055.