Abstract 387: Pyrimethamine inhibits STAT3 transcriptional activity via dihydrofolate reductase

Abstract Cancer is often characterized by aberrant gene expression patterns that alter cellular function. Such alterations are commonly caused by the inappropriate activation of transcription factors. STAT3, in particular, is a key transcriptional regulator of many pro-tumorigenic processes, including inflammation, proliferation, and survival, and is persistently phosphorylated and activated in more than 70% of breast tumors. Accordingly, it is a promising therapeutic target for the treatment of breast cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we performed an unbiased, high-throughput screen of the Prestwick collection, a library of 1,120 small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity at concentrations known to be safely achieved in humans. Intriguingly, unlike STAT3 phosphorylation inhibitors and STAT3 SH2 domain inhibitors, pyrimethamine does not affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of STAT3 inhibition. To elucidate this mechanism and identify the direct molecular target of pyrimethamine, we performed thermal proteomic profiling. We detected 4,462 proteins, four of which demonstrated a statistically significant shift in thermal stability in the presence of pyrimethamine. We further characterized these hits by assessing the effect of siRNA-mediated knockdown on STAT3 transcriptional activity. Of the four hits, only knockdown of dihydrofolate reductase (DHFR) consistently reduced STAT3 transcriptional activity. Likewise, treatment with methotrexate, a known inhibitor of DHFR, also reduced STAT3 activity. The combination of pyrimethamine and methotrexate had no additive effect on STAT3 inhibition, suggesting that both compounds were acting through a common mechanism. Finally, we tested whether folinic acid, a reduced form of folate, could rescue pyrimethamine-dependent loss of STAT3 activity. Strikingly, folinic acid, but not folic acid, eliminated the effect of pyrimethamine on STAT3 transcriptional activity in a dose-dependent manner. Together, these results suggest that DHFR is the molecular target responsible for the STAT3-inhibitory effects of pyrimethamine. Moreover, these findings implicate folate metabolism in the regulation of STAT3 transcriptional activity, revealing a previously unknown node within the STAT3 pathway that may be important for the development and treatment of STAT3-driven breast cancer. Citation Format: Lisa N. Heppler, Sarah R. Walker, Sanaz Attarha, Brent D. Page, David A. Frank. Pyrimethamine inhibits STAT3 transcriptional activity via dihydrofolate reductase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 387.