Abstract

Cancer remains a leading cause of mortality worldwide, with treatment resistance and recurrence posing major barriers to long-term survival. Despite advancements in targeted therapies, many cancers develop resistance to chemotherapy, radiation, and immunotherapy, significantly limiting their effectiveness. One key driver of therapeutic resistance is nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2), a transcription factor that regulates antioxidant responses and drug detoxification. While NRF2 protects normal cells from oxidative stress, its aberrant activation in cancer promotes tumor progression, metabolic reprogramming, and resistance to standard treatments. Alarmingly, NRF2 hyperactivation is implicated in some of the most aggressive and refractory malignancies, yet no FDA-approved selective NRF2 inhibitors currently exist. This underscores the urgent need for novel therapeutic strategies. This dissertation aims to address this challenge by investigating a new therapeutic approach to NRF2 suppression by inhibiting one-carbon metabolism. Specifically, it explores the potential of Pyrimethamine (PYR), an FDA-approved antiparasitic drug, and its more potent derivative, WCDD115, as NRF2 inhibitors through their suppression of dihydrofolate reductase (DHFR). Chapter 1 establishes NRF2's critical role in cancer resistance, explores its background, highlights the lack of effective inhibitors, and introduces a rationale for targeting metabolic vulnerabilities to suppress NRF2. Chapter 2 presents original preclinical research demonstrating that PYR and WCDD115 inhibit NRF2 by targeting DHFR, linking one-carbon metabolism to NRF2 regulation. Structure-activity relationship (SAR) studies reveal that WCDD115 is a 22-fold more potent NRF2 inhibitor than PYR, with an IC50 of 57 nM compared to 1.2 μM for PYR. Metabolomic and proteomic analyses show that DHFR inhibition disrupts NRF2-dependent antioxidant responses, activates TP53-mediated DNA damage pathways, and induces tumor cell death, positioning DHFR inhibitors as a novel class of NRF2-targeting agents. Chapter 3 describes a first-in-human phase I clinical trial evaluating PYR in 18 patients with HPV-negative head and neck squamous cell carcinoma (HNSCC). The trial assesses safety, tolerability, and biological activity, focusing on tumor DHFR expression, and NRF2 suppression. Using protein quantification techniques, the study measures DHFR protein expression pre- and post-treatment as a proxy for DHFR inhibition, providing preliminary data supporting the potential of repurposing PYR to treat NRF2-driven cancer through folate pathway inhibition. Chapter 4 synthesizes the findings from previous chapters, exploring the broader implications of NRF2 suppression via DHFR inhibition. It discusses potential combination therapies with chemotherapy, radiation, and immunotherapy, the development of next-generation antifolates, and expanding NRF2-targeting strategies to other malignancies. This dissertation is significant because it identifies a novel mechanistic link between NRF2 and one-carbon metabolism, introducing DHFR inhibitors as a promising class of NRF2-targeting agents. By providing preclinical evidence and clinical trial insights into the potential of repurposing PYR for NRF2-driven cancers, this work offers a new therapeutic strategy to overcome resistance in aggressive malignancies. Furthermore, it paves the way for future combination therapies and the development of next-generation antifolates, expanding the therapeutic landscape for NRF2-driven cancers.

Committee Chair

Michael Major

Committee Members

Carmen Bergom; Gary Patti; Grant Challen; Zhongsheng You

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology and Biomedical Sciences

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-8-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-5761-3567

Download

Included in

Biology Commons

Share

COinS