Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia
Chorlada Paiboonrungruang,
Zhaohui Xiong,
David Lamson,
Yahui Li,
Brittany Bowman,
Julius Chembo,
Caizhi Huang,
Jianying Li,
Eric W. Livingston,
Jon E. Frank,
Vivian Chen,
Yong Li,
Bernard Weissman,
Hong Yuan,
Kevin P. Williams,
M. Ben Major,
Xiaoxin Chen
Affiliations
Chorlada Paiboonrungruang
Coriell Institute for Medical Research, Camden, NJ, 08103, USA; Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA
Zhaohui Xiong
Coriell Institute for Medical Research, Camden, NJ, 08103, USA; Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA
David Lamson
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, 27707, USA
Yahui Li
Coriell Institute for Medical Research, Camden, NJ, 08103, USA; Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA
Brittany Bowman
Department of Cell Biology and Physiology, Department of Otolaryngology, Washington University in St. Louis, St. Louis, MO, 63110, USA
Julius Chembo
Department of Cell Biology and Physiology, Department of Otolaryngology, Washington University in St. Louis, St. Louis, MO, 63110, USA
Caizhi Huang
Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA
Jianying Li
Euclados Bioinformatics Solutions, Cary, NC, 27519, USA
Eric W. Livingston
Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, 277599, USA
Jon E. Frank
Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, 277599, USA
Vivian Chen
Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA
Yong Li
Department of Thoracic Surgery, National Cancer Center, Cancer Hospital of Chinese Academy of Medical Sciences, Beijing, 100021, China
Bernard Weissman
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 277599, USA
Hong Yuan
Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, 277599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 277599, USA; Department of Radiology, University of North Carolina, Chapel Hill, NC, 277599, USA
Kevin P. Williams
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, 27707, USA; Corresponding author. Department of Pharmaceutical Sciences and Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, 27707, USA.
M. Ben Major
Department of Cell Biology and Physiology, Department of Otolaryngology, Washington University in St. Louis, St. Louis, MO, 63110, USA; Corresponding author. Department of Cell Biology and Physiology, Washington University in St. Louis, St. Louis, MO, 63110, USA.
Xiaoxin Chen
Coriell Institute for Medical Research, Camden, NJ, 08103, USA; Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA; Surgical Research Lab, Department of Surgery, Cooper University Health Care, Camden, NJ, 08103, USA; MD Anderson Cancer Center at Cooper, Camden, NJ, 08103, USA; Cooper Medical School of Rowan University, Camden, NJ, 08103, USA; Corresponding author. Coriell Institute for Medical Research, 403 Haddon Avenue, Camden, NJ, 08103, USA.
Objective: NRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. Design: We performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. Results: Using high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30 mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. Conclusion: We have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic for NRF2high ESCC.
