Abstract The Nrf2-Keap1-ARE pathway, a master defense mechanism against oxidative and electrophilic stresses, has emerged as a target for cancer therapy. Activation of Nrf2 has been a common strategy for cancer prevention, as Nrf2 deficiency enhances susceptibility to carcinogens in a wide variety of preclinical models. However, accumulating evidence suggests a tumor-promoting role for Nrf2 in established tumors. Constitutive activation of Nrf2 because of gain of functionmutations in NFE2L2 or loss of function mutations in its negative regulator Keap1, has been found in many cancers, especially lung cancer, and is associated with chemoresistance and poor survival in human patients. Nrf2 inhibitors, therefore, provide a potential novel strategy for treating cancer. Here, we report a novel Nrf2 inhibitor, MSU225, identified from an existing chemistry library. MSU225 dose-dependently downregulated tBHQ (tert-butyl hydroquinone)-induced Nrf2 transcriptional activity in a luciferase reporter assay without cytotoxicity. MSU225 also decreased the expression of Nrf2 downstream targets, including HO-1, NQO1, GST1A1, GCLC, GCLM, and UGT1A6 in A549 human lung cancer cells, in which Keap1 is mutated and Nrf2 activation is constitutive. Notably, MSU225 induced a striking and significant (p<0.05) decrease of Nrf2 protein in A549 cells. The effect of MSU225 on Nrf2 protein levels can be blocked with the proteasome inhibitor MG132, suggesting MSU225 enhances Nrf2 degradation through the proteasome system. Ubiquitination of Nrf2 was also increased after MSU225 treatment. Interestingly, MSU225 also decreased Nrf2 protein levels in Keap1 knockout and βTrCP knockout cells, which are the two major pathways inducing Nrf2 degradation. Nrf2 involvement in cancer cell proliferation is well known. MSU225 inhibited the growth of human lung cancer cells (A549, H460, A427) in both 2D cell culture and soft agar. Cells addicted to Nrf2 were more susceptible to MSU225 suppression of cell proliferation than cells not addicted to Nrf2. MSU225 also sensitized A549 cells to chemotherapies including doxorubicin, carboplatin, 5-fluorouracil and Topotecan. Our results suggest that MSU225 is a novel Nrf2 inhibitor that enhances the degradation of Nrf2 in a Keap1 and βTrCP independent manner. In vivo studies to assess the efficacy of MSU225 for treating experimental lung cancer are ongoing. Citation Format: Di Zhang, Kelly Aldrich, Thomas Dexheimer, Edmund Ellsworth, Aaron Odom, Karen Liby. A novel Nrf2 inhibitor suppresses proliferation and enhances the sensitivity of cancer cells to chemotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C063. doi:10.1158/1535-7163.TARG-19-C063