Mechanistic studies of the NRF2-Methylosome interaction in lung cancer.

ABSTRACT Lung cancer (LC) is the leading cause of cancer death, making up nearly 25% of all cancer deaths. Although some advances in personalized treatments have improved survival rates, there is an ongoing clinical need to develop novel targeted therapies against LC subtypes with particularly poor prognosis. However, our ability to

develop these treatments is currently limited by a lack of compelling actionable targets. Publicly available screening data, along with a multitude of prior studies, have validated nuclear factor erythroid 2-related factor 2 (NRF2) as a strong and selective dependency in lung cancer. According to patient data, it is estimated that 15%

to 30% of LC tumors have loss of function mutations in either NRF2 or KEAP1, and these mutations are associated with resistance to conventional treatments and poor prognosis. Furthermore, despite its importance in sustaining tumor growth, germline NRF2 knockout mouse models produce generally normal mice with no

significant developmental or health-related issues. Combined, these results suggest NRF2 has a wide therapeutic index and merits further investigation as a potential drug target for LC. Therefore, the proposed research explores the potential for NRF2 to be a novel therapeutic target in lung cancer by biochemically

characterizing its functional binding partners. To overcome the inherent challenges of targeting NRF2, we used a high-throughput reporter screen and biochemical assays to identify PRMT5 as a putative modulator of NRF2 activity. We hypothesize that the activity of NRF2 in KEAP1-mutant LC is negatively regulated by a PRMT5-

NRF2 interaction, and that loss of this interaction leads to intolerable levels of NRF2 activity that promote tumor growth. This hypothesis will be addressed by mapping the biochemical interaction between PRMT and NRF2 (Aim 1), characterizing the PRMT5-mediated repression of NRF2 transcriptional activity (Aim 2), and evaluating

the impact of methylosome perturbations on KEAP1-mutant LC growth (Aim 3). To accomplish the first aim, we will use reconstituted NRF2 and PRMT5 to carryout advanced biochemical assays designed to identify critical residues necessary for this interaction. For the second aim, we will employ both nascent RNA-seq and a

combination ChIP-seq analysis to uncover the mechanism of PRMT5-mediated repression of NRF2 activity. Finally, using available small molecule inhibitors we will assess how disruption of the PRMT5-NRF2 interaction influences tumor growth in KEAP1-mutant LCs. The information gained from this project will provide a solid

foundation for the advancement of future drug development efforts focused on targeting NRF2.