Identifying the effects of NRF2 signaling on lung squamous cell carcinoma development

PROJECT SUMMARY: The NRF2 transcription factor is mutated and activated in more than 30% of lung and upper aerodigestive squamous cell carcinomas. NRF2 drives a gene expression program that mitigates oxidative and electrophilic stress, reprograms and enables cancer cell metabolism, and suppresses lymphocyte infiltration. In lung squamous cell carcinoma (LUSC) patients, NRF2 activity portends a poor prognosis and resistance to radiation therapy, chemotherapy, and immune checkpoint inhibitors (ICI). Though frequently active in human cancers, mouse models suggest that NRF2 mutational activation is not sufficient for oncogenesis—the temporal and contextual underpinnings of how NRF2 contributes to cancer progression remain ill-defined. This research proposal focuses on a major research objective of our laboratories: to understand the impact of NRF2 activation in LUSC initiation and progression. To meet this goal, we have developed a novel genetically- engineered mouse model (GEMM) to test the impact of an activated NRF2E79Q on LUSC incidence, and on tumor histology, gene expression and metabolism. Because NRF2 activity correlates with resistance to ICI, our proposed studies will also assess how NRF2 alters the tumor microenvironment (TIME) in LUSC. Thus, we meet a research objective of PAR-22-216, “Development and early validation of in vivo or in vitro models for the assessment of immunoprevention or immunotherapy efficacy”. Based upon our previous studies along with other published reports, we hypothesize that NRF2 activation will increase LUSC incidence and progression, while decreasing mouse survival and altering the TIME landscape. To test this hypothesis, we propose two Specific Aims. In the first specific aim, we will define and understand NRF2’s oncogenic functions as a ‘second hit’ in LUSC initiation and progression in our novel Rosa26LSL-Sox2-IRES-GFP;Nkx2-1fl/fl, LSL-Nrf2E79Q (SNN) GEMM. In the second specific aim, we will assess the impact of NRF2 activation on TIME of LUSC developed in our GEMM. We will characterize the incidence and pathology of LUSC arising in our GEMM along with overall survival. We will apply state-of-the-art proteomics and mass spectrometry imaging to molecularly characterize NRF2-wildtype and NRF2-mutant tumors. We will also comprehensively define changes in the composition of pro- and anti-tumorigenic immune cell types and cell-intrinsic immunomodulatory molecules of the TIME using high dimensional spectral flow cytometry, OIS-PRM proteomics, IHC, and transcriptomics. We will use the data from this grant to support a subsequent R01 application identifying underlying mechanisms and therapeutic targets for NRF2-active cancers and determine the efficacy of pharmacological agents to suppress NRF2 as well as their abilities to sensitize LUSC to ICI therapy.