Identification of Cellular Processes Regulated by NRF2 and Dependencies of NRF2-hyperactivated Tumor Cells

Abstract

Genetic alterations that lead to hyperactivation of NRF2, the master regulator of the cellular antioxidant response, are prevalent in lung cancers, suggesting that oxidative stress defense programs are critical for these cancers. However, how NRF2 promotes tumor progression is not well understood. The goal of the studies described herein was to interrogate the biological consequences of loss of NRF2 and identify dependencies of NRF2-hyperactivated tumor cells using three-dimensional (3D) spheroid models which recapitulate the architecture and high oxidative stress environment of tumors. Reduction of NRF2 affected two distinct biological processes in tumor cell spheroids: (i) it suppressed cell proliferation in an antioxidant-independent manner and (ii) it promoted cell death in the inner luminal space in an antioxidant- dependent manner. The ability of tumor cell lines to form spheroids, but not their proliferation in monolayer culture, was associated with high NRF2 signature scores. To identify dependencies of NRF2-hyperactivated cells specifically in 3D, we performed parallel CRISPR-Cas9 screens targeting a custom set of genes associated with NRF2-hyperactivated tumors or implicated in NRF2 or antioxidant function in standard monolayer and 3D spheroid cultures. sgRNAs targeting the mTOR negative regulator, TSC1, were highly enriched, while sgRNAs targeting GPX4, a phospholipid hydroperoxidase that specifically protects against lipid peroxidation, were depleted. Loss of TSC1 increased spheroid size, whereas loss of GPX4 enhanced death of inner cells of spheroids via ferroptosis, a non-apoptotic form of cell death. Interestingly, reduction of NRF2 phenocopied the ferroptotic cell death of inner cells observed with GPX4 loss and was rescued by a ferropotosis inhibitor, indicating that NRF2 protects the inner cells from ferroptotic death. Inner cells displayed increased lipid peroxidation compared to outer cells, and the combined loss of both NRF2 and GPX4 caused death of both inner and outer cells of spheroids, suggesting that inner and outer cells have different oxidative stress thresholds. Altogether, this thesis presents novel insights into the role of NRF2 in promoting tumor cell proliferation and survival and suggests that bypassing multiple oxidative stress defenses is required to efficiently kill tumor cells.