Heterogeneous expression of immunotherapy response markers in non-small cell lung cancer with hyperactive NRF2 pathway: PD-L1 and beyond

Activating mutations in the NRF2 pathway characterize an aggressive, molecularly distinct subtype of non-small cell lung cancer (NSCLC) associated with poor prognosis and resistance to immune checkpoint inhibitors (ICI). In this study, we used a validated NRF2 transcriptional signature along with a literature-curated, immune-related 47-gene set associated with favorable responses to ICI (the "ICI-Immune" signature) to demonstrate the heterogeneous expression of ICI response markers and tumor-infiltrating immune cells in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) with hyperactive NRF2. Protein association network and gene expression correlation analyses revealed that NRF2 and ICI-Immune gene sets operate as independent, loosely connected transcriptional modules, wherein the ICI-Immune genes show no significant correlation with NRF2 activity. Dichotomous categorization of NSCLC into NRF2-low and NRF2-high signature groups indicated an overall immune-depleted tumor microenvironment in NRF2-high cases, as well as identified a tumor subset exhibiting the NRF2-high phenotype without documented NFE2L2/KEAP1 mutations. Interestingly, clustering of NSCLC based on NRF2 and ICI-Immune scores showed that while most NRF2-high cases express low levels of ICI-Immune genes and tumor-immune infiltrates, a subset termed "NRF2-high/ICI-Immune-high", which makes up approximately one-third of NRF2-high cases, exhibits molecular features characteristic of ICI-responsive cancers. This encompasses CD274/PD-L1 and ICI-Immune signature upregulation, an increased leukocyte fraction, augmented immune scores and tumor-infiltrating immune cells, as well as a greater frequency of the C2/IFNγ-dominant tumor-immune subtype. Histopathological analysis of n = 103 NSCLC cases identified a subgroup of NRF2-high/PD-L1+ tumors with increased tumor-infiltrating lymphocytes (TIL), which may histologically represent the NRF2-high/ICI-Immune-high subset. Experiments in KEAP1-mutated cells and omics data from a panel of cell lines indicated that PD-L1 mRNA/protein levels are not dictated by constitutive NRF2 activation in vitro. These results advance our understanding of the tumor microenvironment diversity in NSCLC with hyperactive NRF2 and offer a potential histology-based method to identify ICI responders within this subset.