Fig. 1
Smoking alters the tumor immune microenvironment and selects for mutations activating the NRF2 pathway. A Two-way hierarchical clustering for the presence of 14 different immune subtypes based on ssGSEA scores identifies a cluster of immunologically cold patient tumors from the OCSCC TCGA cohort that were enriched for patients who smoke or recently quit. The number of patients in the hot and cold clusters according to smoking status are shown in the table directly underneath, along with the numbers expected by chance if there was complete independence and the p value (p < 0.0013) obtained after χ2 analysis. B The Nrf2 pathway measured by ssGSEA scores is significantly elevated in OCSCC patients with a smoking history (p < 0.003), with a similar trend observed in LUSC tumors. C Stratification of patient tumors by KEAP1 (red symbols) and NFE2L2(NRF2) (orange symbols) mutations demonstrated they were significantly associated with elevated Nrf2 pathway activation compared to mutated tumors from non-smokers in OCSCC (p = 0.0005) or patients who were wild-type (WT) for both genes (D), regardless of smoking history in LUSC (p < 0.0001). Both smoking and mutation were associated with elevated Nrf2 scores in OCSCC (e.g., p < 0.00001 for both variables) and showed a significant interaction (p = 0.013) with a large effect of smoking on mutation-driven Nrf2 pathway elevation (i.e., Cohen’s d = 1.03). E Pooling of TCGA data from smoking-related cancers (OCSCC, LUSC, LUAD) demonstrates that both NFE2L2(NRF2) and KEAP1 mutations are significantly associated with elevated Nrf2 pathway activation on average compared to WT tumors, but for a minority of mutations (e.g., 10 percentile values indicated with a dashed line) the differences are marginal. F NFE2L2(NRF2) mutations in amino acid motifs involved in Keap1 binding (ETGE and DLG) or their immediate vicinity occurred in 90% of cases when Nrf2 scores were elevated compared to just 30% when Nrf2 activation was marginal (i.e., Nrf2 scores in the lower 10 percentile), which was found to be significant by χ2 analysis (p < 0.00001)