Figure 6: Inducing NADH-reductive stress selectively blocks proliferation of NRF2-activated NSCLCs.

(A) IACS-010759 (IACS) a Complex-I inhibitor, selectively increases NADH/NAD⁺ ratio in KEAP1-dependent (red) and KEAP1-mutant cells (blue) but not KEAP1-independent cells. NSCLC cells stably expressing SoNar were treated with IACS and analyzed by immunofluorescence. The representative NADH/NAD⁺ ratiometric image was constructed by taking the ratio of the emission intensity of 405 nm (NADH binding) vs 488 nm (NAD⁺ binding) for SoNar (see also Figure S6A). (B) IACS selectively blocks NSCLC proliferation following NRF2 activation. IC₅₀ values were calculated for each cell line and where indicated cells were also treated with KI696 (1 μM) (Data are represented as a mean ± SEM, n= 6 biological replicates). (C) IACS selectively inhibits the anchorage-independent growth of NSCLCs with hyperactivate NRF2 signaling. Representative images of NSCLC cell lines grown in soft agar following treatment with IACS-017509 (200 nM) or co-treated with KI696 (1 μM) as indicated (Data are represented as a mean ± SEM, n= 6–8 biological replicates) (see also Figure S6C). (D) Schematic for in vivo study. (E) IACS selectively blocks the growth of KEAP1-mutant tumors. Relative tumor growth of subcutaneous patient-derived xenografts (PDX) WT or mutant (MUT) for KEAP1 receiving vehicle or IACS (5 mg/kg) (Data are normalized to first treatment, n=10 KEAP1-WT, Vehicle; 12 KEAP1-WT IACS, 14 KEAP1-MUT Vehicle, 18 KEAP1-MUT IACS (see also Figure S6E). (F-G) Representative immunohistochemistry staining (F) and quantification of Ki67 serial sections taken from KEAP1-WT and KEAP1-MUT PDX tumors treated with IACS or vehicle. (H) Model. NRF2 activation following pharmacologic inhibition or mutation of KEAP1 increases ALHD3A1 resulting in NADH reductive stress. KEAP1-dependent and -independent cells utilize different NADH oxidation pathways to counter reductive stress. * indicates p-values < 0.05, ** indicates p-values < 0.01, *** indicates p-values < 0.0001. One-way ANOVA with Sidak’s post-hoc correction was used to determine statistical significance. Scale-bar: 25 μm for immunofluorescence and 50 μM for soft agar and immunohistochemistry.