Fig. 6.

KHS101 sensitivity is related to NSCLC metabolic state. A) Bar graph showing IC₅₀ values (μM) of all the 26 NSCLC cells belonging to CL-100 ProLiFiler screening and (on the right panel) dose–response curves to KHS101 treatment of the indicated two cell lines. Points are average values of replicates ± SD. IC₅₀ values (μM) are shown with 95% confidence intervals. B) Bar graph showing the average IC₅₀ for each NSCLC histotype and SCLC. Bars are average ± SD. P-values are from one-way ANOVA. C) Dose–response curves of the 4 most sensitive cell lines (H460, NCI-H1581, LOU-NH91, A549) compared to the 4 most resistant ones (NCI-H838, BEN, NCI-H1563, SK-LU-1) resulting from the sensitivity screening. Points are average of biological replicates ± SD. D) Correlation between HSPD1 gene expression and KHS101 IC₅₀ values in a panel of NSCLC cell lines from GSE47206 (n = 13). E) Genes with most differential expression between KHS101-sensitive and KHS101-resistant groups (log₂ fold change cut-off of 4 and p-value < 0.05). F) Western blot analysis of NLRC5 protein level in A549 cells overexpressing NLRC5 or empty vector. β-Actin was used as loading control. G) Dose–response curves of A549 overexpressing NLRC5 compared to empty control cells. H) Dose–response curves of A549 SLC6A8 KO compared to control cells. I) Metabolite analysis between KHS101-sensitive and KHS101-resistant cells with a log₁₀ fold change of 2 and p-value < 0.05. J) Metabolic phenogram showing OCR (basal respiration) and ECAR (indicative glycolysis) values of creatine high (H460, A549) and creatine low (BEN, H838) cells. K) Metabolic phenogram showing OCR and ECAR levels in A549 treated for 24 h with creatine-kinase inhibitor (DNFB). L) Dose–response curves of A549 and H460 cells in presence of creatine-kinase inhibitor DNFB. In G, H and L IC₅₀ values (μM) are shown. P-values are from two-way ANOVA and points are average of biological replicates ± SD. * < 0.05, *** < 0.001, **** < 0.0001. All dose–response curves are normalized to the DMSO control