Fig. 3: NOXA downregulation induced by ALK inhibition leads to anti-apoptotic MCL-1 resistance.

A Contribution of each anti-apoptotic protein on the induction of cytochrome c release in response to 1 µM of crizotinib, alectinib, brigatinib, and lorlatinib in H3122 cell line. BAD BH3 peptide correlates with BCL-2/BCL-xL tumor dependence; HRK BH3 peptide, with BCL-xL dependence; and MS1 BH3 peptide, with MCL-1 dependence. Results are expressed as ∆% priming, representing the higher difference in cytochrome c released compared with control condition. The final concentrations of each peptide solution were: 10 µM of BAD BH3 peptide, 100 µM of HRK BH3 peptide, 10 µM or 1 µM of MS1 BH3 peptide. B Representative images from Western blot analysis of H3122 cell lysates after 1 µM of crizotinib, alectinib, brigatinib, and lorlatinib for 16 h. C Optical density quantification normalized to tubulin and represented as fold change compared to control. D External validation using RNA-seq data of four EML4-ALK-positive patient-derived NSCLC cell lines after 24 h of incubation with alectinib, brigatinib, and lorlatinib. E Graphical scheme of MCL-1 release through NOXA downregulation and sensitization to specific MCL-1 inhibitors. Created in BioRender. Montero, J. (2025) https://BioRender.com/c82p948. F Results of cell death assay in H3122 cell line carried out with Annexin V and DAPI staining after 96 h of incubation with crizotinib 0.1 µM, alectinib 0.1 µM, brigatinib 0.01 µM, lorlatinib 0.01 µM, ABT-199 0.1 µM, A-133 0.1 µM, DT2216 0.1 µM, S63845 1 µM, and dinaciclib 0.01 µM. Values indicate mean values ± SEM from at least three independent experiments. *p < 0.05 and **p < 0.01, and # indicates CI < 1 and statistically significant difference (p < 0.05) between the combination treatment and single agents. The value above the # represents the CI. For RNA-seq data, values indicate Log₂ (FC) ± IfcSE, where IfcSE represents the Standard Error Estimate for the Log₂ Fold Change Estimate; adjusted *p < 0.05.