Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors Verdura, Sara Segura Carretero, Antonio ALK Crizotinib Brigatinib Lorlatinib Silibinin EMT TGF-beta Lung cancer Epithelial-to-mesenchymal transition (EMT) may drive the escape of ALK-rearranged non-small-cell lung cancer (NSCLC) tumors from ALK-tyrosine kinase inhibitors (TKIs). We investigated whether first-generation ALK–TKI therapy-induced EMT promotes cross-resistance to new-generation ALK–TKIs and whether this could be circumvented by the flavonolignan silibinin, an EMT inhibitor. ALK-rearranged NSCLC cells acquiring a bona fide EMT phenotype upon chronic exposure to the first-generation ALK–TKI crizotinib exhibited increased resistance to secondgeneration brigatinib and were fully refractory to third-generation lorlatinib. Such cross-resistance to new-generation ALK–TKIs, which was partially recapitulated upon chronic TGF stimulation, was less pronounced in ALK-rearranged NSCLC cells solely acquiring a partial/hybrid E/M transition state. Silibinin overcame EMT-induced resistance to brigatinib and lorlatinib and restored their efficacy involving the transforming growth factor-beta (TGF )/SMAD signaling pathway. Silibinin deactivated TGF -regulated SMAD2/3 phosphorylation and suppressed the transcriptional activation of genes under the control of SMAD binding elements. Computational modeling studies and kinase binding assays predicted a targeted inhibitory binding of silibinin to the ATP-binding pocket of TGF type-1 receptor 1 (TGFBR1) and TGFBR2 but solely at the two-digit micromolar range. A secretome profiling confirmed the ability of silibinin to normalize the augmented release of TGF into the extracellular fluid of ALK–TKIs-resistant NSCLC cells and reduce constitutive and inducible SMAD2/3 phosphorylation occurring in the presence of ALK–TKIs. In summary, the ab initio plasticity along the EMT spectrum may explain the propensity of ALK-rearranged NSCLC cells to acquire resistance to new-generation ALK–TKIs, a phenomenon that could be abrogated by the silibinin-driven attenuation of the TGF /SMAD signaling axis in mesenchymal ALK-rearranged NSCLC cells. 2023-01-26T13:35:23Z 2023-01-26T13:35:23Z 2022-12-11 info:eu-repo/semantics/article Verdura, S... [et al.]. Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors. Cancers 2022, 14, 6101. [https://doi.org/10.3390/cancers14246101] https://hdl.handle.net/10481/79391 10.3390/cancers14246101 eng http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess Atribución 4.0 Internacional MDPI