Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors

SIMPLE SUMMARY: Epithelial-to-mesenchymal transition (EMT) is a cellular plasticity program that can confer invasiveness, dissemination, and therapy resistance to cancer cells. Although inhibitors of this cellular process are expected to work as good “partners” for chemotherapy, immunotherapy or targeted therapy drugs, direct targeting of the EMT phenomenon is, in most cases, pharmacologically challenging. The objective of this work was twofold: On the one hand, to determine if the mere process of EMT is sufficient to foster the resistance of lung cancer cells to various generations of ALK tyrosine kinase inhibitors (TKIs); on the other hand, to test the capacity of the natural compound silibinin to re-sensitize lung cancer cells that gained a mesenchymal phenotype to the anti-tumor activity of ALK–TKIs. Our findings show that not all ALK-aberrant lung cancer cells exhibit the same propensity to undergo an EMT process, thereby determining whether they are able to acquire multi-resistance to various ALK–TKIs. We have also discovered the ability of silibinin to decrease the hypersecretion of the EMT-driver TGFβ, to directly block, to some extent, the activity of purified TGFβ receptors, and to attenuate the activation status of the SMAD pathway in response to ALK–TKIs. Since there exist bioavailable formulations of silibinin with proven clinical activity in oncology patients, our results suggest a new therapeutic strategy that would merit exploration to prevent or reverse resistance to ALK–TKIs induced by the EMT phenomenon. ABSTRACT: 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 second-generation brigatinib and were fully refractory to third-generation lorlatinib. Such cross-resistance to new-generation ALK–TKIs, which was partially recapitulated upon chronic TGFβ 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 (TGFβ)/SMAD signaling pathway. Silibinin deactivated TGFβ-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 TGFβ 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 TGFβ 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 TGFβ/SMAD signaling axis in mesenchymal ALK-rearranged NSCLC cells.