P-glycoprotein Mediates Resistance to the Anaplastic Lymphoma Kinase Inhiitor Ensartinib in Cancer Cells

SIMPLE SUMMARY: P-glycoprotein (P-gp; ABCB1) is the most well-characterized ATP-binding cassette (ABC) multidrug efflux transporter known to actively transport a wide variety of cytotoxic and molecularly targeted drugs out of cancer cells, causing multidrug resistance (MDR) and poor prognosis in cancer patients. In this study, we investigate whether P-gp overexpression can contribute to reduced susceptibility of cancer cells to the anaplastic lymphoma kinase (ALK) inhibitor ensartinib. We discovered that P-gp-overexpressing cancer cells and cells with ectopic expression of P-gp are significantly less sensitive to ensartinib than the respective parental cells. By blocking the drug transport activity of P-gp, the intracellular accumulation and cytotoxic activity of ensartinib were fully restored in P-gp-overexpressing cancer cells. Moreover, in silico molecular docking analysis of ensartinib with the inward-open structure of P-gp provided additional information on the potential binding orientation of ensartinib in the substrate-binding pocket of P-gp. ABSTRACT: Ensartinib (X-396) is a promising second-generation small-molecule inhibitor of anaplastic lymphoma kinase (ALK) that was developed for the treatment of ALK-positive non-small-cell lung cancer. Preclinical and clinical trial results for ensartinib showed superior efficacy and a favorable safety profile compared to the first-generation ALK inhibitors that have been approved by the U.S. Food and Drug Administration. Although the potential mechanisms of acquired resistance to ensartinib have not been reported, the inevitable emergence of resistance to ensartinib may limit its therapeutic application in cancer. In this work, we investigated the interaction of ensartinib with P-glycoprotein (P-gp) and ABCG2, two ATP-binding cassette (ABC) multidrug efflux transporters that are commonly associated with the development of multidrug resistance in cancer cells. Our results revealed that P-gp overexpression, but not expression of ABCG2, was associated with reduced cancer cell susceptibility to ensartinib. P-gp directly decreased the intracellular accumulation of ensartinib, and consequently reduced apoptosis and cytotoxicity induced by this drug. The cytotoxicity of ensartinib could be significantly reversed by treatment with the P-gp inhibitor tariquidar. In conclusion, we report that ensartinib is a substrate of P-gp, and provide evidence that this transporter plays a role in the development of ensartinib resistance. Further investigation is needed.