We identified an acquired ceritinib resistance mechanism cau

We identified an acquired ceritinib-resistance mechanism caused by P-gp overexpression in an ALK-rearranged NSCLC patient who was receiving ceritinib therapy. We also demonstrated that ceritinib is a P-gp substrate and that P-gp inhibition reversed ceritinib and crizotinib resistance. While our manuscript was under review, it was reported that hdac inhibitor accumulation of ceritinib is restricted by P-gp and BCRP (Kort et al., 2015). For ALK-rearranged NSCLC patients, treatment with ALK-TKIs, such as crizotinib, ceritinib, or alectinib often leads to conspicuous tumor shrinkage; however, tumors inevitably relapse because of acquired resistance approximately within 1–2years of treatment, similar to what is known for imatinib-treated CML with BCR-ABL- (Branford et al., 2002) or EGFR-TKI-treated EGFR-mutated NSCLC (Engelman and Janne, 2008; Kobayashi et al., 2005). Unlike lung cancers with EGFR mutations, various crizotinib resistance mutations have been identified in crizotinib-treated ALK-rearranged NSCLC. In addition, ALK fusion gene amplification has also been identified as a crizotinib resistance mechanism. Ceritinib is a potent oral ALK inhibitor, which has been shown to be effective for the treatment of naïve and crizotinib-treated ALK-rearranged NSCLC patients (Shaw et al., 2014). However, acquired ceritinib resistance also occurs, such as G1202R or F1174C/V mutations in the ALK kinase domain (Friboulet et al., 2014). Similarly, G1202R or I1171T/N/S mutations in ALK have been identified as an alectinib resistance mutation. In addition, MET gene amplification has also been identified in an alectinib-resistant patient in whom the tumor responded to crizotinib, which is a potent MET inhibitor. Apart from alterations in the ALK gene, bypass pathway activation mediated crizotinib resistance, such as EGFR activation, KIT amplification with SCF upregulation, or IGF1R activation has been identified. However, the mechanism underlying crizotinib, ceritinib and alectinib resistance remains largely unknown in many cases (Katayama et al., 2012; Shaw and Engelman, 2013; Gainor et al., 2013; Lovly et al., 2014). Recently, crizotinib was shown to be exported by P-gp, resulting in decreased penetration of the blood–brain barrier by P-gp (Chuan Tang et al., 2014). Because the use of crizotinib following ceritinib treatment showed minimal effect in our patient (JFCR013), we suspected P-gp upregulation as a mechanism of crizotinib resistance. Among the family of ATP-binding cassette proteins, P-gp is most extensively characterized. Its overexpression is shown to be sufficient to confer chemotherapeutic drug resistance to both cell cultures and animal models of human cancers. To date, attempts to improve anticancer therapy by the co-administration of various P-gp inhibitors, including MS209, with chemotherapeutic agents have been disappointing. In addition, the limited success of a combination therapy of a P-gp inhibitor with a chemotherapeutic agent was mainly because of undesirable toxicities. Although we did not observe any severe adverse effects in our mouse experiments, we must carefully consider the benefits and undesirable toxicities of molecular-targeted drugs in combination with the use of a P-gp inhibitor. Many studies have investigated P-gp-associated drug resistance, but very few have described clinical cases of P-gp-mediated drug resistance; in particular, molecular-targeted agent resistance in NSCLC. In the JFCR013 cells, P-gp overexpression was not caused by ABCB1 gene amplification. mRNA expression in JFCR013 cell lines was substantially higher than that in H3122 or ALK-TKI-naïve patient-derived JFCR018-1 cells, in which P-gp expression was barely detectable. When we continuously treated the H3122 tumor-bearing mice with ceritinib more than 2months, we found that one tumor overexpressed ABCB1 mRNA (230 fold higher than the untreated H3122 xenograft tumor) without ABCB1 gene amplification (data not shown). Thus, ABCB1 overexpression may be epigenetically regulated. Hence, further studies are needed to clarify the mechanism of ABCB1 upregulation in ceritinib-resistant cell lines.