Induction of PD-L 1 Expression by the EML 4 – ALK Oncoprotein andDownstreamSignalingPathways in Non – Small Cell Lung Cancer

Purpose: Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non–small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4–ALK fusion gene. ExperimentalDesign: The expressionof PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry andby reverse transcription and real-time PCR analysis, respectively. TheexpressionofPD-L1 in134surgically resectedNSCLCspecimens was evaluated by immunohistochemical analysis. Results: The PD-L1 expression level was higher in NSCLC cell lines positive for EML4–ALK than in those negative for the fusion gene. Forced expression of EML4–ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4–ALK–positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK–ERK and PI3K–AKT signaling pathways in NSCLC cells positive for either EML4–ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4–ALK in NSCLC specimens. Conclusions: Our findings that both EML4–ALK and mutant EGFR upregulate PD-L1 by activating PI3K–AKT and MEK–ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression. Clin Cancer Res; 21(17); 4014–21. 2015 AACR. Introduction The interaction of programmed cell death–ligand 1 (PD-L1, also known as B7-H1 or CD274) expressed on antigen-presenting cells and parenchymal cells with its receptor programmed cell death–1 (PD-1, also known as CD279) on T cells is a physiologic mechanismunderlying escape from immune activity (1). PD-L1 is also expressed by various human tumors, resulting in inhibition of the immune response to the cancer cells and consequent facilitation of tumor progression and metastasis (2–5). A high level of PD-L1 expression in several types of malignancy has been associated with a poor clinical outcome, although some findings remain controversial (6–9). Recent clinical trials have shown that inhibition of the PD-1–PD-L1 interactionwith antibodies specific for these proteins is a promising approach to cancer treatment (10, 11). The expression level of PD-L1 at the tumor cell surface has been found to correlate with the likelihood of a response to PD-1– or PD-L1–targeted therapy (11). The molecular mechanisms responsible for the regulation of PD-L1 expression in tumors remain unclear, however. Oncogenic drivers play central roles in tumorigenesis as well as in tumor cell survival and proliferation. Non–small cell lung cancer (NSCLC) is at the forefront ofmolecularly targeted therapy as a result of rapid advances in our understanding of its genetic drivers (12, 13). Mutations of the EGFR gene that result in constitutive activation of the receptor tyrosine kinase have, thus, been identified as oncogenic drivers in a subset of NSCLC tumors (14–16), and tyrosine kinase inhibitors (TKI) specific for EGFR have had a substantial impact on treatment outcome in patients with such tumors (13, 17, 18). Weandothers recently showed that PD-L1 expression in NSCLC is upregulated as a result of EGFR mutation, implicating oncogenic drivers in regulation of the expression of immunosuppressive molecules (19, 20). Chromosomal rearrangement involving the anaplastic lymphoma kinase (ALK) and echinoderm microtubule–associated protein–like 4 (EML4) genes also defines a distinct molecular subset of NSCLC, with the resulting fusion gene manifesting pronounced transforming activity (21, 22). Treatment with ALK-targeted TKIs has also been established for patients with EML4–ALK fusion–positive NSCLC (13, 23–27). We have now examined the role of EML4–ALK rearrangement in PD-L1 expression inNSCLC cells. In addition, we examined the signaling pathways thatmodulate PDL1 expression in NSCLC cells harboring driver oncogenes, including ALK fusions and mutated EGFR. Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Division of Respirology, Neurology, and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Fukuoka, Japan. Department of Diagnostic Pathology, Kurume University Hospital, Fukuoka, Japan. Biostatistics Center, Kurume University, Fukuoka, Japan. Department of Comprehensive Clinical Oncology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan. Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. Department of Surgery, Kurume University School of Medicine, Fukuoka, Japan. Center for Clinical and Translational Research, Kyushu University Hospital, Fukuoka, Japan. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Isamu Okamoto, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-5378; Fax: 81-92-642-5390; E-mail: okamotoi@kokyu.med.kyushu-u.ac. jp doi: 10.1158/1078-0432.CCR-15-0016 2015 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 21(17) September 1, 2015 4014 on January 13, 2018. © 2015 American Association for Cancer Research. clincancerres.aacrjournals. org Downloaded from Published OnlineFirst May 27, 2015; DOI: 10.1158/1078-0432.CCR-15-0016