title: Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: Implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients.
abstract: Driver mutations were reported to upregulate programmed death-ligand 1 (PD-L1) expression. However, how PD-L1 expression and immune function was affected by ALK-TKIs and anti-PD-1/PD-L1 treatment in ALK positive non-small-cell lung cancer (NSCLC) remains poorly understood. In the present study, western-blot, real-time PCR, flow cytometry and immunofluorescence were employed to explore how PD-L1 was regulated by ALK fusion protein. ALK-TKIs and relevant inhibitors were used to identify the downstream signaling pathways involved in PD-L1 regulation. Cell apoptosis, viability and Elisa test were used to study the immune suppression by ALK activation and immune reactivation by ALK-TKIs and/or PD-1 blocking in tumor cells and DC-CIK cells co-culture system. We found that PD-L1 expression was associated with EGFR mutations and ALK fusion genes in NSCLC cell lines. Over-expression of ALK fusion protein increased PD-L1 expression. PD-L1 mediated by ALK fusion protein increased the apoptosis of T cells in tumor cells and DC-CIK cells co-culture system. Inhibiting ALK by sensitive TKIs could enhance the production of IFNγ. Anti-PD-1 antibody was effective in both crizotinib sensitive and resistant NSCLC cells. Synergistic tumor killing effects were not observed with ALK-TKIs and anti-PD-1 antibody combination in co-culture system. ALK-TKIs not only directly inhibited tumor viability but also indirectly enhanced the antitumor immunity via the downregulation of PD-L1. Anti-PD-1/PD-L1 antibodies could be an optional therapy for crizotinib sensitive, especially crizotinib resistant NSCLC patients with ALK fusion gene. Combination of ALK-TKIs and anti-PD-1/PD-L1 antibodies treatment for ALK positive NSCLC warrants more data before moving into clinical practice.
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Shaodong
authors.initials: S
authors.lastname: Hong
authors.affiliation: Department of Medical Oncology, the Fifth Affiliated Hospital of Sun Yat-sen University , Zhuhai, Guangdong, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Chen
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Fang
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhan
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Shiyang
authors.initials: S
authors.lastname: Kang
authors.affiliation: Department of Medical Oncology, the Fifth Affiliated Hospital of Sun Yat-sen University , Zhuhai, Guangdong, China.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: He
authors.affiliation: Department of Medical Oncology, the Fifth Affiliated Hospital of Sun Yat-sen University , Zhuhai, Guangdong, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Department of Medical Oncology, the Fifth Affiliated Hospital of Sun Yat-sen University , Zhuhai, Guangdong, China.
authors.forename: Jiaxing
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Medical Oncology, the Fifth Affiliated Hospital of Sun Yat-sen University , Zhuhai, Guangdong, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Qin
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Yaxiong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Ma
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
keywords: EML4-ALK; NSCLC; PD-1; PD-L1; immunotherapy