ROS1 Fusion Mediates Immunogenicity by Upregulation of PD-L1 After the Activation of ROS1–SHP2 Signaling Pathway in Non-Small Cell Lung Cancer

Abstract

The drug resistance of first-line crizotinib therapy for ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion non-small cell lung cancer (NSCLC) is inevitable. Whether the administration of immune checkpoint inhibitor (ICI) therapy is suitable for ROS 1 fusion NSCLCs or after the development of crizotinib resistance is still unknown. In this study, five different crizotinib resistant concentration cell lines (HCC78CR1-5) from primary sensitive HCC78 cells were cultured. Ba/F3 cells expressing crizotinib sensitive ROS1 fusion and crizotinib resistant ROS1-G2032R mutation were used to explore the relationship between ROS1 fusion, ROS1-G2032R mutation and programmed death-ligand 1 (PD-L1) expression and the clinical potential of anti-PD-L1 ICI therapy. The signaling pathway net was compared between HCC78 and HCC78CR1-5 cells using RNA sequencing. Anti- PD-L1 ICI therapy was performed on mouse xenograft models with Ba/F3 ROS1 fusion or ROS1-G2032R mutation. HCC78CR1-5 showed more immunogenicity than HCC78 in immune-related pathways. The PD-L1 expression level was remarkably higher in HCC78CR1-5 with ROS1 fusion upregulation than HCC78 primary cell. Furthermore, the expression of PD-L1 was down-regulated by RNA interference with ROS1 siRNAs and up-regulated lower in Ba/F3 ROS1-G2032R resistant mutation than ROS1 fusion. Western blotting analysis showed the ROS1–SHP2 signaling pathway activation in HCC78CR1-5 cells, Ba/F3 ROS1 fusion and ROS1-G2032R resistant mutation. Mouse xenograft models with Ba/F3 ROS1 fusion showed more CD3+PD-1+ T cells both in blood and tissue, and more sensitivity than the cells with Ba/F3 ROS1-G2032R resistant mutation after anti-PD-L1 therapy. Our findings indicate that PD-L1 upregulation depends on ROS1 fusion more than ROS1-G2032R mutation. We share our insights of NSCLCs treatment management into the use of anti-PD-L1 ICI therapy in ROS1 fusion and not in ROS1-G2032R resistant mutation.