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.

The combination of checkpoint immunotherapy and targeted therapy in cancer

“…PD-L1 expression is significantly higher in ALK rearranged NSCLCs compared to NSCLCs with EGFR or KRAS mutation or to those with no genetic alteration of ALK, EGFR or KRAS (triple negative) (45,46). Interestingly, EGFR and ALK tyrosine kinase inhibitors (TKIs) directly inhibit tumor cell viability and indirectly enhance antitumor immunity through the PD-L1 downregulation (40,47,48). PD-L1 expression is related with better response to EGFR and ALK TKIs (45,49,50).…”

The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells

“…6,[16][17][18][19][20] It has been shown that PD-L1 is constitutively expressed in various types of tumor cells and regulated by oncogenes. [18][19][20] Furthermore, PD-L1 expression can be upregulated by inflammatory cytokines such as IFNg [20][21][22][23] In addition to expression in tumor cells, PD-L1 is also expressed on subsets of T cells, NK cells, macrophages, myeloid DCs, B cells, epithelial cells and vascular endothelial cells. [24][25][26][27] PD-L1 is notably expressed on mature macrophages, particularly in activated macrophages.…”

Diagnosis and Treatment of Anaplastic Lymphoma Kinase–Positive Non–Small Cell Lung Cancer

“…Preclinical data suggested that over expression of ALK fusion protein increased PDL-1 expression raising the possibility that PD-L1 antibodies may be effective in the treatment of ALK positive NSCLC. 48,49 Retrospective data, however, indicates that patients with ALK rearrangements have lower response rates to PD-1/PD-L1 inhibitors compared to patients with ALK negative tumors. 50 …”