Galectin9 targeted therapy for aiming cure in oncogenic driver alterations-positive non-small cell lung cancer

Small-molecule tyrosine kinase inhibitors (TKIs) exert dramatic antitumor effect in patients with oncogenic driver alterations-positive non-small cell lung cancer (driver-positive NSCLC). However, it is inevitable for those patients to experience recurrence because of the existence of intrinsic drug-resistant subpopulation called drug-tolerant persisters (DTPs). Therefore, elimination of DTPs is crucial toward complete cure. We examined whether innate immunity could eliminate DTPs for complete cure in driver-positive NSCLC. To identify the key immune checkpoint molecule (ICM) to eradicate DTPs, EGFR mutation-positive NSLC cells (HCC827 and PC-9) and ALK fusion-positive NSLC cells (H3122 and H2228) were exposed to specific TKI osimertinib and alectinib, respectively. Among ICMs, galectin-9 (Gal-9) transcripts were most dramatically upregulated in both driver-positive NSCLC cells via activation of interferon regulatory factor 1 in nucleus. Gal-9 was also overexpressed in both HCC827 osimertinib-resistant cells (HCC827OR) and H3122 alectinib-resistant cells established by continuous exposure to respective TKI. Moreover, TCGA database analysis showed the possibility for Gal-9 to be a negative prognostic factor in lung adenocarcinoma. We next examined whether Gal-9 expression in cancer cells affected the cytotoxicity of natural killer (NK) cells. Cytotoxicity was evaluated by co-incubating cancer cells and NK leukemia cells (NKL) under continuous monitoring using xCELLigence. While exogenous Gal-9 expression weakened NKL-mediated cytotoxicity for HCC827 cells, anti-Gal-9 antibody enhanced the cytotoxicity against HCC827OR cells. We then generated CRISPR/Cas9-mediated Gal-9-knockout (KO) HCC827 cells (HCC827-sgLGALS9) and performed long-term NKL killing assay in the presence of osimertinib for evaluating the cytotoxicity against DTPs. While HCC827-sgLGALS9 cells were completely depleted by osimertinib exposure plus NKL mediated-cytotoxicity, scramble guide RNA-transfected HCC827 cells (HCC827-scr) reproliferated, which suggested that NKL-mediated cytotoxicity failed to eliminate DTPs in HCC827-scr cells. We further engrafted HCC827-scr and HCC827-sgLGALS9 cells into the flank of athymic nude mice and administered osimertinib orally for 30 days. Xenografts in both groups rapidly shrank and disappeared by osimertinib, however, the regrowth rate was higher in HCC827-scr group. Since human Gal-9 has low affinity for mouse immune cells, we transfected mutant human EGFR into NIH 3T3 cells (3T3-EGFRmt) and established allograft model. As expected, osimertinib significantly suppressed the growth of mouse sgLGALS9 transfected 3T3-EGFRmt allografts compared with that of scramble control allografts. Collectively, targeting Gal-9 is promising for aiming cure in driver-positive NSCLC by activating innate immunity against DTPs.