Tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR TKIs) greatly improved clinical outcomes of patients with non-small cell lung cancer (NSCLC). Unfortunately, primary and acquired resistance limits their clinical benefits. To overcome such resistance, new generations of EGFR TKIs have been developed by targeting newly identified mutations in EGFR. However, much less effort has been put into alternative strategies, such as targeting the intrinsic protective responses to EGFR TKIs. In this study, we found that EGFR TKIs, including gefitinib and AZD9291, impaired lysosome-dependent degradation of SQSTM1, thus compromising their anti-cancer efficiency. By accumulating in the lysosome lumen, gefitinib and AZD9291 attenuated lysosomal acidification and impaired autolysosomal degradation of SQSTM1 owing to their intrinsic alkalinity. As a result, SQSTM1 protein was stabilized in response to gefitinib and AZD9291 treatment and conferred EGFR TKI resistance. Depleting SQSTM1 significantly increased the sensitivity of NSCLC cells to gefitinib and AZD9291 both in vitro and in vivo. Furthermore, a chemically modified gefitinib analog lacking alkalinity displayed stronger inhibitory effects on NSCLC cells. Therefore, targeting accumulated SQSTM1 or chemically modified EGFR TKIs may represent new strategies to increase the effectiveness of EGFR targeted therapy.

BackgroundsIn advanced pancreatic ductal adenocarcinoma (PDAC), immune therapy, including immune checkpoint inhibitors, has limited efficacy, encouraging the study of combination therapy.MethodsTumor necrosis factor receptor 2 (TNFR2) was analyzed via immunohistochemistry, immunofluorescence, western blotting, and ELISAs. The in vitro mechanism that TNFR2 regulates programmed cell death 1 ligand 1 (PD-L1) was investigated using immunofluorescence, immunohistochemistry, flow cytometry, western blotting, and chromatin immunoprecipitation (ChIP). In vivo efficacy and mechanistic studies, using C57BL/6 mice and nude mice with KPC cell-derived subcutaneous and orthotopic tumors, employed antibodies against TNFR2 and PD-L1. Survival curves were constructed for the orthotopic model and a genetically engineered PDAC model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre). Mass cytometry, immunohistochemistry, and flow cytometry analyzed local and systemic alterations in the immunophenotype.ResultsTNFR2 showed high expression and is a prognostic factor in CD8+ T cell-enriched pancreatic cancer. TNFR2 promotes tumorigenesis and progression of pancreatic cancer via dual effect: suppressing cancer immunogenicity and partially accelerating tumor growth. TNFR2 positivity correlated with PD-L1, and in vitro and in vivo, it could regulate the expression of PDL1 at the transcription level via the p65 NF-κB pathway. Combining anti-TNFR2 and PD-L1 antibodies eradicated tumors, prolonged overall survival in pancreatic cancer, and induced strong antitumor immune memory and secondary prevention by reducing the infiltration of Tregs and tumor-associated macrophages and inducing CD8+ T cell activation in the PDAC microenvironment. Finally, the antitumor immune response derived from combination therapy is mainly dependent on CD8+ T cells, partially dependent on CD4+ T cells, and independent of natural killer cells.ConclusionsAnti-TNFR2 and anti-PD-L1 combination therapy eradicated tumors by inhibiting their growth, relieving tumor immunosuppression, and generating robust memory recall.

Programmed death-1 (PD-1) and its ligand programmed death-ligand 1 (PD-L1) help tumor cells evade immune surveillance, and are regarded as important targets of anti-tumor immunotherapy. Post-translational modification of PD-L1 has potential value in immunosuppression. Here, we identified that ubiquitin-specific protease 8 (USP8) deubiquitinates PD-L1. Pancreatic cancer tissues exhibited significantly increased USP8 levels compared with those in normal tissues. Clinically, the expression of USP8 showed a significant association with the tumor-node-metastasis stage in multiple patient-derived cohorts of pancreatic cancer. Meanwhile, USP8 deficiency could reduce tumor invasion and migration and tumor size in an immunity-dependent manner, and improve anti-tumor immunogenicity. USP8 inhibitor pretreatment led to reduced tumorigenesis and immunocompetent mice with Usp8 knockdown tumors exhibited extended survival. Moreover, USP8 interacted positively with PD-L1 and upregulated its expression by inhibiting the ubiquitination-regulated proteasome degradation pathway in pancreatic cancer. Combination therapy with a USP8 inhibitor and anti-PD-L1 effectively suppressed pancreatic tumor growth by activation of cytotoxic T-cells and the anti-tumor immunity was mainly dependent on the PD-L1 pathway and CD8 + T cells. Our findings highlight the importance of targeting USP8, which can sensitize PD-L1-targeted pancreatic cancer to immunotherapy and might represent a novel therapeutic strategy to treat patients with pancreatic tumors in the future.