Novel therapeutic strategy to prevent immune escape in metastatic NSCLC through combined PD-1 and Id1 blockade

Primary lung cancer ranks as the most common tumor worldwide and the leading cause of cancer-related mortality. One of the greatest advances over the last years in primary lung cancer diagnosis and treatment is the concept of personalized medicine, in which therapeutic decisions are based on the specific histopathologic and genomic characteristics of the tumor. However, no targeted therapies have been approved for KRAS-mutant lung adenocarcinoma (LUAD) so far, with the exception of sotorasib for LUAD harbouring KRAS p.G12C mutation. The use of PD-1/PD-L1 checkpoint inhibitors in advanced non-small cell lung cancer (NSCLC) is associated with longer survival. However, many patients do not benefit from PD-1/PD-L1 blockade, largely because of immunosuppression. New immunotherapy-based combinations are under investigation to improve outcomes. Inhibitor of differentiation-1 (Id1) is the most extensively studied member of the Id family. Deregulation of Id1 impacts on cancer initiation, maintenance and progression and also plays a role in suppressing the antitumor immune response during tumor progression and metastasis appearance. Id1 mRNA expression is higher in LUAD patients with KRAS mutations and constitutes an independent prognostic marker specifically in this subgroup of tumors. In this study, we explored the potential synergistic effect of the combination of Id1 inhibition and pharmacological PD-L1 blockade in KRAS-mutant LUAD. Id1 mRNA expression levels were inversely correlated with several immune-related factors, including PD-L1 expression, in TCGA, and Id1 inhibition in cell lines led to PD-L1 upregulation after IFN-γ stimulation. Unfortunately, results from TCGA were not consistent in TMAs created from tumor specimens of patients with NSCLC. In vivo experiments using syngeneic and metastatic murine models of KRAS-driven LUAD in which Id1 was silenced in tumor cells, host cells or in both revealed the synergistic effect of Id1 inhibition when combined with an anti-PD-1 monoclonal antibody, which resulted more effective than each strategy alone in terms of tumor growth impairment and overall survival. These results were reproducible and consistent in different murine models. A significant increase in TILs was observed in the tumor tissue from the mice that were treated with the anti-PD-1 agent in which Id1 was inhibited. The antitumor immune response observed may be mediated mainly through CD8+ T lymphocyte tumor infiltration. Co-culture assays demonstrated that Id1 deficiency in tumor cells increased the killing effect of CD8+ T cells. Both multiplex quantification and flow cytometry exposed that activated CD8+ T lymphocytes infiltrating the tumor are the main represented immune cell population and CD8+ T cells depletion led to a tumor growth phenotype restoration due to a significant reduction in the efficacy of PD-1 blockade, despite the inhibition of Id1 and the anti-PD-1 agent administration. Therefore, the most plausible biological mechanism behind the antitumor activity of the combined inhibition of Id1 and the PD-1/PD-L1 axis is that this combination favors the tumor infiltration by activated immune cells. In light of our results, further investigation into the identification of Id1 pharmacological inhibitors and pre-clinical combination studies for Id1-PD-1 blockade are warranted. This combined blockade of Id1 and the PD-1/PD-L1 axis may broaden the benefit of immunotherapy in NSCLC, as it could be a strategy to overcome resistance to immunotherapy in tumors harboring STK11/LKB1 and KRAS co-mutations.