Background: The identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD. Methods: We evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry. Results: We found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells. Conclusions: Our data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers. Graphical

Background: Harnessing the anti-tumor immune system response by targeting the program cell death protein (PD-1) and program cell death ligand protein (PD-L1) axis has been a major breakthrough in non-small cell lung cancer (NSCLC) therapy. Nonetheless, conventional imaging tools cannot accurately assess response in immunotherapy-treated patients. Using a lung cancer syngeneic mouse model responder to immunotherapy, we aimed to demonstrate that [⁸⁹Zr]-anti-PD-1 immuno-PET is a safe and feasible imaging modality to assess the response to PD-1/PD-L1 blockade in NSCLC.Materials and methods: A syngeneic mouse model responder to anti-PD-1 therapy was used. Tumor growth and response to PD-1 blockade were monitored by conventional 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]-FDG) PET scans. Additionally, tumor lymphocyte infiltration was analyzed by the use of an [⁸⁹Zr]-labeled anti-PD-1 antibody and measured as ⁸⁹Zr tumor uptake.Results: Conventional [¹⁸F]-FDG-PET scans failed to detect the antitumor activity exerted by anti-PD-1 therapy. However, [⁸⁹Zr]-anti-PD-1 uptake was substantially higher in mice that responded to PD-1 blockade. The analysis of tumor-infiltrating immune cell populations and interleukins demonstrated an increased anti-tumor effect elicited by activation of effector immune cells in PD-1-responder mice. Interestingly, a positive correlation between [⁸⁹Zr]-anti-PD-1 uptake and the proportion of tumor-infiltrating lymphocytes (TILs) was found (Cor = 0.8; p = 0.001).Conclusion: Our data may support the clinical implementation of immuno-PET as a promising novel imaging tool to predict and assess the response of PD-1/PD-L1 inhibitors in patients with NSCLC.

Background The identification of novel therapeutic strategies to overcome the intrinsic or acquired resistance to trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a major challenge. This study analyzes the effects of trametinib in Id1, a key factor involved in the oncogenic KRAS pathway, and investigates the Id1 role in acquire resistance and trametinib synergy with immunotherapy in KRAS-driven LUAD.Methods We evaluated the effects of trametinib in KRAS-mutant LUAD tumors by western blot, RNA-seq and syngeneic mouse models. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis was measured by flow cytometry. The anti-tumor efficacy of the trametinib and PD-1 blockade combined treatment was investigated in two KRAS-driven LUAD mouse models, and the effects in the tumor immune infiltrate was analyzed by immunohistochemistry.Results We found that trametinib activates the proteasome to downregulate Id1 expression in KRAS-mutant LUAD cells and tumors. Moreover, Id1 inhibition overcome the acquire resistance to trametinib in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-driven LUAD mouse models we found that trametinib synergizes with PD-1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity was associated with an increase of the intratumoral CD8⁺/Treg ratio and PD-L1 expression on LUAD cells surface.Conclusions Our data suggests that Id1 may be involved in the resistance to trametinib and in the synergy with immunotherapy in KRAS-driven LUAD tumors. These findings suggest a potential therapeutic approach for refractory KRAS-mutant lung cancers.