Purpose: The efficacy of immune checkpoint inhibitors such as programmed cell death ligand 1 (PD-L1) antibodies in non-small cell lung cancer (NSCLC) is limited, and combined use with other therapies is recommended. Dipeptidyl peptidase 4 (DPP4) inhibitors, a class of small molecule inhibitors, are highly effective for treating type 2 diabetes. Emerging evidence implicates DPP4 inhibitors as immunomodulators that modify aspects of innate and adaptive immunity. We evaluated the combination of a DPP4 inhibitor (anagliptin) and PD-L1 blockade in an NSCLC mouse model. Methods: The effect of the combination of anti-PD-L1 and anagliptin was evaluated in subcutaneous mouse models of NSCLC. Tumor-infiltrating immune cells were analyzed by flow cytometry. Bone marrow-derived monocytes of C57BL/6 mice were isolated in vitro to examine the underlying mechanism of anagliptin on the differentiation and polarization of macrophage. Results: Anagliptin dramatically improved the efficacy of PD-L1 antibody monotherapy by inhibiting macrophage formation and M2 polarization in the tumor microenvironment. Mechanistically, anagliptin suppressed the production of reactive oxygen species in bone marrow monocytes by inhibiting NOX1 and NOX2 expression induced by macrophage colony-stimulating factor, reduced late ERK signaling pathway activation, and inhibited monocyte-macrophage differentiation. However, the inhibitory effect was reactivated by lipopolysaccharide and interferon-gamma interacting with corresponding receptors during M1 macrophage polarization, but not M2. Conclusions: Anagliptin can enhance PD-L1 blockade efficacy in NSCLC by inhibiting macrophage differentiation and M2 macrophage polarization, and combination therapy may be a promising strategy for treating PD-L1 blockade therapy-resistant patients with NSCLC.

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is the main cause of mortality in lung cancer. This study aimed to investigate the roles of neuropilin 1 (NRP1) in non-small cell lung cancer (NSCLC). NRP1 expression was assessed in tumor tissues from patients with osimertinib-resistant (OR) NSCLC and osimertinib-responsive NSCLC as well as in patients with paracancerous NSCLC tissues who did not undergo radiotherapy or chemotherapy. In vitro experiments were conducted using five cell lines: BEAS-2B, HCC827, and PC9 cells, and the constructed OR cell lines, HCC827-OR and PC9-OR. HCC827-OR cells showing significant differences in osimertinib IC₅₀ were selected for further study. After investigating the effects of altering NRP1 expression on cell sensitivity to osimertinib, NRP1 expression was inhibited to further investigate changes in cell viability, proliferation, migration, invasion, and apoptosis in OR cells. Additionally, bioinformatics techniques were used to detect targets (Integrin β3) and signaling pathways (PI3K/AKT) downstream of NRP1; subsequent cell experiments verified their interactivity. Finally, an orthotopic mouse tumor model was constructed using HCC827-OR cells treated with a PI3K/AKT signaling pathway activator (740Y-P), allowing exploration of the role played by the PI3K/AKT signaling pathway via NRP1 regulation on NSCLC resistance both in vivo and in vitro. Results showed that NRP1 expression was significantly increased in the cells of patients with NSCLC-OR, and increased NRP1 expression reduced HCC827 cell sensitivity to osimertinib. Both in vitro and in vivo experiments showed that NRP1 deficiency mediated by NRP1 inhibitors inhibited HCC827-OR cell proliferation, migration, and invasion, promoted tumor cell apoptosis, and enhanced osimertinib efficacy. In contrast, 740Y-P partially inhibited the effects of NRP1 inhibitors combined with osimertinib on the PI3K/AKT signaling pathway and on tumor growth in vivo and in vitro. Cellular experimental results showed that NRP1 positively regulates the Integrin β3 expression and activation of the PI3K/AKT signaling pathway. Bioinformatics analysis showed that both NRP1 and Integrin β3 may jointly participate in regulating the PI3K/AKT signaling pathway. In conclusion, our findings suggest that elevated NRP1 expression in NSCLC tumor tissues may promote NSCLC resistance to osimertinib by activating the PI3K/AKT signaling pathway, and integrin β3 potentially being involved in this process. These insights may provide a novel strategy for combination therapy for OR NSCLC.

The E3 ubiquitin ligase RING finger protein 6 (RNF6) has been widely recognized for its role in promoting tumorigenesis in multiple cancers. However, we found that it is downregulated in lung adenocarcinoma (LUAD), and the molecular rationale for this discrepancy remains unclear. In the present study, we find that RNF6, but not its ΔRING inactive form, inhibits LUAD cell proliferation and migration and sensitizes LUAD to chemotherapy. To understand the molecular mechanism, we utilize affinity purification/tandem mass spectrometry (MS-MS) to analyze RNF6-interacting proteins and find that cyclin D2 (CCND2), a key regulator of the G1–S transition in the cell cycle. RNF6 physically binds to CCND2 and mediates its K48-linked polyubiquitination and subsequent degradation. However, ΔRING RNF6 fails to mediate CCND2 for ubiquitination and degradation. Moreover, Thr280 is critically important for CCND2 stability. When Thr280 is mutated, CCND2 becomes more stable and less ubiquitinated by RNF6. Furthermore, RNF6 arrests LUAD cell cycle at the G1 phase by inhibiting the CCND2/phospho-Rb signaling pathway, which is consistent with decreased cell proliferation. Lastly, RNF6 curtails the growth of LUAD xenografts in vivo, associated with decreased CCND2 expression. Therefore, RNF6 is a novel E3 ligase of CCND2 and suppresses LUAD cell proliferation. Implications: This study reveals a novel regulation on cell-cycle transition in LUAD and suggests the RNF6/CCND2 axis may represent an alternative therapeutic target for the treatment of LUAD.

Supplemental Fig. S5. RNF6 inhibits the phosphorylation of Rb in vivo. The expression level of pRb/Rb level were analyzed by ImageJ when RNF6 was overexpressed (A) or knocked down (B) based on the WB blots in Fig. 5G-H (Mean±SD, n=3). ***, P < 0.001.

Supplemental Fig. S2. RNF6 decreases the half-life of CCND2. (A) A549 and NCI-H1299 cells were transfected a Flag-RNF6 plasmid for 48 hrs, followed by treatment with MG132 for 8 hrs and IP/IB assays. (B) A549 and NCI-H1299 cells were transfected with siRNF6 for 48 hrs, followed by treatment with MG132 for 8 hrs and IP/IB assays. (C-F) NCI-H1299 cells were transfected with RNF6 plasmid (C-D) or siRNF6 (E-F) for 48 hrs, followed by CHX treatment for indicated periods. The cell lysates were then subjected to IB assays (C and E), followed by quantitative analysis on CCND2 against GAPDH (D and F). Data were presented as mean±SD (n=3). **, P < 0.01.