NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death, and immunotherapy, particularly immune checkpoint blockade targeting PD-1/PD-L1, offers a promising therapeutic avenue. However, PD-1/PD-L1 blockade has shown limited effectiveness in PDAC. Post-translational modifications (PTMs) of PD-L1 significantly impact the efficacy of PD-L1-targeted therapies. This study focuses on the role of never in mitosis gene A-related kinase 2 (NEK2), a serine-threonine kinase involved in cell cycle regulation and frequently upregulated in various cancers, in regulating PD-L1 and impacting the response to immunotherapy in pancreatic cancer. The study investigates whether NEK2 is a critical regulator of PD-L1 PTMs in PDAC and whether its inhibition could improve the efficacy of PD-L1 blockade.

Existing research highlights the critical role of post-translational modifications (PTMs) such as ubiquitination, phosphorylation, and glycosylation in regulating PD-L1 stability, internalization, and localization, ultimately affecting its function and the efficacy of targeted therapies. Studies have shown that N-glycosylation of PD-L1 is crucial for its proper folding, transport, and function. Other studies have demonstrated that EGFR signaling upregulates B3GNT3, promoting N-glycosylation of PD-L1 and stabilizing it by preventing GSK3β binding and subsequent degradation. Conversely, metformin-activated AMP-activated protein kinase phosphorylates PD-L1, inducing its degradation. While NEK2's role in cell cycle regulation and various cancers is well-established, its involvement in cancer immune resistance remained undefined before this study.

The study employed a multi-faceted approach combining bioinformatic analysis of TCGA and other databases, in vitro cell experiments, and in vivo mouse models. Bioinformatic analyses assessed the correlation between NEK family member expression and pancreatic cancer prognosis. Immunohistochemistry (IHC) validated NEK2 expression in paired PDAC tissue samples and tumor tissue microarrays. To investigate the role of NEK2 in anti-tumor immune response, a KPC-NEK2 knockdown (KD) cell line was generated. In vivo studies using immunocompetent and immunodeficient mice compared tumor growth and survival in NEK2 KD versus wild-type (WT) cells. The effects of NEK2 inhibition on pancreatic tumorigenesis were assessed using a NEK2 inhibitor in both immunocompetent and immunodeficient mouse models. The interaction between NEK2 and PD-L1 was investigated using co-immunoprecipitation, GST pull-down assays, and Duolink assays. The mechanisms underlying NEK2's regulation of PD-L1 were explored through examining PD-L1 expression levels under NEK2 inhibition/depletion/overexpression conditions, using proteasome inhibitors, and investigating PD-L1 ubiquitination and phosphorylation. Site-specific antibodies were generated to detect PD-L1 phosphorylation at specific residues. The synergistic effect of combining NEK2 inhibitor and anti-PD-L1 antibody was evaluated in vitro and in vivo using cell-mediated cytotoxicity assays and mouse models. Flow cytometry analyzed tumor-infiltrating lymphocytes (TILs) to assess changes in immune cell composition and activation.

The study revealed several key findings: 1. NEK2 is a significant prognostic factor in immunologically “hot” pancreatic cancer, correlating with poor overall survival. 2. NEK2 deficiency improves pancreatic cancer immunogenicity, leading to reduced tumor growth and increased survival in immunocompetent mice but not in immunodeficient mice. This indicates an immune-dependent mechanism. 3. NEK2 positively correlates with and directly interacts with PD-L1 in pancreatic cancer. 4. NEK2 inhibits ubiquitination-mediated proteasomal degradation of PD-L1, maintaining its stability. 5. NEK2 phosphorylates PD-L1 at T194/T210 residues, preventing its degradation. 6. NEK2 inhibition sensitizes PD-L1-targeted pancreatic cancer immunotherapy, significantly enhancing the anti-tumor immune response both in vitro and in vivo. The combination of NEK2 inhibitor and anti-PD-L1 antibody synergistically suppressed tumor growth and increased the number and activation of tumor-infiltrating T cells.

This study demonstrates a novel mechanism by which NEK2 promotes immune evasion in pancreatic cancer through its regulation of PD-L1. The finding that NEK2 inhibition sensitizes PD-L1 blockade offers a new therapeutic strategy to overcome immune resistance in PDAC. The study highlights the importance of considering the tumor immune microenvironment when developing immunotherapies. The observation that NEK2's prognostic value is particularly relevant in immunologically “hot” tumors suggests a potential for personalized therapeutic strategies based on immune status. The identification of specific phosphorylation sites (T194/T210) on PD-L1 provides potential targets for future therapeutic development. The study's findings are consistent with other research showing that cell cycle regulators play an important role in cancer immune regulation.

This research establishes NEK2 as a critical regulator of PD-L1 stability in pancreatic cancer and a promising therapeutic target. The synergistic effect of combining NEK2 inhibition with anti-PD-L1 antibody therapy provides a potential approach to improve the effectiveness of immunotherapy in PDAC, particularly in immunologically “hot” tumors. Further preclinical studies and clinical trials are needed to validate these findings and explore the clinical translation of this approach.

While the study provides compelling evidence for the role of NEK2 in pancreatic cancer immunotherapy, further research is necessary. The study primarily focused on a specific pancreatic cancer cell line (KPC) and might not fully represent the heterogeneity of PDAC. Additionally, the long-term effects of NEK2 inhibition and the potential for off-target effects require further investigation. Larger clinical studies will be needed to confirm the findings and to establish NEK2 inhibition as a viable therapeutic approach.