Targeting NEK9 to Overcome Immunotherapy Resistance in Hepatocellular Carcinoma by Reprogramming the Tumor Microenvironment

Highlight

• NEK9 kinase is markedly overexpressed in hepatocellular carcinoma (HCC) and correlates with poor patient outcomes.
• NEK9 promotes immune evasion by phosphorylating TRIM28 and USP46, stabilizing NF-κB2 and inducing PD-L1 and CXCL1 expression.
• NEK9 activity leads to CD8+ T cell dysfunction and recruitment of immunosuppressive myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment.
• Two novel NEK9 small-molecule inhibitors, MIPO and FPTP, synergize with anti-PD-L1 immunotherapy, enhancing antitumor immune responses and suppressing HCC growth in vivo.

Study Background

Hepatocellular carcinoma (HCC) ranks among the leading causes of cancer mortality worldwide, often diagnosed at advanced stages with limited treatment options. Immune checkpoint inhibitors (ICIs), particularly those targeting the PD-1/PD-L1 axis, have transformed cancer therapeutics but exhibit modest efficacy in HCC compared to other malignancies. This limited success is primarily attributed to a profoundly immunosuppressive tumor microenvironment (TME) that hinders effective T cell–mediated antitumor responses. Therefore, identifying tumor-intrinsic factors that drive immune evasion and contribute to ICI resistance is critical for developing combinatorial strategies to improve outcomes in HCC patients.

Study Design

This study investigated NEK9, a kinase involved in mitotic control, as a putative tumor-intrinsic immune evasion driver in HCC. The authors analyzed NEK9 expression and clinical correlations in patient-derived HCC cohorts. Functional roles were elucidated by genetic knockdown/knockout and pharmacological inhibition of NEK9 in HCC cell lines and in orthotopic mouse models that faithfully recapitulate human disease.

To comprehensively characterize the TME remodeling associated with NEK9 activity, the team employed single-cell RNA sequencing, flow cytometry, and multiplex immunohistochemistry, enabling detailed profiling of immune cell subsets and phenotypes. Molecular mechanisms underpinning NEK9-mediated immune modulation were explored through co-immunoprecipitation, phosphoproteomics, and in vitro kinase assays. Finally, synergy between NEK9 inhibitors and PD-L1 blockade was quantitatively assessed using zero interaction potency (ZIP) models.

Key Findings

NEK9 was significantly upregulated in HCC tumor tissues compared to adjacent non-tumor liver and was associated with worse overall survival. High NEK9 expression inversely correlated with intratumoral CD8+ T cell infiltration and positively with levels of myeloid-derived suppressor cells (MDSCs), hallmarking an immunosuppressive TME.

Mechanistically, NEK9 directly phosphorylates TRIM28 and USP46, two regulatory proteins that enhance the stability of nuclear factor-κB2 (NF-κB2). Stabilized NF-κB2 upregulates transcription of PD-L1 and the chemokine CXCL1. PD-L1 overexpression contributes to CD8+ T cell dysfunction by engaging PD-1 inhibitory receptors, whereas CXCL1 recruits CXCR2-expressing MDSCs, further suppressing antitumor immunity.

Pharmacological inhibition of NEK9 with two novel small molecules, MIPO and FPTP, led to NF-κB2 destabilization, downregulation of PD-L1 and CXCL1, and reversal of immunosuppression within the TME. In vivo, combination therapy of NEK9 inhibitors with anti-PD-L1 antibodies produced strong synergy, evidenced by enhanced CD8+ T cell effector function, decreased MDSC infiltration, and significant tumor growth inhibition.

Expert Commentary

This study robustly delineates NEK9 as a druggable mediator of immune evasion in HCC, linking kinase-driven oncogenic signaling with immunosuppressive TME remodeling. By unveiling the molecular cascade from NEK9 to TRIM28/USP46 phosphorylation and subsequent NF-κB2 stabilization, it defines novel regulatory nodes that modulate PD-L1 expression and MDSC recruitment.

The use of orthotopic mouse models and single-cell immune profiling strengthens the translational relevance of the findings. Importantly, the discovery of two specific NEK9 inhibitors with efficacy and synergy when combined with PD-L1 blockade addresses an unmet translational need for strategies to overcome ICI resistance in HCC.

Potential limitations include the need for extended toxicity and pharmacokinetic evaluation of these inhibitors and validation in diverse HCC etiologies and patient-derived xenograft models. Future work should also explore biomarkers predicting response to combined NEK9 inhibition and immunotherapy.

Conclusion

NEK9 represents a critical tumor-intrinsic kinase that orchestrates immune evasion in hepatocellular carcinoma by phosphorylating key regulators and stabilizing NF-κB2-driven PD-L1 and CXCL1 expression. Targeting NEK9 reverses the immunosuppressive microenvironment and synergizes with PD-L1 blockade, offering a promising therapeutic approach to enhance immune checkpoint inhibitor efficacy in HCC.

These findings provide a compelling rationale for advancing NEK9 inhibitors into clinical trials for combination immunotherapy in HCC, with potential to improve patient survival outcomes significantly.

Funding and ClinicalTrials.gov

The original study details funding sources and clinical trial information at https://pubmed.ncbi.nlm.nih.gov/42350096/. Further clinical development of NEK9 inhibitors will require formal toxicology assessments and early-phase trials.

References

Lu G, Du R, Wan Y, Dong J, Li B, Liu M, Han Q, He F, Wang Y, Jia L, An Y, Liu Y, Han Y, Shang Y. Targeting NEK9 synergises with immunotherapy in hepatocellular carcinoma by remodelling the immunosuppressive microenvironment. Gut. 2026 Jun 25. PMID: 42350096.

Additional relevant literature:
– El-Khoueiry AB, Sangro B, Yau T, et al. Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet. 2017;389(10088):2492-2502.
– Llovet JM, et al. Immunotherapies for hepatocellular carcinoma. Nat Rev Clin Oncol. 2021 Jun;18(6):375-380.