Targeting HK3 in tumor-associated macrophages enhances antitumor immunity through augmenting antigen cross-presentation in cervical cancer.

Background: Tumor-associated macrophages (TAMs) are among the most prevalent cells within the tumor microenvironment (TME) of cervical cancer (CC). Although TAMs frequently exhibit an immunosuppressive phenotype, their plasticity enables them as an intriguing reprogrammable target for immunotherapy of CC.

Methods: Consensus clustering was employed to delineate immune infiltration patterns in a cohort of 119 patients with CC. Single-cell RNA sequencing, complemented by flow cytometry analysis, was used to characterize hexokinase 3 (HK3)-expressing cell populations. In vivo tumor models were established to assess the functional impact of HK3-expressing cells on the TME, with interventions including knockout and CD8 T-cell depletion. A comprehensive approach involving bulk RNA sequencing, immunoprecipitation assays, confocal microscopy imaging, and in vitro co-culture systems was implemented to elucidate the mechanisms underlying HK3 inhibition-mediated enhancement of antitumor immunity. Furthermore, the therapeutic efficacy of HK3 inhibition, both as a monotherapy and in combination with immunotherapeutic strategies, was systematically evaluated in preclinical tumor models.

Results: We elucidated a cross-regulation between TAMs and CD8 T cells, with HK3 serving as a central regulatory node. Upon HK3 expression was upregulated by CD8 T cells through the IFN-γ-STAT1 signaling axis, TAMs exhibited impaired cross-presentation capacity, which in turn attenuated CD8 T cell-mediated antitumor immunity. Mechanistically, HK3 physically interacted with mechanistic target of rapamycin (mTOR), promoting nuclear translocation of transcription factor EB (TFEB) and resulting in excessive lysosomal activation and antigen degradation. Moreover, targeting HK3 in combination with immune checkpoint blockade yielded a synergistic effect in enhancing antitumor immunity.

Conclusions: Targeting HK3 in TAMs represents a promising therapeutic strategy capable of enhancing antitumor immunity and synergizing with immune checkpoint blockade by restoring efficient antigen cross-presentation.