Natural killer (NK) cells are pivotal in the innate immune response against cancer and viral infections, with their presence in tumors correlating to better patient outcomes in various cancers. However, NK cells in the tumor microenvironment often become functionally exhausted, characterized by decreased numbers and impaired functions due to imbalances in immune regulatory signals. This exhaustion is influenced by immune checkpoint receptors, which are inhibitory cell surface receptors that can suppress antitumor immunity. Notably, intracellular checkpoint molecules within NK cells, such as FBP-1, EZH2, CIS, TIPE2, and HIF-1α, play a significant role in NK cell exhaustion by affecting their metabolism, proliferation, survival, and cytotoxic activity. These molecules offer a universal target for cancer immunotherapy, as their impact on antitumor immunity is consistent across different contexts.

The intricate relationship between NK cells and cancer is further complicated by the expression of HLA class I molecules, which interact with inhibitory KIRs on NK cells to regulate their activity. The polymorphic nature of KIRs and their ligands adds a layer of complexity to NK cell interactions with tumor cells. Moreover, other inhibitory receptors like CD94/NKG2A, PD-1, TIGIT, and TIM-3, which are often expressed in tumor tissues, serve as context detectors, influencing the antitumor immunity based on the presence and levels of their ligands.

Intracellular checkpoint molecules like BIM, which mediates apoptosis in NK cells, can be targeted to enhance antitumor responses. The absence of BIM in NK cells has been shown to increase their resistance to apoptosis and accumulate in later stages of maturation, potentially enhancing their antitumor capabilities. Similarly, Cbl-b, an E3 ubiquitin protein ligase, negatively regulates NK cell cytolytic activity, and its inhibition can improve NK cell-mediated control of tumor metastasis.

CIS, a negative regulator of IL-15 signaling, is upregulated by IL-15 in NK cells, and its absence enhances NK cell sensitivity to IL-15, proliferation, survival, and cytotoxicity against tumor cells. EZH2, a component of the polycomb repressive complex 2, acts as a negative regulator of NK cell effector functions, and its targeting may promote NK cell immunotherapy. FBP1, a gluconeogenesis enzyme, suppresses glycolysis in NK cells, and its inhibition can recover NK cell activity, suggesting a role in NK cell exhaustion.

TIPE2, a negative regulator of IL-15 signaling, is upregulated in various conditions and suppresses downstream AKT and Ral activation. The absence of TIPE2 in NK cells results in enhanced functional maturation, cytotoxicity, and IFN-γ production, indicating its potential as a checkpoint for NK cell immunotherapy. HIF-1α, a transcription factor involved in cellular responses to hypoxia, negatively regulates IL-18-driven NF-κB signaling and antitumor activity of tumor-infiltrating NK cells. Inhibition of HIF-1α in NK cells may improve treatment of solid tumors.

The therapeutic potential of targeting intracellular checkpoint molecules in NK cells is more universal than targeting checkpoint receptors on the cell surface, as the latter's efficacy is often tumor-specific and dependent on ligand interactions. Targeting intracellular checkpoints could be combined with other strategies, such as tumor sensors, immune checkpoint blockade, and synthetic gene circuits, to enhance NK cell immunotherapy. Emerging intracellular checkpoint molecules offer new targets for improving NK cell antitumor activity, and understanding their mechanisms of action is crucial for identifying targetable checkpoints.

Strategies for targeting intracellular checkpoint molecules include CRISPR/Cas9, shRNA, and small-molecule inhibitors, each with its challenges and limitations. The development of technologies for NK cell genetic editing and the improvement of viral transfection platforms are essential for stable gene overexpression in NK cells. The discovery and targeting of these intracellular checkpoints hold promise for enhancing the efficacy of NK cell immunotherapy in cancer treatment.
DOI: 10.1007/s11684-024-1090-6