Inositol hexakisphosphate kinase 1 (IP6K1)-driven tumor polyphosphate may contribute to immune checkpoint inhibitor-associated thrombosis Free

Background: Cancer-associated thrombosis (CAT) is driven by multiple procoagulant mechanisms. Immune checkpoint inhibitors (ICI), while enhancing antitumor immunity, are linked to an increased risk of thrombosis (ICI-associated thrombosis, IAT). In a mouse model of colorectal cancer, we have demonstrated that tumor-derived tissue factor is a key driver of IAT. We also observed elevated plasma levels of cleaved high molecular weight kininogen (cHK) in ICI-treated mice, suggesting that contact activation may also contribute to IAT, as we observed in previous studies of CAT not attributable to ICI. In these studies, cleavage of HK was mediated by polyphosphate (polyP) expressed on extracellular vesicles (EV). PolyP can serve as a negatively charged surface to trigger activation of FXII. However, there is little information available on the pathways of polyP synthesis by tumor cells.

Objective: To further characterize mechanisms of contact system activation in IAT, we examined whether ICI treatment promotes polyP synthesis and release through increased expression of inositol hexakisphosphate kinase 1 (IP6K1), a known regulator of intracellular polyP levels, in tumor cells.

Methods: We utilized a CT26 murine colorectal tumor model of IAT induced by inferior vena cava ligation. Contact activation was assessed by measuring cHK levels in mice (n=7/group, ICI vs IgG) and in paired plasma samples from cancer patients pre- and post-ICI therapy (n=5) using semi-quantitative LI-COR immunoblotting. To assess polyP synthesis, we examined IP6K1 protein expression in tumors from ICI-treated mice and CT26 cells treated with TNF-α by LI-COR immunoblotting. The functional role of IP6K1 in polyP production was tested using N2-(m-(trifluoromethyl)benzyl), N6-(p-nitrobenzyl)purine (TNP), an IP6K1 inhibitor, in the presence of TNF-α. Intracellular and EV-associated polyP were isolated via phenol/chloroform extraction and size exclusion chromatography, respectively, and quantified using JC-D7 dye. T cell involvement was tested via CD4⁺/CD8⁺ T cell depletion prior to ICI treatment. Cytokines (measured by MSD U-PLEX), along with cHK and IP6K1 expression, were assessed in T cell depleted mice.

Results: In mice treated with ICI, plasma levels of cHK were significantly higher than in mice treated with isotype control IgG (12.73% vs. 8.79%, p=0.0175). Similarly, in cancer patients receiving anti-PD-1 antibodies (pembrolizumab or nivolumab), cHK levels increased after treatment (p=0.0412). Tumors from these ICI-treated mice also showed an increased expression of IP6K1. When CT26 cells were treated with TNF-α, IP6K1 expression was increased in a dose-dependent manner. Blocking IP6K1 with the pharmacological inhibitor TNP reduced polyP in the cells, suggesting a role for IP6K1 in polyP synthesis. Increased IP6K1 and intracellular polyP suggest that ICI may also enhance polyP release via EV, potentially activating the contact system. Mice depleted of T cells before ICI treatment had lower levels of TNF-α and other inflammatory cytokines (IFN-γ, BLC, MIP-1β, and RANTES) in tumors, as well as reduced levels of IFN-γ, IL-6, MIP-2, and SDF-1α in plasma. In the T cell-depleted mice, cHK levels, which were elevated by ICI treatment (27.9%), were reduced to control levels (19.6%), and the mice developed less thrombosis.

Conclusion: ICI treatment upregulates IP6K1 in tumors, leading to polyP production in a T cell dependent manner. Tumor cell-derived polyP may contribute to contact system activation in IAT, enhancing thromboinflammation in ICI-treated patients. Targeting the IP6K1-polyP axis may offer new strategies to reduce thrombotic risk during cancer immunotherapy.