IDDF2021-ABS-0189 Metabolic inhibitor of PFKFB3 and its implication in immune evasion by upregulating pd-l1 expression via the PHOSPHOPFKFB3–HIF1Α–PD-L1 axis

BackgroundPFKFB3 is a crucial metabolic enzyme which is highly upregulated in cancer cells, prompting much research on potential selective inhibitors of PFKFB3 which all have shown promising tumor inhibitive effects. However, clinical trials such as NCT02044861 on PFKFB3 inhibitors seem resultless. With recent implications of cancer metabolism and tumor immune evasion, we hypothesized that PFKFB3 inhibition and subsequent metabolic reprogramming might affect tumor immune evasion.MethodsWe examined the function of PFK-15 on tumor cells in vitro and in vivo in immune-deficient nude-mice models and immune-competent mouse models such as C57/BALB/c-CDX models and huPBMC-CDX and PBX models. CO-IP, fluorescent immunohistochemistry, CHIP and dual-luciferase assays were used to investigate the underlying mechanism.ResultsInhibition of PFKFB3 using PFK-15 suppressed cell growth in human esophageal cell lines in vitro and in vivo in immune-deficient xenografts. However, inhibition of PFKFB3 caused a marked upregulation in PD-L1 which inactivated cocultured T-cells in vitro and compromised anti-tumor immunity in immune-competent mouse, an effect which could be reversed when PFK-15 was combined with PD-1 mab. Mechanistically, we identified ERK pathway upregulation after treatment with PFK-15, which increased PFKFB3 phosphorylation levels, causing its transformation and increased its binding with HIF-1α, then Phospho-PFKFB3 co-translocated into the nucleus together with HIF-1α by binding with importin α5, whereby nuclear HIF-1α attaches itself to HRE regions on PD-L1 promoter, upregulating PD-L1 expression. Clinically, we observed higher Phospho-PFKFB3 in tumor tissues from non-responders to PD-1 mab treated ESCC patients.ConclusionsThis study shows that inhibition of PFKFB3 increases immune evasion via the phospho-PFKFB3–HIF-1α–PD-L1 axis. The translational significance of this study lies in the fact that in-vivo experiments in immune-competent mice using the closest to human immune tumor microenvironment model huPBMC-CDX/PDX combining PFKFB3 inhibitors and PD-1 mab resulted in a marked decrease in tumor development. These findings may be of relevance for the design of anti-cancer treatment trials with PFKFB3 inhibitors.