Fig. 7.

The PFKFB inhibitor KAN0438757 regulate metabolic activity in UM cells. The homodimeric 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) are bifunctional enzymes that synthesize and degrade fructose-2,6-bisphosphate in glycolysis. PFKFB2 is the major cytosolic isoform of PFKFB in UM cells. A, expression of PFKFB isoforms in MP41 and MP46 cells based on previous RNAseq analyses (19) and this proteomics analysis. PFKFB1 expression is not detectable (n.d.) in UM cells. PFKFB2 shows the highest expression followed by PFKFB3 and then PFKFB4 has the lowest expression. B, schematic diagram of PFKFBs showing the locations of phosphorylation sites in PFKFB2 (above) and PFKFB3 (below). Boxes show log₂(fold-change) in phosphorylation of each site in response to FR in the indicated cell line. S32 phosphorylation was not detectable in either cell line, and S24 phosphorylation was not detectable in MP46 cells. C, KAN0438757 is a PFKFB inhibitor with inhibitory activities against PFKFB3 (IC₅₀ 0.2 μM) and PFKFB2 (IC₅₀ ∼ 50 μM). Responses of MP41 and MP46 cells to 50 μM KAN0438757 were compared to FR in Agilent Seahorse assays for glycolytic stress. D, dosage responses to KAN0438757 were measured in glycolysis and glycolytic capacity. Both cell lines showed significant changes in glycolysis and glycolytic capacity only at 50 μM KAN0438757. E, responses of MP41 and MP46 cells to 50 μM KAN0438757 were likewise compared to FR in Agilent Seahorse assays for mitochondrial respiration stress. F, dosage responses to KAN0438757 were measured in basal and maximal respiration and demonstrated highly similar responses in both cell lines. UM, uveal melanoma.