Abstract
PIKfyve, an evolutionarily conserved kinase synthesizing PtdIns5P and PtdIns(3,5)P2, is crucial for mammalian cell proliferation and viability. Accordingly, PIKfyve inhibitors are now in clinical trials as anti-cancer drugs. Among those, apilimod is the most promising, yet its potency to inhibit PIKfyve and affect endomembrane homeostasis is only partially characterized. We demonstrate here for the first time that apilimod powerfully inhibited in vitro synthesis of PtdIns5P along with that of PtdIns(3,5)P2. HPLC-based resolution of intracellular phosphoinositides (PIs) revealed that apilimod triggered a marked reduction of both lipids in the context of intact cells. Notably, there was also a profound rise in PtdIns3P resulting from arrested PtdIns3P consumption for PtdIns(3,5)P2 synthesis. As typical for PIKfyve inhibition and the concomitant PtdIns(3,5)P2 reduction, apilimod induced the appearance of dilated endomembrane structures in the form of large translucent cytoplasmic vacuoles. Remarkably, bafilomycin A1 (BafA1) fully reversed the aberrant cell phenotype back to normal and completely precluded the appearance of cytoplasmic vacuoles when added prior to apilimod. Inspection of the PI profiles ruled out restoration of the reduced PtdIns(3,5)P2 pool as a molecular mechanism underlying BafA1 rescue. Rather, we found that BafA1 markedly attenuated the PtdIns3P elevation under PIKfyve inhibition. This was accompanied by profoundly decreased endosomal recruitment of fusogenic EEA1. Together, our data demonstrate that apilimod inhibits not only PtdIns(3,5)P2 but also PtdIns5P synthesis and that the cytoplasmic vacuolization triggered by the inhibitor is precluded or reversed by BafA1 through a mechanism associated, in part, with reduction in both PtdIns3P levels and EEA1 membrane recruitment.
Highlights
Seven phosphoinositides (PIs), all regulating critical cellular processes, are produced in mammalian cells [1–5]
Our data demonstrate that apilimod inhibits PtdIns(3,5)P2 and PtdIns5P synthesis and that the cytoplasmic vacuolization triggered by the inhibitor is precluded or reversed by bafilomycin A1 (BafA1) through a mechanism associated, in part, with reduction in both PtdIns3P levels and early endosomal antigen 1 (EEA1) membrane recruitment
Apilimod-dependent inhibition of the PIKfyve-catalyzed PtdIns5P synthesis in vitro has not been tested in the original study characterizing the drug as a PIKfyve inhibitor [22]
Summary
Seven phosphoinositides (PIs), all regulating critical cellular processes, are produced in mammalian cells [1–5] They differ by the number and/or position of the phosphate in the inositol headgroup, and are: phosphatidylinositol (PtdIns) 3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(4,5)P2, PtdIns(3,5)P2 and PtdIns(3,4,5)P3 [1, 2]. The PIKfyve dysfunction achieved by means of pharmacological inhibition, genetic inactivation or dominant-negative interference, is manifested by the appearance of massive and progressively enlarging cytoplasmic vacuoles in proliferating mammalian cells [15, 18–20]. This phenomenon is due to selective reduction in PtdIns(3,5)P2 but not in PtdIns5P, evidenced by complementation experiments with exogenous delivery of either lipid [21]. Such information is important in light of observations for increased cell toxicity and death in several cancer types through cell vacuolization
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