Research Article: Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation

Date Published: September 21, 2018

Publisher: Public Library of Science

Author(s): Diego Sbrissa, Ghassan Naisan, Ognian C. Ikonomov, Assia Shisheva, Shulin Ju.


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.

Partial Text

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]. Position-specific kinases and phosphatases govern the PI metabolism and are subject to tight intracellular regulation. The phosphoinositide kinase PIKfyve, an evolutionarily conserved and a sole enzyme in mammals, synthesizes two of the seven PIs, i.e., PtdIns(3,5)P2 and PtdIns5P, by phosphorylating the 5-hydroxyl in inositol headgroups of PtdIns3P and PtdIns, respectively [6–10]. The best characterized cellular functions of PIKfyve are linked to regulating various aspects of both degradative and recycling endosomal trafficking, cytoskeletal rearrangement and autophagy [8, 11–14]. The two PIKfyve products appear to be differentially connected to these processes. Thus, PtdIns5P regulates F-actin remodeling and the non-canonical Vps34-independent autophagy whereas PtdIns(3,5)P2 controls fusion and fission events in the endosomal system thereby maintaining endomembrane homeostasis [13, 15–17]. Phenotypically, 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]. Under these conditions only PtdIns(3,5)P2 is capable of restoring the normal endomembrane morphology. Concordantly, only PIKfyve mutants with disrupted PtdIns(3,5)P2, but with intact PtdIns5P, synthesis trigger aberrant cytoplasmic vacuolation [21]. Mechanistic understanding of the defects eliciting the cell vacuolization is still incomplete. Such information is important in light of observations for increased cell toxicity and death in several cancer types through cell vacuolization.

Apilimod {3-methyl-2-[6-(4-morpholinyl)-2-[2-(2-pyridinyl)ethoxy]-4-pyrimidinyl] hydrazone, benzaldehyde}, obtained from Axon Medchem LLC (USA), and YM201636 {[6-amino-N-(3-(4-(4-morpholinyl)-pyrido[3’,2’:4,5]furo[3,2-d]pyrimidin-2-yl)phenyl)-3-pyridinecarboxamide]}, purchased from Symansis NZ (Timaru, New Zealand), were used as recommended by the manufacturers. BafA1 was purchased from Enzo Life Sciences, Inc., USA. Thin layer chromatography (TLC) 20 x 20 cm glass plates (K6 silica gel 60Å, 250 μm layer thickness) and an HPLC 5-micron Partisphere SAX column were from Whatman. Methylamine (40% w/w solution in water), n-propoanol and tetrabutylammonium bisulfate (TBAS) were from Sigma-Aldrich, USA. Glucose- and inositol-free DMEM was prepared in house in sterile distilled deionized water from amino acids and vitamins purchased from Gibco Laboratories (Life Technologies, Inc., USA) or Sigma (Sigma-Aldrich, USA), and inorganic salts from various commercial sources. [γ-32P]ATP (6000 Ci/mmol) and myo-[2-3H]inositol (22.5 Ci/mmol) were from NEN Du-Pont (Boston, MA) and Perkin Elmer (Boston, MA), respectively. Natural soybean PtdIns was from Avanti Polar Lipids, Inc. (USA). Polyclonal anti-PIKfyve antibody was previously described [39]. Goat polyclonal anti-early endosomal antigen 1 (EEA1) antibodies (N-19) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Data for early embryonic lethality of the systemic pikfyve knockout in mice has unveiled the critical role of PIKfyve in cell proliferation and viability [19, 54, 55]. Consequently, several PIKfyve inhibitors have been found to exhibit anti-proliferative capacity and cytotoxicity, bringing to light a potential new modality in anti-cancer therapy [27, 56]. Among those, apilimod appears to be the most promising and is currently in clinical trials as an anti-cancer drug [28] yet its potential as a PIKfyve inhibitor remains only partially characterized. In this study we took advantage of our ability to unambiguously quantify alterations of the two PIKfyve lipid products in vitro and in a cellular context. We report for the first time that apilimod powerfully inhibits PIKfyve kinase activity not only for PtdIns(3,5)P2 but also for PtdIns5P synthesis. The IC50 for both products was estimated to be in the subnanomolar range (Fig 1). Furthermore, both PtdIns(3,5)P2 and PtdIns5P lipid products were markedly and similarly reduced in different cell lines treated with 100 nM apilimod (Figs 2 and 3). These new data provide clarification of the apilimod inhibitory potency and might further enlighten the molecular basis of its usage in cancer therapy.

Our study characterizes for the first time that apilimod inhibits not only PIKfyve- catalyzed PtdIns(3,5)P2 but also PtdIns5P production and reveals that the BafA1 rescue of the endomembrane vacuolation triggered by the drug is mechanistically associated with attenuated PtdIns3P elevation and suppressed recruitment of fusogenic EEA1. In addition to basic impact, the data in this study have also clinical relevance. For example, if apilimod is approved as a drug in cancer treatment, cautions should be taken in combined therapy with medications that affect PtdIns3P levels as such conditions might impair apilimod cytotoxicity.




0 0 vote
Article Rating
Notify of
Inline Feedbacks
View all comments