PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells
Ajit Roy1, Arup R Chakraborty1, Tyzoon Nomanbhoy2
1Division of Developmental Biology, National Institute of Child Health & Human Development, National Institutes of Health, Bethesda, MD, USA.
Abstract:
Although PIKFYVE phosphoinositide kinase inhibitors can selectively eliminate PIKFYVE-dependent human cancer cells in vitro and in vivo, the basis for this selectivity has remained elusive. Here we show that the sensitivity of cells to the PIKFYVE inhibitor WX8 is not linked to PIKFYVE expression, macroautophagic/autophagic flux, the BRAFV600E mutation, or ambiguous inhibitor specificity. PIKFYVE dependence results from a deficiency in the PIP5K1C phosphoinositide kinase, an enzyme required for conversion of phosphatidylinositol-4-phosphate (PtdIns4P) into phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2/PIP2), a phosphoinositide associated with lysosome homeostasis, endosome trafficking, and autophagy. PtdIns(4,5)P2 is produced via two independent pathways. One requires PIP5K1C; the other requires PIKFYVE and PIP4K2C to convert PtdIns3P into PtdIns(4,5)P2. In PIKFYVE-dependent cells, low concentrations of WX8 specifically inhibit PIKFYVE in situ, thereby increasing the level of its substrate PtdIns3P while suppressing PtdIns(4,5)P2 synthesis and inhibiting lysosome function and cell proliferation. At higher concentrations, WX8 inhibits both PIKFYVE and PIP4K2C in situ, which amplifies these effects to further disrupt autophagy and induce cell death. WX8 did not alter PtdIns4P levels. Consequently, inhibition of PIP5K1C in WX8-resistant cells transformed them into sensitive cells, and overexpression of PIP5K1C in WX8-sensitive cells increased their resistance to WX8. This discovery suggests that PIKFYVE-dependent cancers could be identified clinically by low levels of PIP5K1C and treated with PIKFYVE inhibitors.Abbreviations: DMSO: dimethylsulfoxide; ELISA: enzyme-linked immunosorbent assay; LC3-I: microtubule associated protein light chain 3-I; LC3-II: microtubule associated protein light chain 3-II; MS: mass spectrometry; PtdIns: phosphatidylinositol; PtdIns3P: PtdIns-3-phosphate; PtdIns4P: PtdIns-4-phosphate; PtdIns5P: PtdIns-5-phosphate; PtdIns(3,5)P2: PtdIns-3,5-bisphosphate; PtdIns(4,5)P2/PIP2: PtdIns-4,5-bisphosphate; PtdIns(3,4,5)P3/PIP3: PtdIns-3,4,5-trisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PI4KA: phosphatidylinositol 4-kinase alpha; PI4KB: phosphatidylinositol 4-kinase beta; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PI4K2B: phosphatidylinositol 4-kinase type 2 beta; PIP4K2A: phosphatidylinositol-5-phosphate 4-kinase type 2 alpha; PIP4K2B: phosphatidylinositol-5-phosphate 4-kinase type 2 beta; PIP4K2C: phosphatidylinositol-5-phosphate 4-kinase type 2 gamma; PIP5K1A: phosphatidylinositol-4-phosphate 5-kinase type 1 alpha; PIP5K1B: phosphatidylinositol-4-phosphate 5-kinase type 1 beta; PIP5K1C: phosphatidylinositol-4-phosphate 5-kinase type 1 gamma; WX8: 1H-indole-3-carbaldehyde (4-anilino-6-[4-morpholinyl]-1,3,5-triazin-2-yl)hydrazone.
Insights
PIKFYVE inhibitors selectively kill cancer cells by targeting a deficiency in PIP5K1C, an enzyme crucial for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) production. This discovery enables identification and treatment of PIKFYVE-dependent cancers.
Area of Science:
- Molecular Biology
- Cancer Therapeutics
- Cell Signaling
Background:
- PIKFYVE phosphoinositide kinase inhibitors show selective cancer cell elimination, but the mechanism remains unclear.
- Previous hypotheses involving PIKFYVE expression, autophagy, or BRAF mutations did not explain this selectivity.
Purpose of the Study:
- To elucidate the molecular basis for cancer cell selectivity by PIKFYVE inhibitors.
- To identify biomarkers for predicting sensitivity to PIKFYVE inhibitors.
Main Methods:
- Investigated cellular sensitivity to the PIKFYVE inhibitor WX8.
- Analyzed phosphoinositide levels, including phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2).
- Assessed the role of PIP5K1C and PIKFYVE in PtdIns(4,5)P2 synthesis pathways.
Main Results:
- PIKFYVE dependence correlates with a deficiency in PIP5K1C, a key enzyme for PtdIns(4,5)P2 synthesis.
- WX8 inhibits PIKFYVE, leading to PtdIns3P accumulation and suppressed PtdIns(4,5)P2 production, impacting lysosome function and proliferation.
- Inhibition of PIP5K1C confers WX8 sensitivity, while PIP5K1C overexpression confers resistance.
Conclusions:
- PIKFYVE-dependent cancer cell selectivity is driven by a deficiency in the PIP5K1C-mediated pathway of PtdIns(4,5)P2 synthesis.
- Low PIP5K1C levels can serve as a clinical biomarker for identifying patients who may benefit from PIKFYVE inhibitors.
- This finding opens new avenues for targeted cancer therapy using PIKFYVE inhibitors.
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