Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

Choy, Christopher H.; Saffi, Golam T.; Gray, Matthew A.; Wallace, Callen T.; Dayam, Roya M.; Ou, Zhen-Yi Andy; Lenk, Guy M.; Puertollano, Rosa; Watkins, Simon C.; Botelho, Roberto J.

doi:10.1242/jcs.213587

Cited by 122 publications

(188 citation statements)

References 82 publications

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“…These results confirmed that loss of PIKfyve activity, and consequent diminished PI(3,5)P2 synthesis, caused accumulation of endosomes and inhibition of lysosomal formation in osteoclasts. Our results are consistent with previous reports in various cells where, if PIKfyve is inhibited, membrane recycling is disrupted and vesicles become massively enlarged, cells have impaired degradative capacity, ion dysregulation, and abated autophagic flux resulting in different physiological defects and overt inflammation . Upon PIKfyve inhibition, fewer but enlarged endo/lysosomes were formed, suggesting endo/lysosome fusion was being favored over fission and that this imbalance resulted in endo/lysosome enlargement …”

Section: Discussionsupporting

confidence: 92%

“…PIKfyve has multiple roles, for example, in the conversion of early endo/phagosomes into mature lysosomes, lysosomal acidification, trafficking to late endosomes/lysosomes, and interleukin‐12 secretion in monocyte‐derived dendritic cells. In addition, this kinase plays a key role in T‐cell activation and in metastasis, and it has emerged as a potential target for treating solid tumors because it promotes cancer cell migration and invasion…”

Section: Introductionmentioning

confidence: 99%

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Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Sultana

Morse

Picotto

et al. 2019

J of Cellular Biochemistry

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Bone resorption and organelle homeostasis in osteoclasts require specialized intracellular trafficking. Sorting nexin 10 (Snx10) is a member of the sorting nexin family of proteins that plays crucial roles in cargo sorting in the endosomal pathway by its binding to phosphoinositide(3)phosphate (PI3P) localized in early endosomes. We and others have shown previously that the gene encoding sorting Snx10 is required for osteoclast morphogenesis and function, as osteoclasts from humans and mice lacking functional Snx10 are dysfunctional. To better understand the role and mechanisms by which Snx10 regulates vesicular transport, the aim of the present work was to study PIKfyve, another PI3P‐binding protein, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is known to be required for endosome/lysosome maturation, and the inhibition of PIKfyve causes endosome enlargement. Overexpression of Snx10 also induces accumulation of early endosomes suggesting that both Snx10 and PIKfyve are required for normal endosome/lysosome transition. Apilimod is a small molecule with specific, nanomolar inhibitory activity on PIKfyve but only in the presence of key osteoclast factors CLCN7, OSTM1, and Snx10. This observation suggests that apilimod's inhibitory effects are mediated by endosome/lysosome disruption. Here we show that both Snx10 and PIKfyve colocalize to early endosomes in osteoclasts and coimmunoprecipitate in vesicle fractions. Treatment with 10 nM apilimod or genetic deletion of PIKfyve in cells resulted in the accumulation of early endosomes, and in the inhibition of osteoclast differentiation, lysosome formation, and secretion of TRAP from differentiated osteoclasts. Snx10 and PIKfyve also colocalized in gastric zymogenic cells, another cell type impacted by Snx10 mutations. Apilimod‐specific inhibition of PIKfyve required Snx10 expression, as it did not inhibit lysosome biogenesis in Snx10‐deficient osteoclasts. These findings suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2 and may point to a new strategy to prevent bone loss.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Sultana

Morse

Picotto

et al. 2019

J of Cellular Biochemistry

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“…As shown in Figure E,F, the lysosomes were significantly enlarged in PIPKIγi5 knockdown cells compared with those in control cells. Enlarged lysosomes always indicate impaired lysosomal degradative capacity and lysosome dysfunction . This is consistent with the reduction in hydrolase sorting to lysosomes (Figure C).…”

Section: Resultssupporting

confidence: 80%

Control of Rab7a activity and localization through endosomal type Igamma PIP 5‐kinase is required for endosome maturation and lysosome function

et al. 2019

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The small GTPase Ras‐related protein Rab‐7a (Rab7a) serves as a key organizer of the endosomal‐lysosomal system. However, molecular mechanisms controlling Rab7a activation levels and subcellular translocation are still poorly defined. Here, we demonstrate that type Igamma phosphatidylinositol phosphate 5‐kinase i5 (PIPKIγi5), an endosome‐localized enzyme that produces phosphatidylinositol 4,5‐bisphosphate, directly interacts with Rab7a and plays critical roles in the control of the endosomal‐lysosomal system. The loss of PIPKIγi5 blocks Rab7a recruitment to early endosomes, which prevents the maturation of early to late endosomes. PIPKIγi5 loss disturbs retromer complex connection with Rab7a, which blocks the retrograde sorting of Cation‐independent Mannose 6‐Phosphate Receptor from late endosomes. This leads to the decreased sorting of hydrolases to lysosomes and reduces the autophagic degradation. By modulating the retromer‐Rab7a connection, PIPKIγi5 is also required for the recruitment of the GTPase‐activating protein TBC1 domain family member 5 to late endosomes, which controls the conversion of Rab7a from the active state to the inactive state. Thus, PIPKIγi5 is critical for the modulation of Rab7a activity, localization, and function in the endosomal‐lysosomal system.

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“…Because of THZ-P1-2's high potency on PIKfyve in the biochemical context, we took a step further to investigate its cellular activity on PIKfyve in a vacuolar enlargement assay. THZ-P1-2 produced the established PIKfyve-inhibitory phenotype in Vero cells only at a high concentration of 10 M, 1000 x that of positive control apilimod (Cai et al, 2013;Choy & Saffi et al, 2018;Sharma & Guardia et al, 2018) which causes substantial vacuolar enlargement at 10 nM ( Fig. S3D), but failed to show inhibition of PIKfyve at 1 M.…”

Section: Kinase Selectivity and Cellular Target Engagement By Thz-p1-2mentioning

confidence: 94%

Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

Sivakumaren

Shim

Zhang

et al. 2019

Preprint

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The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4K//. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

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Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

Cited by 122 publications

References 82 publications

Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Control of Rab7a activity and localization through endosomal type Igamma PIP 5‐kinase is required for endosome maturation and lysosome function

Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

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