Abstract
The Hippo pathway plays a key role in development, organ size control and tissue homeostasis, and its dysregulation contributes to cancer. The LATS tumor suppressor kinases phosphorylate and inhibit the YAP/TAZ transcriptional co-activators to suppress gene expression and cell growth. Through a screen of marine natural products, we identified microcolin B (MCB) as a Hippo activator that preferentially kills YAP-dependent cancer cells. Structure–activity optimization yielded more potent MCB analogs, which led to the identification of phosphatidylinositol transfer proteins α and β (PITPα/β) as the direct molecular targets. We established a critical role of PITPα/β in regulating LATS and YAP. Moreover, we showed that PITPα/β influence the Hippo pathway via plasma membrane phosphatidylinositol-4-phosphate. This study uncovers a previously unrecognized role of PITPα/β in Hippo pathway regulation and as potential cancer therapeutic targets.
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Data availability
The RNA-seq data are available at the Gene Expression Omnibus (GEO) database with accession ‘GSE198890’. The PITPα receptor model was prepared from available structures of human PITPα (PDB ID: 1UW5). All the other data are available within the article and its Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank E. Fatti and F.-X. Yu for assistance in the initial chemical library screen. This work was supported by National Institutes of Health (NIH) grants GM51586, CA217642 and CA268179 to K.-L.G. J.M.F. is supported by NIH grant T32CA009523.
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F.-L.L., V.F., G.L. and K.-L.G. conceived the study. F.-L.L., V.F., G.L., J.E.D. and K.-L.G. conducted experiments and data analyses. T.T., A.K. and X.P. performed the compound optimization. W.H.G. provided the marine compound library and advice. J.M.F. assisted in cell imaging experiments. Z.W. assisted in RNA-seq experiments and gene set enrichment analysis. E.K. and B.F.C. assisted in mass spectrometry experiments. J.M. performed the docking simulation. V.F., F.-L.L. and K.-L.G. wrote the manuscript with input from all other authors.
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K.-L.G. is a cofounder of and has equity interest in Vivace Therapeutics. T.T., A.K. and X.P. are Vivace Therapeutics employees. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 MCB induce YAP inactivation, phosphorylation and cytoplasmic localization.
a, MCB inhibits the YAP transcription reporter. HEK293A clone, which has the 8X TBD-luciferase reporter stably integrated in the genome, was treated with increasing concentration of MCB. Luciferase activity was determined to measure YAP transcriptional activity. n = 4 biologically independent samples. b, MCB induces YAP phosphorylation in a dose-dependent manner. HEK293A cells were treated with indicated concentration of MCB for 2 hours. Western blot was performed with indicated antibodies. c, MCB induce YAP phosphorylation in a time-dependent manner. HEK293A cells were treated with MCB (270 nM) for the indicated times. Cell lysates were analyzed by Western blot with indicated antibodies. d, MCB induces YAP phosphorylation in 92.1 cells. Cells were treated with 0.7 μM MCB for the indicated times. e, MCB induces YAP cytoplasmic translocation. HEK293A cells were treated with 270 nM MCB for 2 hours and subjected to immunofluorescence for YAP localization. Scale bar = 10 μm. The right panel shows quantification of a representative experiment.
Extended Data Fig. 2 MCB and derivatives induce YAP phosphorylation and cytoplasmic localization through the Hippo pathway.
a, VT01454 induces YAP phosphorylation in a dose- or time- dependent manner. HEK293A cells were treated with indicated concentration of VT01454 for 1 hour or 50 nM VT01454 for indicated time. b, Structures of MCB and derivatives. c, VT01454 inhibits the expression of YAP target genes CTGF, ANKRD, and Cyr61. RT-PCR experiment was performed with HEK293A cells treated with indicated concentrations of VT01454 for 2 hours. Results are mean ± SD of three biologically independent samples. d, MCB induces the phosphorylation of YAP, but not the YAP-5SA mutant. HEK293A cells were transfected with Flag-YAP or YAP-5SA, which has all LATS phosphorylation sites mutated to alanine residues. Cells were treated with MCB and then analyzed by Western Blot on phos-tag gel. e, LATS1/2 are required for MCB to induce YAP phosphorylation. HEK293A WT, LATS1 KO, LATS2 KO, and LATS1/2 dKO cells were treated with 270 nM MCB for 2 hours and Western blotted with indicated antibodies. f, LATS1/2 double knockout blocks the effect of MCB on YAP localization. Scale bar = 10 μm. Quantification of YAP subcellular localization is shown in the right. g, VT01454 has little effect on the expression of key Hippo pathway components. Heatmap analysis of the RNA-seq data in Fig. 1d. h, VT01454 does not regulate NF2 plasma membrane localization. Flag-tagged NF2 construct was transfected into HEK293A cells. After 24 hours, cells were treated with indicated concentrations of VT01454 for 2 hours before subject to immunofluorescence staining. Scale bar = 10 μm. i, VT01454 does not affect LATS-NF2 interaction. Flag-tagged NF2 and HA-tagged LATS1 was co-transfected into HEK293A cells. After 24 hours, cells were treated with indicated concentrations of VT01454 for 2 hours before subjecting to immunoprecipitation with Flag antibody.
Extended Data Fig. 3 MCB and VT01454 preferentially kills YAP dependent cancer cells.
a and b, MCB induces cell death in the GNAQ/11 mutant 92.1 and OMM1, but not the B-Raf mutant OCM1 and OCM8 cells where VP displays general toxicity. The concentrations of MCB and VP were 2.7μM and 2 μM, respectively. Scale bar = 50 μm. Results are mean ± SD of three biologically independent samples. c, 92.1 cells are more sensitive than OCM1 cells to MCB-induced apoptosis. The MCB concentrations were 2.7 and 5.4 μM. d, MCB is more selective than verteporfin to induce PARP cleavage. Concentration gradients (0, 0.27, 0.68, 1.36, 2.72, 6.8 μM) of MCB or verteporfin (0, 0.28, 0.70, 1.39, 2.78, 6.9 μM) were used to treat cells. PARP cleavage was determined by Western blot. e, MCB (2.7 μM) preferentially inhibits colony formation in 92.1 cells over OCM1 cells. Both images and quantification of colonies in soft agar assays are shown. Results are mean ± SD of three biologically independent samples. f, VT01454 does not induce YAP-5SA phosphorylation. The stable 92.1 cell pools were generated by infection with lentivirus expressing YAP-5SA or empty-vector. These cells were treated with 20 nM or 50 nM of VT01454 for 1 hours before subjecting to western blot with indicated antibodies. g, VT01454 sensitivity in multiple cancer cell lines. 1.5 × 105 cells were seeded and treated with VT01454 (20 nM) or DMSO for 48 hours. Scale bar = 100 μm. Representative images were taken by a phase-contrast microscope. h, Quantification of live cell numbers (left panel) was performed by cell counting after trypan blue staining. The right panel was the normalized result of the left panel by setting the DMSO control as 1. Experiments were similar to panel g. Results were mean ± SD of three biologically independent samples.
Extended Data Fig. 4 VT01454 covalently modifies Cys94 in PITPβ.
a, Chemical structure of VT01702. b, VT01702 induces YAP phosphorylation and inhibits YAP reporter activity. c, Structure of the trifunctional 545-biotin-azide compound used to conjugate VT01702. d, Optimization of purification conditions for the VT01702 modified protein by streptavidin beads. e. VT01454 covalently modifies Cys94 in PITPβ. HA-tagged wild-type or indicated mutants of PITPβ were transfected into HEK293A cells. After 24 hours, cells were treated with 50 nM VT01454 for 1 hour before being subjected to western blot with indicated antibodies.
Extended Data Fig. 5 PITPα/β knockout induces YAP phosphorylation independent of cell types.
a, Genomic characterization of the PITPβ knockout cell clone #1.12. The HEK293A cell line has three alleles of PITPβ. DNA sequence confirms mutations of the three alleles. b, The PITPα siRNA knockdown efficiency was measured by Quantitative Real-time PCR. Results are mean ± SD of three technically replicates. c, PITPα/β knockout induces YAP phosphorylation independent of cell types. These knockout cell pools were generated by co-infecting lentiviruses stably expressing Cas9 and guide RNA targeting PITPNA and PITPNB. Cells were lysed and subjected to Western Blot with indicated antibodies. d, PITPα/β knockout blocks LPA-induced YAP de-phosphorylation. HEK293A wild-type and PITPα/β dKO cell pool were treated with Latrunculin B or LPA and then analyzed by Western blot with indicated antibodies.
Extended Data Fig. 6 VT01454 preferentially depletes plasma membrane PI4P.
a, Volume (combined) view of the Z-section confocal images in Fig. 5a. Scale bar = 10 μm. b, Zoomed out view of area with multiple cells to indicate that the single cell images in Fig. 5a are representative. Scale bar = 10μm. c, VT01454 treatment preferentially depletes plasma membrane PI4P. Scale bar = 10 μm. d, VT01454 has little effect on PI3P. HEK293A cells were transfected with RFP-p40px, which is a reporter for PI3P, then treated with VT01454. Scale bar = 10 μm. e, Expression of Sac1-GRIP does not induce YAP cytoplasmic translocation. Scale bar = 20 μm.
Extended Data Fig. 7 PI4KA overexpression dampens the effect of VT01454.
a, Inhibition of PI4KA increases YAP cytoplasmic localization. HEK293A cells were treated with PI4KA inhibitor GSK-A1 or PI4KB inhibitor PI4KIIIb-IN-10 prior to YAP staining. Scale bar = 20 μm. Quantification is shown on the right. b, GSK-A1 stimulates phosphorylation of LATS and YAP. c, PI4KA overexpression dampens the effect of VT01454 on YAP phosphorylation. Flag-YAP was co-expressed with HA-PI4KA or vector in HEK293A cells. After 24 hours, these cells were treated with DMSO or VT01454 for 1 hour, then Western blotted with the indicated antibodies.
Extended Data Fig. 8 NF2 is required for the phosphorylation of LATS1 and YAP induced by VT01454.
a, Characterization of FERM protein (Protein 4.1/ Ezrin/ Radixin/ Moesin). knockout efficiency in HEK293A. The knockout cell pool was generated with individual or combinatory lentivirus stably expressing Cas9 and specific guide RNAs. These cells were selected with puromycin for 48 hours before subjecting to western blot with indicated antibodies. b, Knockout of Protein 4.1, Ezrin, Radixin, and Moesin did not affect YAP phosphorylation. The cells as described in panel a were treated with DMSO or 20 nM VT01454 for 1 hour before western blot with indicated antibodies. c, NF2 is required for the phosphorylation of LATS1 and YAP induced by VT01454. The wild-type or NF2 knockout cells were treated with 20 nM VT01454 for indicated times before subjecting to western blot with indicated antibodies.
Supplementary information
Supplementary Information
Supplementary Fig. 1 and Notes 1 and 2.
Supplementary Data
Supplementary Dataset 1. Information of the marine compounds library; Supplementary Dataset 2. List of proteins enriched by VT01702 over DMSO; Supplementary Dataset 3. Proteins list of excised gel identified by MS.
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Li, FL., Fu, V., Liu, G. et al. Hippo pathway regulation by phosphatidylinositol transfer protein and phosphoinositides. Nat Chem Biol 18, 1076–1086 (2022). https://doi.org/10.1038/s41589-022-01061-z
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DOI: https://doi.org/10.1038/s41589-022-01061-z
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