Cancer Biology

Targeting the Hippo pathway in cancers via ubiquitination dependent TEAD degradation

Departments of Discovery Oncology, Genentech, Inc., South San Francisco, USA
Discovery Chemistry, Genentech, Inc., South San Francisco, USA
Structural Biology, Genentech, Inc., South San Francisco, USA
Microchemistry, Proteomic & Genomic Technologies, Genentech, Inc., South San Francisco, USA
Bioinformatics, Genentech, Inc., South San Francisco, USA
Immunology and Regenerative Medicine, Genentech, Inc., South San Francisco, CA 94080, USA
Cheminformatics, Genentech, Inc., South San Francisco, USA
Biochemical and Cell Biology, Genentech, Inc., South San Francisco, USA
Translational Oncology, Genentech, Inc., South San Francisco, USA
Oregon Health and Science University, Portland, USA
Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, USA

https://doi.org/10.7554/eLife.92450.2

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Figures and data

TEAD transcription factors are ubiquitinated and targeted for proteasome-dependent degradation.
(A) TEADs are ubiquitinated. HEK293 cells were transfected with a control vector, TEAD1-Myc, TEAD2-Myc, TEAD3-Myc, or TEAD4-Myc and V5-ubiquitin plasmids for 48 hours, followed by immunoprecipitation with an anti-V5 antibody. TEAD expression and ubiquitinated TEAD were detected with anti-Myc antibody (n=3). (B) MG132 induces accumulation in TEAD ubiquitination. HEK293 cells were transfected with TEAD4-Myc and V5-ubiquitin plasmids for 48 hours, followed by immunoprecipitation with anti-V5 antibody after an additional treatment with 10 µM MG132. TEAD4 expression and ubiquitinated TEAD4 were detected with anti-Myc antibody (n=3). (C-E) Ubiquitination sites on TEAD4 were mapped using KGG motif immunoprecipitation, followed by LC-MS/MS. C, experimental schematic: TEAD4-Myc was overexpressed in HEK293 cells for 48 hours, MG132 was added 6 hours prior to cell lysis where indicated. KGG motif immunoprecipitation was then performed, followed by LC-MS/MS. D, each ubiquitination site was represented by * and summarized in the table. E, ubiquitination for TEAD4 across conditions. Red line represents the aggregate of individually quantified K-GG peptides (black lines) using a linear mixed-effect model. Each quantification event represents the non-redundant, absolute peak area (converted to log2) under a given condition for a confidently assigned K-GG peptide spectral match.

RNF146 is a putative ubiquitin ligase for TEAD.
(A) RNF146 was identified from an E3 ligase screen using siRNA libraries targeting 1088 ubiquitin-related genes. All seven siRNAs of RNF146 were identified from the screen. The x-axis represents the calculated robust Z-score and the y-axis represents the number of siRNAs per bin. (B) RNF146 is a negative regulator of the Hippo pathway. DepMap dependency network of YAP/TAZ. Nodes are significantly correlated genes, significantly correlated genes are connected by a line. Colors of nodes denote 2 distinct clusters based on a random walk algorithm. (C) The RNF146 Chronos score is negatively correlated with the Chronos score of WWTR1 and positively correlated with the Chronos scores of NF2. (D) TEAD ubiquitination is reduced upon knockdown of RNF146. After 24 hours of siRNF146 treatment, TEAD2-Myc or TEAD4-Myc and V5-ubiquitin were transfected into HEK293 cells for an additional 48 hours, and a ubiquitination assay was performed as described in Figure 1. TEAD expression and ubiquitinated TEAD were detected with anti-Myc antibody. RNF146 knockdown was confirmed via Taqman RT-qPCR (n=3). (E) RNF146 negatively regulates the Hippo signature. OVCAR-8 cells were transfected with siNTC, siRNF146, or siYAP1 as indicated for 48 hours. RNA-seq was performed, and the Hippo signature score was calculated. (F) TEAD interacts with RNF146. HEK293 cells were transfected with TEAD4-Myc for 48 hours. TEAD4 pull-down was then performed with an antibody that recognizes the Myc-tag. TEAD4 expression and RNF146 interaction were detected using Myc antibody and RNF146 antibody, respectively.

The ubiquitination of TEADs is dependent on their PARylation state.
(A) TEAD4 interacts with PARP1. HEK293 cells were transfected with TEAD4-Myc plasmid for 48 hours, followed by immunoprecipitation with anti- Myc antibody. PARP1 expression was detected with an anti-PARP1 antibody (n=3). (B) TEAD2 and TEAD4 are PARylated. HEK293 cells were transfected with duo-tagged TEAD2-Myc-Flag or TEAD4-Myc-Flag and V5-ubiquitin plasmids for 48 hours, followed by immunoprecipitation with anti-Flag antibody. TEAD expression and PARylation were detected with anti-Myc and anti-PAR antibodies, respectively (n=3). (C) TEAD4 ubiquitination is significantly reduced upon knockdown of PARP1. HEK293 cells were transfected with specific guide RNAs to knock down PARP1. The control and PARP1 knockdown cell lines were transfected with TEAD4-Myc and V5-ubiquitin plasmids for 48 hours, followed by immunoprecipitation with anti-V5 antibody. TEAD4 expression and ubiquitinated TEAD4 were detected with anti-Myc antibody (n=3). (D) PARylation of TEAD is located in the DNA binding domain (Asp70 site on TEAD4). Structure of TEAD4 DNA binding domain in complex with DNA (Shi et al., 2017) with labeled sites for PARylation (green). (E) TEAD4(D70A) mutation affects PARylation. HEK293 cells were transfected with duo-tagged TEAD2-Myc-Flag, TEAD4-Myc-FLAG or TEAD4(D70A)-Myc-FLAG and V5-ubiquitin plasmids for 48 hours, followed by immunoprecipitation with anti-Flag antibody. TEAD expression and PARylation were detected with anti-Myc and anti-PAR antibodies, respectively (n=2). (F) The ubiquitination of TEAD is significantly reduced in the absence of PARylation. HEK293 cells were transfected with TEAD4-Myc or TEAD4(D70A)-Myc and V5-ubiquitin plasmids for 48 hours, followed by immunoprecipitation with an anti-V5 antibody. TEAD4 expression and ubiquitinated TEAD4 were detected with anti-Myc antibody (n=3).

Overexpression of RNF146 E3 ligase antagonizes the overgrowth phenotype caused by the Hpo mutant.
(A) PARylation site for TEAD is conserved in humans and Drosophila melanogaster, as shown in the alignment of TEAD1/2/3/4 and Sd protein sequences. (B, C) Overexpression of RNF146 can rescue the overgrowth phenotype caused by Hpo RNAi. B, the first group shows the flies with a control RNAi (ey:: Gal4>UAS:: luciferase RNAi) (n=7). The second group shows knockdown of Hpo (w1118; ey::Gal4/+: UAS::hpoRNAi/+) resulted in a significantly larger eye (n=10). The third group shows flies over expressing RNF146 (ey::Gal4>UAS::RNF146) (n=9). The fourth group, overexpression of RNF146 in a hyperactive Yki background (Hpo RNAi) rescues the overgrowth phenotype resulting in a significantly smaller eye (n=13). C is the quantification of B. Means +/- S.D. of eye area quantified per genotype are shown in arbitrary units (A.U.). One-way ANOVA with Sidak’s multiple comparisons. Numbers (n) of eye area quantified and P-values are indicated. (D, E) Overexpression of RNF146 can rescue the overgrowth phenotype caused by a Hpo loss of function mutation. D, Wild-type (wt) control clones were generated using the eyFLP system (genotype: w1118; neoFRT42D, +/neoFRT42D, GMRHid; ey::Gal4, UAS::FLP/+) (Newsome et al., 2000) (n=11, both eyes). hpo^KC202 mutant clones (genotype: w¹¹¹⁸; FRT42D, hpoKC202/FRT42D, GMRHid; ey::Gal4,UAS::FLP/+) result in a large, folded eye with cuticle overgrowth around the eye. This increase in eye area was significant compared to the eyes of WT clones. (p<0.0001; n=9, both eyes). Clones overexpressing RNF146 showed no phenotype compared to control (genotype: w¹¹¹⁸; FRT42D, + /FRT42D, GMRHid; eyGal4, UAS::FLP/UAS::RNF146^OE; n=11, both eyes). Combining hpo^KC202 and RNF146 overexpression (genotype: w¹¹¹⁸; FRT42D, hpoKC202/FRT42D, GMRHid; ey::Gal4,UAS::FLP/ UAS::RNF146^OE) resulted in a significantly smaller eye as compared to the hpo^KC202 mutant only. (p=0.0027; n=6, both eyes). E is the quantification of the eye area of all eyes in D. Means +/- S.D. of eye area quantified per genotype are shown in arbitrary units (A.U.). One-way ANOVA with Sidak’s multiple comparisons. Numbers (n) of eye areas quantified and P-values are indicated.

TEAD knockdown induces robust Hippo pathway suppression.
(A) Cell viability was assessed by Cell Titer-Glo upon siRNA treatment of TEAD1-4 in Hippo/TEAD sensitive (NF2 null/YAP1 amplified) and insensitive cell lines (no YAP1/WWTR1). Cells were treated with siNTC (dark gray bars), siTox (light gray bars) and siTEAD1-4 (red bars) for 72 hours before the cell viability assay. (B) In vivo xenograft growth (measured as tumor volume over time) of MDA-MB-231 cells expressing TEAD1-4 shRNA in the presence of sucrose (red) or doxycycline (green), or expressing shNon-Targeting Control (NTC) in the presence of sucrose (black) or doxycycline (blue). (C) Chemical structures, biochemical and cellular activities of all CIDEs screened. (D) Crystal structure of Compound A bound to TEAD. (E) Protein abundance measurements were made using tandem mass tag quantitative mass spectrometry and plotted as a log fold change. The scatterplot depicts the change in relative protein abundance in MDA-MB-231 cells treated with Compound D (x-axis) and Compound E (y-axis) relative to Compound A. Cells were treated with 0.5 µM of the compounds for 8 hours before harvesting.

CIDE-mediated TEAD degradation is dependent on CRBN and the ubiquitin-proteasome pathway.
(A) Immunoblot for pan-TEAD, PDE68, RNF166, YAP, TAZ, and β-Actin in MDA-MB-231 cells after 24 hours of Compound D treatment at the indicated concentrations (0.1 µM, 0.8 µM, 5 µM). (B) Immunoblot for pan-TEAD, YAP, TAZ, and β-Actin in OVCAR-8 cells after 16 hours of treatment with Compound A/Compound E/Compound D at the indicated concentrations of 0.1 μM, 1 µM and 10 µM. (C) Immunoblot for pan-TEAD, YAP, TAZ, and β-Actin in OVCAR-8 cells upon co-treatment with DMSO/Compound E /Compound D (1 µM) for 5 hours and MG132 (10 µM) for 6 hours. (D) Immunoblot for pan-TEAD, YAP, TAZ, and β-Actin in MDA-MB-231 cells upon co-treatment with DMSO/Compound D/Compound E (1 µM) for 5 hours and MLN-7243 (1 µM) for 6 hours. (E) Immunoblot for pan-TEAD, CRBN, YAP, TAZ, and β-Actin in MDA-MB-231 cells upon treatment with DMSO/Compound E/Compound D (0.1 µM and 0.5 µM) for 16 hours. siRNA was transfected 3 days prior to compound treatment as indicated.

TEAD degradation inhibits cell proliferation and downstream Hippo pathway signaling.
(A, B) Cell viability was assessed using CellTiter-Glo in (A) OVCAR-8 cells, or in (B) SK-N-FI cells after 6 days of treatment with Compound D (red), Compound E (green), Compound A (purple), Compound B (blue), or DMSO (black). Error bars represent the standard deviation of five technical replicates. (C, D) Crystal violet growth assay in OVCAR-8 cells (Hippo dependent) and SK-N-FI cells (Hippo independent) after 6 days treatment with Compound D at indicated concentrations. (E, F) RNA-seq of Hippo signature genes after Compound D/Compound A treatment in MDA-MB-231 cells. Hippo signature genes exhibit significant downregulation in a concentration dependent manner upon treatment with Compound D. Cells were treated with 0.1 µM and 0.5 µM of Compound A or Compound D for 24 hours before harvesting.

Compound D specifically reduces chromatin accessibility at TEAD motifs.
(A) Differential analysis of ATAC-seq of OVCAR-8 cells treated with DMSO or Compound D for 48 hours using DiffBind. (B) Genomic annotations of the differential ATAC-seq peaks by ChIP-Annotate. (C) UCSC browser view of ATAC-seq at known targets of TEAD including ANKRD1 and CCN1/CYR61. (D) Lost ATAC-seq peaks were visualized by Deeptools and analyzed for motif enrichment by HOMER. (E) Lost peaks were assigned to genes using known distal enhancer-gene target links in Poly-Enrich, and the 10 most significant pathways are shown.

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