Targeting the Hippo pathway in cancers via ubiquitination dependent TEAD degradation

Reviewer #1 (Public review):

Summary:

In the first half of this study, Pham et al. investigate the regulation of TEAD via ubiquitination and PARylation, identifying an E3 ubiquitin ligase, RNF146, as a negative regulator of TEAD activity through an siRNA screen of ubiquitin-related genes in MCF7 cells. The study also finds that depletion of PARP1 reduced TEAD4 ubiquitination levels, suggesting a certain relationship between TEAD4 PARylation and ubiquitination which was also explored through an interesting D70A mutation. Pham et al. subsequently tested this regulation in D. melanogaster by introducing Hpo loss-of-function mutations and rescuing the overgrowth phenotype through RNF146 overexpression.

In the second half of this study, Pham et al. designed and assayed several potential TEAD degraders with a heterobifunctional design, which they term TEAD-CIDE. Compounds D and E were found to effectively degrade pan-TEAD, an effect which could be disrupted by treatment with TEAD lipid pocket binders, proteasome inhibitors, or E1 inhibitors, demonstrating that the TEAD-CIDEs operate in a proteasome-dependent manner. These TEAD-CIDEs could reduce cell proliferation in OVCAR-8, a YAP deficient cell line, but not SK-N-FI, a Hippo pathway independent cell line. Finally, this study also utilizes ATAC-seq on Compound D to identify reductions in chromatin accessibility at the regions enriched for TEAD DNA binding motifs.

Strengths:

The study provides compelling evidence that the E3 ubiquitin ligase RNF146 is a novel negative regulator of TEAD activity. The authors convincingly delineate the mechanism through multiple techniques and approaches. The authors also describe the development of heterobifunctional pan-degraders of TEAD, that could serve as valuable reagents to more deeply study TEAD biology.

Weaknesses:

The scope of this study is extremely broad. The first half of the paper highlights the mechanisms underlying TEAD degradation; however, the connection to the second half of the paper on small molecule degraders of TEAD is jarring, and it seems as though two separate stories were combined into this single massive study. In my opinion, the study would be stronger if it chose to focus on only one of these topics and instead went deeper.

Additionally, the figure clarity needs to be substantially improved, as readability and interpretation was difficult in many panels. Lastly, there are numerous typos and poor grammar throughout the text that need to be addressed.

Comments on revisions:

The authors have addressed most of our critiques. The manuscript has improved significantly, particularly in the clarity of the figures and the flow of the text. The findings of this study contribute valuable insights into TEAD biology in cancer and provide useful resources for further research into TEAD.

However, as noted by other reviewers, the manuscript still feels somewhat disjointed, despite the attempt to connect the two parts on RNF146-mediated TEAD degradation and the development of TEAD degraders. Certain data inconsistencies and technical limitations may have made some aspects of the data hard to interpret accurately and could benefit from further clarification.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In the first half of this study, Pham et al. investigate the regulation of TEAD via ubiquitination and PARylation, identifying an E3 ubiquitin ligase, RNF146, as a negative regulator of TEAD activity through an siRNA screen of ubiquitin-related genes in MCF7 cells. The study also finds that depletion of PARP1 reduced TEAD4 ubiquitination levels, suggesting a certain relationship between TEAD4 PARylation and ubiquitination which was also explored through an interesting D70A mutation. Pham et al. subsequently tested this regulation in D. melanogaster by introducing Hpo loss-of-function mutations and rescuing the overgrowth phenotype through RNF146 overexpression.

In the second half of this study, Pham et al. designed and assayed several potential TEAD degraders with a heterobifunctional design, which they term TEAD-CIDE. Compounds D and E were found to effectively degrade pan-TEAD, an effect which could be disrupted by treatment with TEAD lipid pocket binders, proteasome inhibitors, or E1 inhibitors, demonstrating that the TEAD-CIDEs operate in a proteasome-dependent manner. These TEAD-CIDEs could reduce cell proliferation in OVCAR-8, a YAP-deficient cell line, but not SK-N-FI, a Hippo pathway independent cell line. Finally, this study also utilizes ATAC-seq on Compound D to identify reductions in chromatin accessibility at the regions enriched for TEAD DNA binding motifs.

Strengths:

The study provides compelling evidence that the E3 ubiquitin ligase RNF146 is a novel negative regulator of TEAD activity. The authors convincingly delineate the mechanism through multiple techniques and approaches. The authors also describe the development of heterobifunctional pan-degraders of TEAD, which could serve as valuable reagents to more deeply study TEAD biology.

Weaknesses:

The scope of this study is extremely broad. The first half of the paper highlights the mechanisms underlying TEAD degradation; however, the connection to the second half of the paper on small molecule degraders of TEAD is jarring, and it seems as though two separate stories were combined into this single massive study. In my opinion, the study would be stronger if it chose to focus on only one of these topics and instead went deeper.

We thank the reviewer for the thoughtful feedback. In our mind, the two parts of the paper are inherently related as they both focus on proteasome-mediated degradation of TEADs. We first demonstrated that TEAD can be turned over by the ubiquitin proteasome system under endogenous conditions and identified a PARylation-dependent E3 ligase RNF146 as a major regulator of TEAD stability. Intriguingly, we observed that the four TEAD paralogs show different levels of polyubiquitination with some of them being highly stable in cells. These observations raised the question of whether the activity of the ubiquitin-proteasome system could be further enhanced pharmacologically to effectively target TEADs. We then tackled this question by providing a proof-of-concept demonstration of engineered heterobifunctional protein degraders can effectively degrade TEADs in cells and can be exploited as a therapeutic strategy for treating Hippo-dependent cancers.

Additionally, the figure clarity needs to be substantially improved, as readability and interpretation were difficult in many panels. Lastly, there are numerous typos and poor grammar throughout the text that need to be addressed.

We appreciate the suggestions from the reviewer and have updated the figures with high resolution images. We also corrected typos and grammatical errors in the text.

Reviewer #2 (Public Review):

The paper is made of two parts. One deals with RNF146, the other with the development of compounds that may cause TEAD degradation. The two parts are rather unrelated to each other.

The main limit of this work is the lack of evidence that TEAD factors are in fact regulated by the proteasome and ubiquitylation under endogenous conditions. Also lacking is the demonstration that TEADs are labile proteins to the extent that such quantitative regulation at the level of stability can impact on YAP-TAZ biology. Without these two elements, the relevance and physiological significance of all these data is lacking.

As for the development of new inhibitors of TEAD, this is potentially very interesting but underdeveloped in this manuscript. Irrespectively, if TEAD is stable, these molecules are likely lead compounds of interest. If TEAD is unstable, as entertained in the first part of the paper, then these molecules are likely marginal.

We thank the reviewer for evaluating our manuscript. As the reviewer pointed out, the paper aimed to address 1) whether TEAD is being regulated by the proteasome and ubiquitination under endogenous conditions, and 2) whether TEAD can be inhibited through pharmacologically-induced degradation. First, we demonstrated that TEAD is ubiquitinated in cells and mapped the lysine residues that are poly-ubiquitinated (Fig. 1). Next, we identified RNF146 as a major E3 ligase that ubiquitinates TEADs and reduces their stability. Third, we show that RNF146-mediated TEAD ubiquitination is functionally important: RNF146 suppresses TEAD activity, and RNF146 genetically interacts with Hippo pathway components in fruit flies. Furthermore, as we showed in Fig. S2H, RNF-146 does not affect TEAD1 and TEAD4 to the same extent. Across all four cell lines evaluated, TEAD1 is more stable than TEAD4, raising the question of whether more consistent degradation of different TEAD paralogues could be achieved. To this end, we demonstrated that while the TEAD family of proteins is labile under endogenous conditions, more complete degradation of the TEAD proteins could be achieved using a heterobifunctional CRBN degrader. We further characterized these TEAD degraders in a series of cellular and genomic assays to demonstrate their cellular activity, selectivity, and inhibitory effects against YAP/TAZ target genes. We believe these degrader compounds would be of great interest to the Hippo community. We have revised the main text to clarify these points.

Here are a few other specific observations:

(1) The effect of MG is shown in a convoluted way, by MS. What about endogenous TEAD protein stability?

We thank the reviewer for the question. The MS experiment shown in Figure 1 is a standard KGG experiment, where we used MS to map ubiquitination sites on TEADs. The graphical representation of the process is included in Fig. 1C, and the details of the procedure are included in the Methods section. Fig. 1D shows the different KGG peptides detected with or without MG-132 treatment. Fig. 1E shows the quantified abundance of each of the peptides across the four conditions indicated at the bottom of the plot. Regarding endogenous TEAD stability, ​​we conducted cycloheximide chase experiments to assess the stability of endogenously expressed TEAD isoforms upon RNF146 knockdown (Fig. S2G and S2H). Using isoform-specific antibodies, we demonstrated that siRNF146 significantly stabilized TEAD4 in multiple cell lines, including H226, PATU-8902, Detroit-562, and OVCAR-8 (Fig. S2G, S2H, and S2I), supporting the notion that RNF146 is a negative regulator of TEAD stability. Notably, the effect of siRNF146 on TEAD1 stability was less pronounced, and TEAD1 is more stable than TEAD4 across all four cell lines. These results are consistent with the lower level of ubiquitination of TEAD1 (Fig. 1A) and are corroborated by various biochemical, molecular, and genetic characterizations (Fig. 3A-C and S3E).

(2) The relevance of siRNF on YAP target genes of Fig.2D is not statistically significant.

We thank the reviewer for this comment. We have now removed the statistically significant claim.

(3) All assays are with protein overexpression and Ub-laddering

We thank the reviewer for the comment. To examine the ubiquitination level of TEAD proteins, we adopted an in vivo ubiquitination assay as described in our Materials and Methods section. To our knowledge, this assay is very standard in the ubiquitination field. Furthermore, as mentioned above, we have included in our revised manuscript cycloheximide chase experiments to assess the stability of endogenously expressed TEAD isoforms upon RNF146 knockdown (Fig. S2G and S2H). In addition to the overexpression system, we also assessed endogenously expressed TEAD using isoform-specific antibodies. We demonstrated that siRNF146 firmly stabilized TEAD4 in multiple cell lines, including H226, PATU-8902, Detroit-562, and OVCAR-8 (Fig. S2G with quantification and t-test), supporting the notion that RNF146 is a negative regulator of TEAD stability.

(4) An inconsistency exists on the only biological validation (only by overexpression) on the fly eye size. RNF gain in Fig4C is doing the opposite of what is expected from what is portrayed here as a YAP/TEAD inhibitor: RNF gain is shown to INCREASE eye size, phenocopying a Hippo loss of function phenotype. According to the model proposed, RNF addition should reduce eye size. The authors stated that " This is in contrast to the anti-growth effect of RNF-146 in the Hpo loss-of-function background and indicates RNF146 may regulate other genes/pathways controlling eye sizes besides its role as a negative regulator of Sd/yki activity". This raises questions on what the authors are really studying: why, according to the authors, these caveats should occur on the controls, and not when they study Hpo mutants?

We thank the reviewer for the comment. We acknowledge the complexity of the fly phenotype compared to tumor growth. TEAD (Sd) isn’t the only substrate of RNF146 in the fly. For instance, RNF146 is known to positively regulate Wnt signaling by degrading Axin. Previous studies have shown that activation of the Wnt signaling pathway by removal of the negative regulator Axin from clones of cells results in an overgrowth phenotype (Legent and Treisman, 2008). The overgrowth phenotype that we observed with overexpressing RNF146 only, therefore, likely is due to the role of RNF146 in regulating other signaling pathways. Importantly, we showed that upon Hippo loss of function, overexpression of RNF146 can rescue the Hippo overgrowth phenotype (Fig 4B). This differential outcome of RNF146 expression in wildtype versus Hippo-deficient flies indicates that the genetic interactions between RNF146 and Hippo pathway components altered the phenotypic outcome, and the phenotype we get with RNF146 overexpression in a Hippo loss of function background is not simply due to additive effects of functional loss of either component alone.

Complementary to these overexpression data, we showed that knockdown of RNF146 increased the eye size further (Fig. S4A, B) in Hippo loss of function background, further supporting the role of RNF146 as a negative regulator of the overall pro-growth signals induced by yki upon Hippo loss of function.

(5) The role of TEAD inactivation on YAP function is already well known. Disappointingly, no prior literature is cited. In any case, this is a mere control.

We thank the reviewer for the suggestion. We have cited several published reviews that touch upon this aspect of the TEAD-YAP function, including Calses et al., 2019; Dey et al., 2020; Halder and Johnson, 2011; Wang et al., 2018. We are open to your suggestions on additional citations.

(6) The second part of the paper on the Development and Screening of pan-TEAD lipid pocket degraders is interesting but unconnected to the above. The degradation pathway it involves has nothing to do with the enzyme described in the first figures.

We thank the reviewer for the comment. We acknowledge that our paper broadly covers two aspects. We believe that they are inherently connected as they both address ubiquitin/proteasome-mediated TEAD degradation and the functional consequences of TEAD degradation. Given the increasing interest in targeting TEAD/YAP/TAZ in cancers, we think the pharmacological approaches to enhance TEAD degradation using orthogonal E3 ligases provide an important toolbox to understand how this pathway can be regulated under both physiological and pathological conditions. While RNF146 appears to be a major E3 ligase responsible for TEAD turnover under physiological conditions, we showed that the four TEAD paralogs have different poly-ubiquitination levels (Fig. 1A), and are differentially labile in cells (Fig. S2G-I). These observations raised the question of whether the activity of the ubiquitination-proteasome system could be further enhanced to allow more complete removal of TEADs. To this end, we demonstrated that E3 ligases that do not regulate TEAD under endogenous conditions can be leveraged pharmacologically to achieve deep TEAD degradation, thus providing a proof of concept that TEADs can be targeted simultaneously using such approaches. Finally, in addition to establishing the basic biological concept linking RNF146 to TEAD degradation, the compounds we engineered will serve as valuable chemical tools for future studies of TEAD biology and the Hippo pathway in cancers and beyond.

(7) The role of CIDE on YAP accessibility to Chromatin is superficially executed. Key controls are missing along with the connection with mechanisms and prior knowledge of TEAD, YAP, chromatin, and other TEAD inhibitors, just to mention a few.

We used ATAC-seq to assess chromatin accessibility comparing cells treated with DMSO and two different concentrations of compound D. We acknowledge there are small molecule inhibitors of TEADs that can modulate accessibility of YAP binding sites. Potential mechanistic differences between TEAD degraders versus TEAD small molecule inhibitions will be a future area of investigation.

(8) The physiological relevance and the mechanistic interpretation of what should be in the ATAC seq in ovcar cells is missing.

We showed in Fig. 7A-D the dose response of OVCAR cells to the TEAD degraders. As evident from those experiments, TEAD degraders inhibit the proliferation of OVCAR cells as expected from their dependencies on the TEAD/YAP/TAZ transcription complex. In the ATAC-seq experiment, we showed that the canonical TEAD/YAP/TAZ target genes ANKRD1 and CCN1 have reduced chromatin accessibility at their promoter/enhancer regions (Fig. 8C). By unbiased motif and pathway analyses, we show that TEAD binding sites and YAP signatures are most significantly downregulated in OVCAR-8 cells (Fig. 8D-E). These results are incorporated into the results section of the manuscript.

Reviewer #3 (Public Review):

Summary

Pham, Pahuja, Hagenbeek, et al. have conducted a comprehensive range of assays to biochemically and genetically determine TEAD degradation through RNF146 ubiquitination. Additionally, they designed a PROTAC protein degrader system to regulate the Hippo pathway through TEAD degradation. Overall, the data appears robust. However, the manuscript lacks detailed methodological descriptions, which should be addressed and improved before publication. For instance, the methods used to analyze the K48 ubiquitination site on TEAD and the gene expression analysis of Hippo Signaling are unclear. Furthermore, the multiple proteomics, RNA-seq, and ATAC-seq data must be made publicly available upon publication to ensure reproducibility. Most of the main figures are of low resolution, which needs addressing.

We thank the reviewer for evaluating our manuscript. All of the data will be uploaded to public databases. We apologize for the low figure resolution and have updated the figures in the revised manuscript. We also expanded the methods section with more details.

Strengths:

- A broad range of assays was used to robustly determine the role of RNF146 in TEAD degradation.

- Development of novel PROTAC for degrading TEAD.

Weaknesses:

- An orthogonal approach is needed (e.g., PARP1 inhibitor) to demonstrate PARP1's dependency in TEAD ubiquitination.

We thank the reviewer for the suggestion. We had attempted to assess the effect of PARP inhibitors (including veliparib and olaparib) on TEAD ubiquitination, but the data is relatively complex to interpret. Besides inhibiting PARP1/2 catalytic activities, these PARP inhibitors also trap PARP on chromatin. Hence, these inhibitors could induce other cellular changes in addition to inhibiting the catalytic activities of PARP1/2. Given these potential pitfalls, we decided not to include these inconclusive data. Even though the experiments with PARP inhibitors were inconclusive, our study supports that TEAD2 and TEAD4 are PARylated in cells using an anti-PAR antibody (Fig. 3B). Furthermore, we show that mutation of the D70 PARsylation site to alanine greatly abolished TEAD4 ubiquitination in cells, suggesting PARylation is important for TEAD4 ubiquitination. In addition, PARP1 depletion by siRNA and CRISPR guide RNA reduced TEAD2 and TEAD4 ubiquitination levels, indicating PARP1 is one of the PARPs responsible for TEAD PARylation in cells.

- The data from Table 2 is unclear in illustrating the association of identified K48 ubiquitination with TEAD4, especially since the experiments were presumably to be conducted on whole cell lysates with KGG enrichment. This raises the possibility that the K48 ubiquitination could originate from other proteins. Alternatively, if the authors performed immunoprecipitation on TEAD followed by mass spectrometry, this should be explicitly described in the text and materials and methods section.

We thank the reviewer for this question. The experiment was an IP-mass spectrometry study in a TEAD4 amplified cell line model (PATU-8902) after IP with a pan-TEAD antibody. Here, we observed K48 ubiquitin and other ubiquitin linkages as shown in the Supplementary Table S2 of the original submission. Although it is possible that the IP wash steps could be more stringent, we did enrich for TEAD protein prior to mass spectrometry. While the ubiquitin linkage signals may come mainly from TEAD protein (mainly TEAD4), we recognized that some signals may come from other proteins. Given the caveat, we have now removed the table from our paper and updated the text accordingly.

- Figure 2D: The methodology for measuring the Hippo signature is unclear, as is the case for Figures 7E and F regarding the analysis of Hippo target genes.

We apologize for the lack of clarification. In short, we previously developed the Hippo signature using machine learning and chemogenomics as described previously (Pham et al. Cancer Discovery 2021). In the revised version of the manuscript, we added the methodology for measuring the Hippo signature and cited our previous publication where we developed the Hippo signature.

- Figure S3F requires quantification with additional replicates for validation.

We thank the reviewer for the suggestion. We added the quantification for the blot and indicated the replication in the figure legend. Note that Figure S3F is now S3G.

- There is a misleading claim in the discussion stating "TEAD PARylation by PAR-family members (Figure 3)"; however, the demonstration is only for PARP1, which should be corrected.

We apologize for the statement. We observed both PARP1 and PARP9 in our TEAD IP-mass spec (now Figure S3E), which suggest both PARP-family members could be invovled. Nonetheless, we primarily focus on PARP1, which is widely expressed aross cell line models and present in higher abundance. Thus, our study only experimentally validated PARP1's role in regulating TEAD.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

General comments:

(1) Please provide a smoother transition and well-defined connection between the first and second parts of the manuscript. The manuscript reads as two papers that were combined into one, without much attempt to disguise the fact.

We thank the reviewer for the suggestion. We have added a transition paragraph to smoothen the transition. We acknowledge that our paper broadly covers two aspects. However, they both touch upon TEAD ubiquitination and degradation. In the first part of the manuscript, we described TEAD biology and showed that TEADs are post-translationally modified and subsequently regulated through PARylation-dependent RNF146-mediated ubiquitination. In the second part, we highlighted our abilities to leverage the PROTAC system for degrading such labile oncogenic proteins like TEADs. In addition to the biological concept, the compounds we engineered will serve as valuable chemical tools for future studies of TEAD biology and the Hippo pathway in cancers and beyond.

(2) To confirm the proteasome mechanism of action, viability assays should be conducted with a CRBN KO.

We thank the reviewer for the comment. In Figure 6E, we measured TEAD protein levels under CRBN knockdown and observed an expected change in TEAD stability. This observation and the other data presented in Figure 6 suggest that TEAD proteins are targeted for proteasomal degradation under compound D treatment.

(3) As a control, sgPARP1 or PARP1 inhibitors should be used to confirm TEAD PARylation reduction.

We thank the reviewer for the suggestion. We had attempted to assess the effect of PARP inhibitors (including veliparib and olaparib) on TEAD ubiquitination, but the data is relatively complex to interpret. Besides inhibiting PARP1/2 catalytic activities, PARP inhibitors also trap PARP on chromatin. Hence, these inhibitors could induce other cellular changes in addition to inhibit the catalytic activities of PARP1/2. Given these pitfalls, we decided not to include these inconclusive data. Even though the experiments with PARP inhibitors were inconclusive, our study supports that TEAD2 and TEAD4 are PARylated in cells using an anti-PAR antibody (Fig. 3B). Furthermore, we show that mutation of the D70 PARsylation site to alanine greatly abolished TEAD4 ubiquitination in cells, suggesting PARylation is important for TEAD4 ubiquitination. In addition, PARP1 depletion by siRNA and CRISPR guide RNA reduced TEAD2 and TEAD4 ubiquitination levels, indicating PARP1 is one of the PARPs responsible for TEAD PARylation in cells.

(4) MS data looks convincing but an FDR of 1% should be applied - this is the accepted standard in the proteomics field. Please research the data with the more stringent filter.

We thank the reviewer for the suggestion. Our IP-MS experiment comparing siNTC versus siYAP1/WWTR1 in Patu-8902 cells did not have replicates and FDR could not be derived. Therefore, we listed the raw data in Supplemental Table 3 without showing statistics. To validate the putative interactions identified by IP-MS, we performed IP-Western experiments to confirm that TEAD4 interacts with PARP1 (Figure 3A). It is important to note that in addition to our report, the interaction between PARP1 and TEADs has been observed in other publications (Calses et al., 2023; Yang et al., 2017). We have included more details of the IP-MS experiment reported in Supplemental Table 3 in the revised manuscript and cited previous work reporting TEAD-PARP1 interaction.

(5) Proofread the manuscript more thoroughly for typos and grammatical errors.

We thank the reviewer for raising this issue and have addressed it in the revision.

(6) Improve figure clarity (e.g., clearly labeling graph axes).

We apologize for the oversight. The revised manuscript contains high resolution figures.

Specific points:

Generally, the manuscript could use additional proofreading for grammar and clarity. It would not be practical to list all, but some representative examples are listed below:

Run-on: "They act through an event-driven mechanism instead of conventional occupancy-driven pharmacology, in addition, target protein degradation removes all functions of the target protein and may also lead to destabilization of entire multidomain protein complexes."

Typo: "Compound D exhibits significant inhibition of cell proliferation and downstream signaling compared to compound A, a reversible TEAD lipid pocket binder that lack the ubiquitin ligase binding moiety."

Typo: "Thus, we sought to deplete TEAD proteins by directly target them for ubiquitination and proteasomal degradation via pharmacologically inducing interactions between TEAD and other abundantly expressed and PARylation-independent E3 ligases."

Typo: "Compound A is a close in analog of Compound B as described previously (Holden et al., 2020)."

We have revised the manuscript and corrected the typos and grammatical errors listed above and beyond.

Specific comments on the figures are listed below:

Figure 2:

• Figures 2B and 2C should be separated into separate panels for clarity.

We have updated the Figures 2B and 2C as suggested.

• Figure 2C - "To further assess the function of RNF146, we depleted RNF146 by either sgRNA or siRNA." This should say either CRISPR-Cas9 KO or siRNA-mediated knockdown.

We thank the reviewer for the suggestion. We revised the text to address this issue.

• Figure 2D - y-axis is not labeled well/clearly. Additionally, there are different resolutions for the p-values on the graph (the top p-value is slightly clearer than the other two, suggesting either a different font was used or the value was pasted on top of a picture of the graph at a different resolution).

We updated the figures according to the suggestions.

• Figure S2A - "We identified three ubiquitin ligases - RNF146, TRAF3, and PH5A - as potential negative regulators for the Hippos pathway from the primary screen using the luciferase reporter." However, the siPHF5A data appears to decrease luciferase levels whereas siRNF146 and siTRAF3 increase it.

We thank the reviewer for catching this error. We removed PH5A from this list.

Figure 3:

• Figure 3A - label more clearly. Is this an endogenous TEAD4 co-IP?

We thank the reviewer for the suggestion. The experiment was an IP-mass spectrometry study in a TEAD4 amplified cell line model (PATU-8902) with pan-TEAD antibody. We have included the details to in the figure legends. Figure 3A is now Figure S3E in the revised manuscript.

• Figure 3C - why are the dark and light exposures not matching/corresponding? In the dark exposure, there are two particularly dark bands, the darkest of which is at the top of the gel. However, this darkest band disappears in the light exposure gel. Additionally, the last lane is marked as +TEAD2 and +TEAD4. Not sure if this is a typo, and meant to be only +TEAD4? Seems a bit strange to have a double TEAD lane.

We thank the reviewer for this comment and apologize for the oversight. There was a typo in the label. The light exposure image was from a replicate run instead of the same run, therefore the lanes didn’t all match up. We have removed the light exposure panel to resolve the confusion. (Figure 3B).

Figure 5:

• Figure 5B - why is shTEAD1-4/Sucrose a much higher tumor volume than shNTC/Sucrose negative control? Additionally, should the legend say "sNTC/Sucrose" as it does or "shNTC/Sucrose"?

The labels for shTEAD1-4/Sucrose and shNTC/Sucrose are correct. We do not understand why there is a slight increase in tumor volume for shTEAD1-4/Sucrose and suspect that is due to the considerable variation in the experiment. This slight change, however, doesn’t influence our observation of tumor regression in shTEAD1-4 under the Doxycycline treatment.

"sNTC/Sucrose" is a typo. We apologize for the oversight and have revised the figure.

• Figure 5E - cited in text after Figures 6 and 7.

We have updated the text accordingly.

Figure 6:

• Figure 6B - it is very interesting how this clearly shows the Hook effect for Compound D, but it's a bit harder to see for compound E that the compound degrades pan-TEAD. Would it be possible to quantify the blots to reinforce claims about protein degradation here?

We thank the reviewer for the question. There may seem to be some hook effect across the three concentrations of compound D treatment in Fig. 6B. However, in Fig. 6C-E, we observed pretty consistent TEAD degradation levels across a variety of concentrations. In addition, these experiments have been repeated in multiple cell lines with consistent results. We respectfully argue that more detailed investigation of the hook effect is beyond the scope of our study.

Figure 7:

• Figure 7F - this heat map is extremely difficult to interpret. Are there any interesting clusters? What are the darker/lighter bands for Compound D compared to DMSO control?

We thank the reviewer for the comment and apologize for the lack of information on the figure. These are genes from a Hippo signature derived from our earlier work (Pham et al. Cancer Discovery). As a result of degrading TEAD when treating the cells with Compound D, we observed an expected downregulation of most of these genes compared to compound A.

Figure 8:

• Figure 8B - these two pie charts are also difficult to interpret. Perhaps try to present the data in a form other than encircling pie charts?

We thank the reviewer for the suggestion. However, this is a very descriptive pie chart, we used this format to save space.

• Figure 8C - what is GNE-6915? Is this Compound D?

Yes, this is compound D. The text is updated accordingly.

Reviewer #3 (Recommendations For The Authors):

Figure 3A would benefit from explicitly stating the conditions within the figure, rather than referring to the legend. This clarity is also needed for Figure 8C, indicating whether the treatment was with compound D or GNE-6915.

We thank the reviewer for the suggestion. We have added the details to the figures and made the suggested edits.

Standardize the terms "ubiquitination" and "ubiquitylation" throughout the paper for consistency.

We now use the term “ubiquitination” throughout the manuscript.

The statement "In this study, we show that the activity of TEAD transcription factors can be post-transcriptionally regulated via the ubiquitin/proteasome system" should be corrected to "post-translationally regulated."

We have update the manuscript accordingly.

There is an additional exclamation mark above Figure 5E that should be removed.

We have revised Figure 5E.