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, 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.

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.

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.

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?

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

3 All assays are with protein overexpression and Ub-laddering

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?

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.

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.

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.

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

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. 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.

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.

- 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.

- 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.

- Figure S3F requires quantification with additional replicates for validation.

- 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.