Efficiency and localisation of AURKA degradation by PROTACs is modulated by deubiquitinases UCHL5 and target-selective OTUD6A

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate the role of deubiquitinases (DUBs) in modulating the efficacy of PROTAC-mediated degradation of the cell-cycle kinase AURKA. Using a focused siRNA screen of 97 human DUBs, they identify UCHL5 and OTUD6A as negative regulators of AURKA degradation by PROTACs. They further offer a mechanistic explanation of enhanced AURKA degradation in the nucleus via OTUD6A expression being restricted to the cytosol, thereby protecting the cytoplasmic pool of AURKA. These findings provide important insight into how subcellular localization and DUB activity influence the efficiency of targeted protein degradation strategies, which could have implications for therapy.

Strengths:

The manuscript is well-structured, with clearly defined objectives and well-supported conclusions.

The study employs a broad range of well-validated techniques-including live-cell imaging, proximity ligation assays, HiBiT reporter systems, and ubiquitin pulldowns - to dissect the regulation of PROTAC activity.

The authors use informative experimental controls, including assessment of cell-cycle progression effects, rescue experiments with siRNA-resistant constructs to confirm specificity, and the application of both AURKA-targeting PROTACs with different warheads and orthogonal degrader systems (e.g., dTAG-13 and dTAGv-1) to differentiate between target- and ligase-specific effects.

The identification of OTUD6A as a cytosol-restricted DUB that protects cytoplasmic but not nuclear AURKA is novel and may have therapeutic relevance for selectively targeting oncogenic nuclear AURKA pools.

Weaknesses:

Although UCHL5 and OTUD6A are shown to limit AURKA degradation, direct physical interaction was not assessed.

While the authors suggest that combining PROTACs with DUB inhibition could enhance degradation, this was not experimentally tested.

The authors acknowledge the apparent discrepancy between the enhanced degradation observed with CRBN-recruiting PROTACs and the lack of change in ubiquitination following UCHL5 knockdown, yet they do not propose any mechanistic explanation.

Reviewer #2 (Public review):

Summary:

In this study, the authors present a screening approach to identify deubiquitylases that may impact PROTAC efficacy/potency, specifically in this case using a previously reported AURKA PROTAC as an initial model. The authors claim that UCHL5 is able to control the level of degradation of both AURKA and dTAG when using CRBN mediated PROTACs, however that VHL is not impacted by UCHL5 activity. They additionally claim that OTUD6A is able to control extent of AURKA degradation in a target protein-specific manner and that this effect is specific to cytoplasm located AURKA.

Overall, the endeavour is of interest and important. Some of the claims made were overly generalised, and in the main effects observed when knocking down the respective DUBs were small. In addition, the systems used are highly artificial, and the data is not presented in a way that makes understanding absolute (rather than relative) changes easy to understand.

Strengths:

The topic is of high interest and relevance and explores an underappreciated and understudied area of the PROTAC mechanism of action. If further supported and understood, they would certainly bring value to the field.

Weaknesses:

The overall effects observed are sometimes limited in real terms. The data provided often omits the absolute changes in protein abundance observed. Data on endogenous/less engineered systems and/or with higher resolution read-outs would
greatly strengthen some conclusions.

Author response:

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

We are grateful for the insightful and constructive feedback received from reviewers. As outlined in our previous response to the public reviews of the manuscript, we have made only minor changes to the manuscript to clarify some points noted by Reviewers 1 and 3. Firstly, we identify the DUB shown in the correlation plot (Fig 3B) - whose knockdown enhances PROTAC sensitivity without significantly altering cell cycle progression - as BAP1. Secondly, we explain in more detail how we selected DUB hits for further study, and thirdly, we acknowledge that the result in Figure 5G is unexpected given prevailing knowledge in the field.

Please see below the detailed list of changes we have made to the manuscript.

In response to Reviewer 1 (Point 2 of public review and Point 2 in recommendations to author)

We have labelled one of the hits (as BAP1) in Figure 3B

In response to Reviewer 1 (Point 2 of public review and Point 2 in recommendations to author) and Reviewer 3 (Point 6 in recommendations to authors)

We have rewritten our description of Figure 3 in order to make clarifications about how we selected which hits to take forwards in our study

In response to Reviewer 3 (Point 1 in the recommendation to authors)

We corrected a typo in the first subtitle of the results section

In response to Reviewer 3 (Point 2 in the recommendation to authors)

We added information requested about how we selected our top hits

In response to Reviewer 1 (Point 4 in public review and Point 4 in recommendation to authors)

We pointed out the seemingly contradictory nature of the UCHL5 result in Figure 5G for the reader

All of the changes have been aimed at clarifying our narrative, without any change to data content, analysis or interpretation, and we hope these improvements can be agreed by editorial review.