Efficiency and localisation of AURKA degradation by PROTACs is modulated by deubiquitinases UCHL5 and target-selective OTUD6A
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eLife Assessment
This study describes a genetic screen to identify deubiquitinases (DUBs) that counteract the activity of small-molecule degraders (PROTACs). The presented data are valuable, identifying OTUD6A and UCHL5 as DUBs that impact the efficacy and potency of PROTACs. While the conclusions are broadly supported and the methods employed are solid, the mechanistic depth and validation are incomplete. Overall, these findings merit further evaluation by the targeted protein degradation community when developing and optimizing PROTACs.
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Abstract
Abstract
Proteolysis-targeting chimeras (PROTACs) represent a promising new drug modality for novel therapeutics. However, the cellular mechanisms and regulatory pathways underlying their activity are not fully understood. Here, we unveil the role of deubiquitinases (DUBs) in regulating PROTAC activity, by screening 97 human DUBs for their influence on degradation of cell-cycle kinase AURKA using siRNA-mediated knockdown. Our findings reveal that DUBs OTUD6A and UCHL5 counteract degradation of AURKA by small molecule PROTACs. Further investigation using orthogonal dTAG PROTACs indicates that the PROTAC-opposing effect of OTUD6A is target-specific for AURKA, while UCHL5 counteracts degradation triggered by other PROTACs dependent on ubiquitin ligase adaptor CRBN, but not VHL. Furthermore, we show that differential sensitivity of the nuclear pool of AURKA to PROTAC-mediated degradation is fully explained by the specific subcellular localisation pattern of OTUD6A. These findings enhance our understanding of cellular pathways underpinning the action of PROTACs and indicate that combinations of DUB inhibitors and PROTACs will lead to enhanced target degradation and potential improvement in therapeutic outcomes.
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eLife Assessment
This study describes a genetic screen to identify deubiquitinases (DUBs) that counteract the activity of small-molecule degraders (PROTACs). The presented data are valuable, identifying OTUD6A and UCHL5 as DUBs that impact the efficacy and potency of PROTACs. While the conclusions are broadly supported and the methods employed are solid, the mechanistic depth and validation are incomplete. Overall, these findings merit further evaluation by the targeted protein degradation community when developing and optimizing PROTACs.
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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:
(1) The manuscript is well-structured, with clearly defined objectives and well-supported conclusions.
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:
(1) The manuscript is well-structured, with clearly defined objectives and well-supported conclusions.
(2) 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.
(3) 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.
(4) 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:
(1) Although UCHL5 and OTUD6A are shown to limit AURKA degradation, direct physical interaction was not assessed.
(2) Although the authors identify a correlation between DUB knockdown-induced cell cycle progression and enhanced PROTAC activity, only one DUB (USP36) is excluded on this basis. In addition, one DUB is shown in the correlation plot (Figure 3B) whose knockdown enhances PROTAC sensitivity without significantly altering cell cycle progression, but it is not identified/discussed.
(3) While the authors suggest that combining PROTACs with DUB inhibition could enhance degradation, this was not experimentally tested.
(4) The study identifies UCHL5 as a general antagonist of CRBN-recruiting PROTACs, yet the ubiquitin pulldown experiments (Figure 5G, H) show no change in AURKA ubiquitination upon UCHL5 knockdown. This raises questions about the precise step or mechanism by which UCHL5 exerts its protective effect.
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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, VHL is not impacted by UCHL5 activity. They additionally claim that OTUD6A is able to control the extent of AURKA degradation in a target protein-specific manner and that this effect is specific to cytoplasm-located AURKA.
Overall, whilst the endeavour is of interest and importance, we found that the claims made were overly generalised, the effects observed when knocking down the respective DUBs were very small, the systems used are highly artificial, …
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, VHL is not impacted by UCHL5 activity. They additionally claim that OTUD6A is able to control the extent of AURKA degradation in a target protein-specific manner and that this effect is specific to cytoplasm-located AURKA.
Overall, whilst the endeavour is of interest and importance, we found that the claims made were overly generalised, the effects observed when knocking down the respective DUBs were very small, the systems used are highly artificial, and the data is not presented in a way that makes understanding absolute changes transparent.
Strengths:
The topic is of high interest and relevance and explores an underappreciated and understudied area of the PROTAC mechanism of action. If findings could be better supported, they would certainly bring value to the field.
Weaknesses:
The overall effects observed are sometimes limited in real terms. Even if statistically significant, the data presented does not fully support that changes in degradation due to UCHL5 activity represent changes of functional relevance. 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.
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Reviewer #3 (Public review):
Summary:
Cardno et al. "test the hypothesis that DUBs could oppose PROTAC-mediated degradation of cellular targets, using AURKA as a model target". A screen with a panel of siRNA that depleted 97 DUBs in the presence and absence of AURKA targeted PROTAC-D identified DUBs that regulated AURKA and those that affected the sensitivity of PROTAC-D. Validation studies with DUBs, UCHL5, and OTU6A yielded mixed results. UCHL5 not only affected PROTAC-mediated AURKA degradation but also affected CRBN-associated substrates, OTUD6A, more specifically, affected PROTAC-mediated AURKA degradation, and the effects of OTUD6A were associated with the localisation of AURKA. The findings are interesting; the impact of the findings would be strengthened if the key results are validated in one or more cancer cell lines that have …
Reviewer #3 (Public review):
Summary:
Cardno et al. "test the hypothesis that DUBs could oppose PROTAC-mediated degradation of cellular targets, using AURKA as a model target". A screen with a panel of siRNA that depleted 97 DUBs in the presence and absence of AURKA targeted PROTAC-D identified DUBs that regulated AURKA and those that affected the sensitivity of PROTAC-D. Validation studies with DUBs, UCHL5, and OTU6A yielded mixed results. UCHL5 not only affected PROTAC-mediated AURKA degradation but also affected CRBN-associated substrates, OTUD6A, more specifically, affected PROTAC-mediated AURKA degradation, and the effects of OTUD6A were associated with the localisation of AURKA. The findings are interesting; the impact of the findings would be strengthened if the key results are validated in one or more cancer cell lines that have not been modified.
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Author response:
We therefore plan to make only a minor change to the manuscript to clarify a point raised by Reviewer 1: the DUB shown in the correlation plot in Fig 3B - whose knockdown enhances PROTAC sensitivity without significantly altering cell cycle progression - is BAP1. Since BAP1 subsequently showed no significant effect on endogenous AURKA levels (Fig 3E) it was excluded from further analysis.
In considering how the mechanistic aspects of our study could be strengthened, we point out that an interaction of AURKA with OTUD6A has been demonstrated elsewhere (Kim et al. 2021). We also argue that an interaction of AURKA with UCHL5 would not be expected since UCHL5 is a proteasomal DUB shown to act on substrates recruited to the proteasome via capture of ubiquitin chains by the ubiquitin receptors of the proteasome lid. We agree …
Author response:
We therefore plan to make only a minor change to the manuscript to clarify a point raised by Reviewer 1: the DUB shown in the correlation plot in Fig 3B - whose knockdown enhances PROTAC sensitivity without significantly altering cell cycle progression - is BAP1. Since BAP1 subsequently showed no significant effect on endogenous AURKA levels (Fig 3E) it was excluded from further analysis.
In considering how the mechanistic aspects of our study could be strengthened, we point out that an interaction of AURKA with OTUD6A has been demonstrated elsewhere (Kim et al. 2021). We also argue that an interaction of AURKA with UCHL5 would not be expected since UCHL5 is a proteasomal DUB shown to act on substrates recruited to the proteasome via capture of ubiquitin chains by the ubiquitin receptors of the proteasome lid. We agree that mechanistically we have not provided complete evidence for a direct deubiquitinating activity of UCHL5 on AURKA. We cannot explain why there is no change in AURKA ubiquitination upon UCHL5 knockdown in our ubiquitin pulldown experiment, but indeed there is considerable uncertainty in the scientific literature on the precise role of UCHL5 at the proteasome.
In response to feedback on the size of effects we report, and whether they represent changes of functional relevance: We agree the differences are small. Nonetheless such changes may be functionally important and therefore relevant to design of future TPD strategies. Our previous characterization of PROTAC-D (Wang et al. 2021) provides evidence that differential degradation of subcellular pools can have functional relevance. We showed in our study that the lack of degradation of the centrosomal pool (even if this represents only a small fraction of the total pool) led to unexpected phenotypic consequences that were distinct from those observed upon treatment with ATP-competitive inhibitor or siRNA. Therefore we believe our specific finding of spatially restricted action of AURKA-selective OTUD6A to be of clear functional relevance to AURKA TPD strategies and of conceptual importance in establishing the paradigm of TPD modulation by DUBs.
As Reviewer 1 notes, we do not directly test our hypothesis that combining PROTACs with DUB inhibition could enhance degradation. We would have done so had there been suitable small molecule inhibitors available for OTUD6A or UCHL5 at the time of our study. We plan a broader study of OTUD6A mechanisms and its role in PROTAC sensitivity in cancer cell lines, and appreciate Reviewer 3’s suggestion that the impact of our findings would be strengthened if key results were validated in one or more cancer cell lines. The scope of this new study means we plan to report it in a separate, future publication.
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