A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

Use the terms sex (biological attribute) and gender (shaped by social and cultural circumstances) carefully in order to avoid confusing both terms.Indicate if findings apply to only one sex or gender; describe whether sex and gender were considered in study design; whether sex and/or gender was determined based on self-reporting or assigned and methods used.Provide in the source data disaggregated sex and gender data, where this information has been collected, and if consent has been obtained for sharing of individual-level data; provide overall numbers in this Reporting Summary.Please state if this information has not been collected.Report sex-and gender-based analyses where performed, justify reasons for lack of sex-and gender-based analysis.
Please specify the socially constructed or socially relevant categorization variable(s) used in your manuscript and explain why they were used.Please note that such variables should not be used as proxies for other socially constructed/relevant variables (for example, race or ethnicity should not be used as a proxy for socioeconomic status).Provide clear definitions of the relevant terms used, how they were provided (by the participants/respondents, the researchers, or third parties), and the method(s) used to classify people into the different categories (e.g.self-report, census or administrative data, social media data, etc.) Please provide details about how you controlled for confounding variables in your analyses.
Describe the covariate-relevant population characteristics of the human research participants (e.g.age, genotypic information, past and current diagnosis and treatment categories).If you filled out the behavioural & social sciences study design questions and have nothing to add here, write "See above."Describe how participants were recruited.Outline any potential self-selection bias or other biases that may be present and how these are likely to impact results.
Identify the organization(s) that approved the study protocol.

nature portfolio | reporting summary
April 2023

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Blinding
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Antibodies
Antibodies used Power calculations using Russ Lenth's power tool (cited in the manuscript) were used to inform in vivo group sizes.For in vitro experiments, no power calculations were undertaken, with sample size taken as the number of repeats of each experiment.
Four data points were excluded as an anomaly in Figure 1B -this has been highlighted in the source data file.No other data has been excluded.Sample size is reported in Figure legends.All attempts at replication were successful.Technical repeats are individual samples prepared and measured on the same day, as part of the same experiment.All figures report n=2, or greater, independent biological repeats except: Figure 1: (A) n=1; representative of four repeats Figure 1: (B) n=1; three technical replicates plotted per A:D ratio; representative of three repeats Figure 1: (D) n=1; four technical replicates plotted per concentration; representative of two repeats Figure 1: (E) n=1; orthogonal confirmation of probe displacement described in Figure 1(D) Figure 1: (F) n=1; repeated independently in HCT116 cells Figure 3: (H-K) n=1; four time points captured (0h, 24h, 48h, 72h); HuR degradation confirmed by western blot (Supplementary Figure 5C-D) observations also independently confirmed by western blot (Supplementary Figure 7A-F; n=2)