Johnson, J. I. | Search bioRxiv

bioRxiv2021

Inhibition of the ubiquitin-proteasome system by a bioactivatable compound

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

cell biology↗

bioRxiv2018

Software-assisted manual review of clinical NGS data:an alternative to routine Sanger sequencing confirmationwith equivalent results in >15,000 hereditary cancer screens

Clinical genomic tests increasingly utilize a next generation sequencing (NGS) platform due in part to the high fidelity of variant calls, yet rare errors are still possible. In hereditary cancer screening, failure to correct such errors could have serious consequences for patients, who may follow an unwarranted screening or surgical-management path. It has been suggested that routine orthogonal confirmation via Sanger sequencing is required to verify NGS results, especially low-confidence positives with depressed allele fraction (<30% of alternate allele). We evaluated whether an alternative method of confirmation--software-assisted manual call review--performed comparably to Sanger confirmation in >15,000 samples. Licensed reviewers manually inspected both raw and processed data at the batch-, sample-, and variant-level, including raw NGS read pileups. Of ambiguous variant calls with <30% allele fraction (1,707 total calls at 38 unique sites), manual call review classified >99% (1,701) as true positives (enriched for long insertions or deletions (\"indels\") and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining <1% (6) being mosaic. Critically, results from software-assisted manual review and retrospective Sanger sequencing were concordant for samples selected from all ambiguous sites. We conclude that the confirmation required for high confidence in NGS-based germline testing can manifest in different ways: a trained NGS expert operating platform-tailored review software achieves quality comparable to routine Sanger confirmation.

genomics↗