Sharp, M. F. | Search bioRxiv

bioRxiv2026

Fanconi Anaemia E3 Ligase complex activity is regulated by a druggable metabolite binding site in FANCX

The Fanconi anaemia (FA) DNA repair pathway is an emerging target for precision cancer therapy. Using a high-throughput FANCD2-monoubiquitination assay, we identified a class of small molecules, including MSG010, that inhibit the FA E3 ligase complex in vitro. Because these molecules, and the metabolite, palmitoyl-CoA, are known to engage allosteric drug and metabolite (ADaM) binding site in AMP-activated kinase (AMPK), we hypothesised that a similar pocket exists within the FA complex. Supporting this, long-chain, but not short-chain, fatty acyl-CoA molecules inhibited the FA E3 ligase complex activity, and sequence analysis revealed similarity between the AMPK ADaM site and a WD40 repeat in the FA subunit FANCX. Targeted mutagenesis of this FANCX region disrupted E3 ligase activity or abolished inhibition by MSG010, suggesting the presence of an ADaM-like site in FANCX. Moreover, MSG010 preferentially killed BRCA1-deficient cells in vitro. These findings identify a putative small-molecule binding site in the FA pathway that may be developed further to test for exploitation as anticancer therapeutics.

cancer biology↗

bioRxiv2025

Differential PARP inhibitor responses in BRCA1-deficient and resistant cells in competitive co-culture

Synthetic lethality describes a genetic relationship where the loss of two genes results in cell death, but the loss of one of those genes does not. Drugs used for precision oncology can exploit synthetic lethal relationships; the best described are PARP inhibitors which preferentially kill BRCA1-deficient tumours preferentially over BRCA1-proficient cells. New synthetic lethal targets are often discovered using genetic screens, such as CRISPR knockout screens. Here, we present a competitive co-culture assay that can be used to analyse drugs or gene knockouts with synthetic lethal effects. We generated new BRCA1 isogenic cell line pairs from both a triple-negative breast cancer cell line (SUM149) and adapted pre-existing non-cancerous BRCA1 isogenic pair (RPE). Each cell line of the isogenic pair was transformed with its own fluorescent reporter. The two-coloured cell lines of the isogenic pair were then grown together in the same vessel to create a more competitive environment compared to when grown separately. We used four PARP inhibitors to validate the ability to detect synthetic lethality in BRCA1-deficient cancer cells. The readout of the assay was performed by counting the fluorescently coloured cells after drug treatment using flow cytometry. We observed preferential targeting of BRCA1-deficient cells, by PARPi, at relative concentrations that broadly reflect clinical dosing. Further we reveal subtle differences between PARPi resistant lines compared to BRCA1-proficient cells. Here, we demonstrate the validation and potential use of the competitive assay, which could be extended to validating novel genetic relationships and adapted for live cell imaging.

genetics↗