Germline APOBEC3B deletion increases somatic hypermutation in Asian breast cancer that is associated with Her2 subtype, PIK3CA mutations and immune activation.

A 30-kb deletion that eliminates the coding region of APOBEC3B (A3B) is >5 times more common in women of Asian descent compared to European descent. This polymorphism creates a chimera with the APOBEC3A (A3A) coding region and A3B 3'UTR, and it is associated with an increased risk for breast cancer in Asian women. Here, we explored the relationship between the A3B deletion polymorphism with tumour characteristics in Asian women. Using whole exome and whole transcriptome sequencing data of 527 breast tumours, we report that germline A3B deletion polymorphism leads to expression of the A3A-B hybrid isoform and increased APOBEC-associated somatic hypermutation. Hypermutated tumours, regardless of A3B germline status, were associated with the Her2 molecular subtype and PIK3CA mutations. Compared to nonhypermutated tumours, hypermutated tumours also had higher neoantigen burden, tumour heterogeneity and immune activation. Taken together, our results suggest that the germline A3B deletion polymorphism, via the A3A-B hybrid isoform, contributes to APOBEC mutagenesis in a significant proportion of Asian breast cancers. In addition, APOBEC somatic hypermutation, regardless of A3B background, may be an important clinical biomarker for Asian breast cancers.

A General in Vitro Assay for Studying Enzymatic Activities of the Ubiquitin System.

The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2, and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies of ubiquitination activities have been challenging. We here report a novel Förster resonance energy transfer (FRET)-based assay for studying the in vitro kinetics of ubiquitination. FRET is established upon binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the free Ub sensor system (FUSS). Using Uba1, UbcH5, and CHIP as model E1, E2, and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3, or substrates and is thus likely compatible with any E1-E2-E3 combinations.