When overexpressed in cancer cells, A3A induces DNA replication stress, DNA double-strand breaks (DSBs), and cell cycle arrest in a catalytic activity-dependent manner 10, 11, 12, 14. A3A is much more catalytically active than A3B, but A3B is typically expressed at higher levels than A3A in tumors. In addition to their differences in substrate specificity, A3A and A3B are also distinct in their catalytic activities. The distinct sequence and structural preferences of A3A and A3B have made it possible to distinguish the contributions of these enzymes to mutagenesis in tumors. Similar to its preference for DNA stem-loops, A3A also recognizes RNA stem-loops in a sequence-specific manner 16.
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Importantly, recurrent APOBEC-signature mutations in different cancer patients are not randomly distributed in the genome but enriched in DNA stem-loops, suggesting that A3A is a major driver of mutations in tumors. In addition, we recently found that A3A but not A3B displays a preference for substrate sites in DNA stem-loops 8. Using yeast as a model to characterize A3A/B-mediated mutagenesis, the Gordenin laboratory identified differences at the substrate sites of A3A and A3B, with A3A preferring the YTCA motif and A3B favoring the RTCA motif (R is a purine and Y is a pyrimidine) 15. While A3A and A3B have similar cytidine deaminase activities, their substrate specificities are not identical.
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In this study, we investigated how to specifically quantify the activity of A3A in tumors. To exploit the A3A/B-induced vulnerability of cancer cells, it is critical to quantitatively measure the A3A/B activities in tumors.
The A3A and A3B activities in cancer cells induce DNA replication stress and DNA breaks 9, 10, 11, 12, 13, offering an opportunity to target the cancer cells harboring high levels of A3A/B activities by disrupting the DNA damage response (DDR) 10, 14. The mutagenesis induced by A3A and A3B in cancer cells may not only contribute to tumor evolution but also confer a vulnerability. In breast, lung, head & neck, esophageal, bladder, and cervical cancers, A3A and A3B are abnormally expressed, which is associated with high levels of APOBEC-signature mutations 2, 4, 8, 10. However, A3A and A3B expression is detected in ~50% of tumors 4, 8, 9.
A3A and A3B are typically expressed after viral infection to defend cells against foreign DNA or RNA, but are not expressed in proliferating normal cells 4. In particular, APOBEC3A (A3A) and APOBEC3B (A3B), which target the TpC motif in single-stranded DNA (ssDNA) 4, 5, 6, 7, are shown to be the main source of APOBEC-signature mutations in patients’ tumors 1, 2, 3. Members of the APOBEC3 family are capable of deaminating cytosine into uracil in DNA and/or RNA 4. Among the 30+ mutational signatures identified to date, at least two are associated with APOBEC (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) proteins 1, 2, 3. Recent cancer genomics studies on tumors of a variety of cancer types have revealed important mechanisms driving tumor evolution and presented opportunities for cancer therapy. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance.
APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells.
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