Mechanisms of APOBEC3 mutagenesis in human cancer cells

Abstract

The APOBEC3 family of cytosine deaminases has been implicated in some of the most prevalent mutational signatures in cancer1–3. However, a causal link between endogenous APOBEC3 enzymes and mutational signatures in human cancer genomes has not been established, leaving the mechanisms of APOBEC3 mutagenesis poorly understood. Here, to investigate the mechanisms of APOBEC3 mutagenesis, we deleted implicated genes from human cancer cell lines that naturally generate APOBEC3-associated mutational signatures over time4. Analysis of non-clustered and clustered signatures across whole-genome sequences from 251 breast, bladder and lymphoma cancer cell line clones revealed that APOBEC3A deletion diminished APOBEC3-associated mutational signatures. Deletion of both APOBEC3A and APOBEC3B further decreased APOBEC3 mutation burdens, without eliminating them. Deletion of APOBEC3B increased APOBEC3A protein levels, activity and APOBEC3A-mediated mutagenesis in some cell lines. The uracil glycosylase UNG was required for APOBEC3-mediated transversions, whereas the loss of the translesion polymerase REV1 decreased overall mutation burdens. Together, these data represent direct evidence that endogenous APOBEC3 deaminases generate prevalent mutational signatures in human cancer cells. Our results identify APOBEC3A as the main driver of these mutations, indicate that APOBEC3B can restrain APOBEC3A-dependent mutagenesis while contributing its own smaller mutation burdens and dissect mechanisms that translate APOBEC3 activities into distinct mutational signatures.

R-loop editing by DNA cytosine deaminase APOBEC3B determines the activity of estrogen receptor enhancers

“…Using the A3B deamination sites detected by WGS, we also added R-loops into the known repository of A3B substrates in addition to lagging DNA synthesis strand 33,35,56 , loop regions of DNA hairpins 10,57 , and ssDNA intermediates during recombination and repair 9,10,58 . The predominant pathway processing A3B-catalysed uracil lesions in the context of R-loops is the BER 24 , which has been observed in previous studies in APOBEC-Cas9-mediated R-loop editing and the AID-catalysed deamination within R-loops during immunoglobulin class switch recombination 27,28 . However, if R-loops are resolved before repair of the uracil lesion, the reannealing of the template strand will lead to a U:G mismatch which can also be repaired by DNA mismatch repair (MMR).…”

“…In order to capture A3B deamination sites that may have a functional impact on protein coding or regulatory DNA sequences, we performed whole-genome sequencing (WGS) of ER-positive T-47D human breast cancer cells co-expressing doxycycline-inducible A3B. and humanised bacteriophage PBS2 uracil glycosylase inhibitor (hUGI) peptide ( Figure 1A , top panel) to inhibit the action of uracil-DNA glycosylase (UDG/UNG) and prevent excision of the A3B-catalysed uracil bases 22, 24 . The T-47D cell line was chosen because it does not express APOBEC3A which may confound the study 6 , and also carries a loss-of-function TP53 mutation that is known to protect the cells from the synthetic lethality caused by ectopic expression of A3B 36 .…”

“…Large-scale whole-genome sequencing (WGS) studies performed on cell lines and primary samples have identified sites of APOBEC editing 10,30 . However, these studies may be confounded by a 'survivorship bias' where the detected A3B-edited sites are limited to those that were not repaired by DDR, as it is known that BER removes U:G mispairing resulting from A3B editing 24,31 . This caveat may also affect studies employing A3Boverexpression cell models [32][33][34][35] .…”

“…In addition to their normal functions as immune defences against DNA viruses and transposons 20, 21 , APOBEC3 catalytic activity has been reported to drive genetic heterogeneity in breast cancer by inducing C>T transitions and C>G transversions at 5’TCW motifs (W = A or T) 9, 22, 23 . Although mounting evidence is pointing APOBEC3A, a highly homologous enzyme to C-terminal A3B, as the main contributor to APOBEC-driven mutation burden and cancer evolution 24 , A3B-driven cytidine deamination still has a prominent role in cancer due to two facts. First, as the only nuclear-localised member of the APOBEC3 family, elevated A3B expression is reported in >50% of primary breast cancers 22, 23 ; and secondly, A3B binds directly to ERα and edits ER binding sites, which triggers DNA damage response (DDR) and are then processed by DNA base excision repair (BER) and non-homologous end joining (NHEJ) into DSBs 6 .…”

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer

“…Two A3H-I expressing TK mutant clones were subjected to WGS and no APOBEC3 mutation signature was evident. In addition, no specific evidence for A3H emerged from sequencing clonally-derived cancer cell lines [88]. However, all of these cell-based studies have limitations as discussed above and have yet to fully eliminate A3H as a source of APOBEC3 signature mutations in cancer.…”