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

Abstract

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which enzyme(s) are responsible. Here we develop a selectable system to quantify genome mutation and compare the mutagenic activities of three leading candidates - APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, was engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Clonal expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggered elevated DNA damage responses and increased frequencies of TK mutation. Mutant TK DNA sequences revealed nearly indistinguishable cytosine mutation patterns. Whole genome sequences from TK mutant clones confirmed these results and enabled broader bioinformatic analyses. Most importantly, comparisons of “pure” APOBEC3A- and APOBEC3B-inflicted mutation signatures from this system and the actual APOBEC3 signature from breast cancer indicated that most tumors manifest a composite signature. These studies help resolve a long-standing etiologic debate in the cancer field and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Targeted cancer therapy induces APOBEC fuelling the evolution of drug resistance

“…The collective data suggest that inhibiting APOBEC3B-driven tumour evolution could suppress the emergence of one key class of resistance mutations and thereby improve response to targeted therapy. It is also possible and likely that other APOBEC3 family members beyond A3B contribute to genetic evolution of resistance 5,52 , although A3B has been shown to be a contributor to mutagenesis in a human cell line model system 6 . We must also not discount the potential contribution of chromosomal instability induced by A3B to TKI resistance.…”

“…There are seven human APOBEC3 family members (APOBEC3A-H) and all but one (G) preferentially deaminate TC dinucleotide substrates 3 . In patients, both APOBEC3A (A3A) 5 and APOBEC3B (A3B) 6 have been implicated to play a major role in NSCLC 3 . In this study, the impact of A3B on epidermal growth factor receptor (EGFR)-driven lung cancer evolution was assessed.…”

Human APOBEC3B promotes tumor heterogeneityin vivoincluding signature mutations and metastases

“…These mutations occur predominantly in TCA, TCC, and TCT trinucleotide motifs, consistent with the established biochemical and structural preferences of A3B in which the target cytosine is most frequently preceded by thymine. 10,[26][27][28][29] Indeed, this TC-focused C-to-T mutation bias is known as SBS2, and the calculated percentage of SBS2 mutations in CAG-A3B tumors associates positively with overall APOBEC mutation signature enrichment score (red data points in Figure S7B). In contrast, neither SBS2 nor significant APOBEC mutation signature enrichment is observed in tumors from WT animals, which is expected because short read WGS predominantly identifies clonal (or near-clonal) somatic mutations (black data points in Figure S7B).…”

“…Although most of these enzymes preferentially deaminate TC motifs in single-stranded (ss)DNA, a number of studies have converged on A3A and A3B as the major sources of APOBEC signature mutations in cancer (refs. 10, 11 and references therein). Specifically, expression of A3A or A3B triggers an abundance of APOBEC signature mutations in human cells, and CRISPR-mediated gene knockouts lower the capacity of cancer cell lines to accumulate both SBS2 and SBS13 mutation signatures.…”

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A

“…In particular, A3A and A3B promote mutagenesis of genomic DNA fueling cancer evolution leading to drug resistance and poor disease outcomes 5-7 . Recent studies show that A3A is capable of driving carcinogenesis in mice 9 and contributing the majority of APOBEC signature mutations to human cancer cells 12-13 . Therapeutic applications of A3 inhibitors have been proposed as a strategy to prevent A3-mediated evolution of primary tumors into lethal metastatic and drug-resisting secondary growths 14-15 .…”