Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B

Abstract

Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80–90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

A rabbit monoclonal antibody against the antiviral and cancer genomic DNA mutating enzyme APOBEC3B

“…The RTqPCR results largely mirrored the immunoblot data in Fig. 1A for 293T, described previously for the T-REx-293-A3B-eGFP system 25,58,60 , and shown above in Fig 3B for the THP18 cell line and its A3Anull and A3A/B-null derivatives. A3A mRNA is undetectable in the 293T cells (regardless of treatment) and induced by 3 orders of magnitude in IFN-α/LPS treated THP18 and, of course, absent from A3A-null and A3A/B-null derivatives (Fig.…”

“…T-REx-293T is a derivative of 293T engineered to constitutively expresses the tetracycline repressor 25,58,60 . THP18 71 was derived from a single cell subclone of the monocyte cell line THP-null derivative have been described 33,36 , and these lines were maintained in IMEM supplemented with 15% FBS and 1% P/S, non-essential amino acids (0.1 mM), and insulin (11.25 nM).…”

Rev1 is a base excision repair enzyme with 5′-deoxyribose phosphate lyase activity

“…Taken together, these results point to the possibility that Rev1 may process some of the AP sites through error-prone BER. It is also interesting that a mutational signature of APOBEC family of cytidine deaminases was found in breast and several other types of cancers genomic DNA (67–70). This suggests that APOBEC-mediated cytosine deaminase activity and Rev1 dCMP insertion could be involved in cancer genome mutagenesis, in addition to SHM.…”

APOBEC3A/B-induced mutagenesis is responsible for 20% of heritable mutations in the TpCpW context

“…Under these assumptions, we infer that ∼15%-30% of TpCpW→K substitutions resulted from APOBEC3A/B deamination, with a lower fraction for the TpCpA→G mutations (∼15%). The differences in strand bias between different APOBEC3A/B contexts are in line with lower frequencies of TpCpA→G among APOBEC3A/B-induced mutations in cancers (Alexandrov et al 2013) and among APOBEC3B-induced mutations in human cells lines (Akre et al 2016). Similar biases were observed for SNPs at all frequencies (Supplemental Table S2) and for interspecies differences accumulated in the human lineage since divergence from the last common ancestor with the chimpanzee, albeit they are slightly less obvious for divergence (Supplemental Table S2; Supplemental Fig.…”

“…While deamination in the TpCpW context excludes some APOBECs from consideration, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3F, and APOBEC3H (Taylor et al 2013;Kim et al 2014;Saraconi et al 2014) all are plausible suspects. Overexpression of APOBEC1, APOBEC3A, and APOBEC3B is associated with increased mutation rate in vertebrate cell lines, with mutations distributed all over the genome (Saraconi et al 2014;Akre et al 2016;Green et al 2016). Notably, however, APOBEC1 causes C→A mutations in experimental systems (Saraconi et al 2014), while the heritable replication asymmetry is largely restricted to C→T and C→G mutations (Table 1).…”