APOBEC3 mutagenesis
The Maciejowski lab is studying the role of the APOBEC3 (A3) family of cytosine deaminases in cancer mutagenesis. The A3s, which target ssDNA and RNA of viruses and retroelements as part of the innate immune defense, are proposed to cause two of the most prominent mutational signatures in human cancers. Progress in testing the mutagenic capacity of individual A3 enzymes in an endogenous setting has been hindered by differences between the human and murine A3 loci and the lack of characterized human cancer cell models with endogenous A3 mutagenesis. We have used our cell model of telomere crisis to demonstrate that the A3B paralog is a source of clustered mutations, or kataegis. More recently, we have used cancer cell line models with active A3 mutagenesis to show that the A3A paralog is a major driver of SBS2 and SBS13, in essence proving that A3A is a major enzymatic source of mutations in cancer. The current goals of the lab are to 1. Dissect the roles of individual A3 enzymes in cancer mutagenesis; 2. Identify causes of A3 dysfunction in cancer; 3. Investigate modulators of A3 activity in cancer.
APOBEC3A is a major driver of cancer mutagenesis
Multiple indirect associations have implicated A3A and/or A3B as major sources of cancer mutagenesis. However, in the absence of direct evidence the identity of the major mutator remains a subject of intense debate. To delineate the roles of A3A and A3B in cancer mutagenesis, we deleted A3A and A3B from two BRCA cell lines (BT-474 and MDA-MB-453), and two BCL cell lines (BC-1 and JSC-1) that acquire A3-associated mutations over time. Wild-type or KO “parent” clones were cultured for 60-143 days before isolating single-cell “daughter” clones. Both parent and daughter clones were subsequently DNA sequenced. This workflow enabled the detection of mutations unique to daughter clones thus identifying mutations acquired de novo over a defined period of in vitro propagation. As expected, A3 mutations accumulated in wild-type clones of all cell lines, often at variable rates. Daughter clones isolated from A3A KO parents exhibited severely diminished genomewide and kataegis-associated SBS2 and SBS13 mutations. 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, while 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. Read more here.
APOBEC3B induces kataegis during telomere crisis
Chromothripsis after telomere crisis is accompanied by kataegis with the hallmark of A3 cytosine deaminase editing: clustered and strand-coordinated mutations in cytosine residues in TCA or TCT triplets. To determine the causes of kataegis in our telomere crisis model system we targeted the A3B locus by CRISPR-Cas9 editing. We focused on A3B because the other likely A3 candidates were not expressed in our telomere crisis model. A3B-deficient cell lines were subjected to telomere crisis alongside A3B-deficient counterparts and clonal post-crisis descendants were isolated for whole-genome sequencing. Deletion of A3B did not affect the prevalence of chromothripsis in this system, however, kataegis was severely diminished in A3B knockout post-crisis clones. A3B mutagenesis occurs independently of A3B transcriptional upregulation during telomere crisis. Instead, A3B appears to target ssDNA formed by TREX1-dependent nucleolytic degradation of the DNA bridges formed during telomere crisis. Collectively, these data provide experimental evidence for the link between A3 activity and the generation of SBS2 and SBS13 in cancer genomes. Read more here.