Jennifer L. McCann

Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B

APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring −1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The −1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5′-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.

The PKC/NF-κB Signaling Pathway Induces APOBEC3B Expression in Multiple Human Cancers

Overexpression of the antiviral DNA cytosine deaminase APOBEC3B has been linked to somatic mutagenesis in many cancers. Human papillomavirus infection accounts for APOBEC3B upregulation in cervical and head/neck cancers, but the mechanisms underlying nonviral malignancies are unclear. In this study, we investigated the signal transduction pathways responsible for APOBEC3B upregulation. Activation of protein kinase C (PKC) by the diacylglycerol mimic phorbol-myristic acid resulted in specific and dose-responsive increases in APOBEC3B expression and activity, which could then be strongly suppressed by PKC or NF-κB inhibition. PKC activation caused the recruitment of RELB, but not RELA, to the APOBEC3B promoter, implicating noncanonical NF-κB signaling. Notably, PKC was required for APOBEC3B upregulation in cancer cell lines derived from multiple tumor types. By revealing how APOBEC3B is upregulated in many cancers, our findings suggest that PKC and NF-κB inhibitors may be repositioned to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes, such as drug resistance and metastasis.

Polyomavirus T-Antigen Induces APOBEC3B Expression using a LXCXE-Dependent and TP53-Independent Mechanism

APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomaviruses and papillomaviruses use dominant oncoproteins to induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T-antigen. First, truncated T-antigen (truncT) is sufficient for APOBEC3B upregulation and the RB interacting motif (LXCXE), but not the TP53 inhibition domain, is required. Second, upregulated APOBEC3B is strongly nuclear and partially localized to virus replication centers. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B. Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B. Fifth, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB binding motif has in addition to RB inactivation at least one additional function for triggering APOBEC3B upregulation in virus-infected cells. IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpresssed in many different cancers and correlated with elevated frequencies of C-to-T and C-to-G mutations in 5’-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T-antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely enables one or more E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to non-viral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.

APOBEC3B Nuclear Localization Requires Two Distinct N-Terminal Domain Surfaces

The APOBEC3 family of cytosine deaminases catalyzes the conversion of cytosines-to-uracils in single-stranded DNA. Traditionally, these enzymes are associated with antiviral immunity and restriction of DNA-based pathogens. However, a role for these enzymes in tumor evolution and metastatic disease has also become evident. The primary APOBEC3 candidate in cancer mutagenesis is APOBEC3B (A3B) for three reasons: (1) A3B mRNA is upregulated in several different cancers, (2) A3B expression and mutational loads correlate with poor clinical outcomes, and (3) A3B is the only family member known to be constitutively nuclear. Previous studies have mapped non-canonical A3B nuclear localization determinants to a single surface-exposed patch within the N-terminal domain (NTD). Here, we show that A3B has an additional, distinct, surface-exposed NTD region that contributes to nuclear localization. Disruption of residues within the first 30 amino acids of A3B (import surface 1) or loop 5/α-helix 3 (import surface 2) completely abolish nuclear localization. These import determinants also graft into NTDs of related family members and mediate re-localization from cell-wide-to-nucleus or cytoplasm-to-nucleus. These findings demonstrate that both sets of residues are required for non-canonical A3B nuclear localization and describe unique surfaces that may serve as novel therapeutic targets.

Abstract P5-06-03: A solution to the APOBEC mutation paradox in breast cancer

Breast cancer has a major genetic component, and individual tumors can have thousands to tens-of-thousands of mutations. Large proportions are cytosine mutations in TCA and TCT trinucleotide motifs. A variety of studies have attributed this mutation signature to the APOBEC family of DNA cytosine deaminases, with previous literature favoring APOBEC3B as the most likely enzyme to bee responsible (from a total of nine active family members). However, breast tumor genomic DNA sequences from patients with a naturally occurring germline deletion of the entire APOBEC3B gene still show an intact APOBEC mutation signature. To resolve this paradox, we tested the hypothesis that the only other functionally dimorphic APOBEC family member, APOBEC3H, may be responsible. First, we performed an unbiased genetic analysis of TCGA data sets and showed that APOBEC3B-null tumors with this mutational bias have at least one copy of the haplotype-I variant of APOBEC3H, despite weak genetic linkage between these two genes (n=14/14). Remarkably, breast tumors without APOBEC3B and APOBEC3H haplotype-I showed no evidence for an APOBEC mutation signature (n=3/3). The proportion of APOBEC signature mutations between the cohort with and the cohort without APOBEC3H haplotype-I was highly significant (p Citation Format: Harris RS, Starrett GJ, McCann JL, Carpenter MA. A solution to the APOBEC mutation paradox in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-06-03.

Abstract B43: APOBEC3B upregulation by the PKC-NFκB pathway in breast cancer

Overexpression of the antiviral DNA cytosine deaminase APOBEC3B has been linked to somatic mutagenesis in breast and other cancer. Human papillomavirus (HPV) infection accounts for APOBEC3B upregulation in cervical and head/neck cancers. However, the responsible mechanisms are unclear for non-viral malignancies such as breast cancer. Here, we demonstrate APOBEC3B upregulation through the PKC-NFκB pathway. PKC activation by the diacylglycerol mimic PMA causes specific and dose-responsive increases in APOBEC3B mRNA, protein, and activity levels, which are strongly suppressed by PKC and NFκB inhibition. Induction correlates with RELB (but not RELA) recruitment to the endogenous APOBEC3B gene implicating non-canonical NFκB signaling. Relevance to tumors is supported by PKC inhibitor-mediated APOBEC3B downregulation in multiple breast cancer cell lines. These data establish the first mechanistic link between APOBEC3B and a common signal transduction pathway, suggesting that existing PKC-NFκB inhibitors could be repurposed to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes such as drug resistance and metastases. Citation Format: Brandon Leonard, Jennifer McCann, Gabriel Starrett, Leah Kosyakovsky, Molan Amy, Michael Burns, Rebecca McDougle, Peter Parker, William Brown, Reuben Harris. APOBEC3B upregulation by the PKC-NFκB pathway in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B43.

Abstract P4-08-02: APOBEC3B upregulation by the PKC-NFκB pathway in breast cancer

Overexpression of the antiviral DNA cytosine deaminase APOBEC3B has been linked to somatic mutagenesis in breast and other cancer. Human papillomavirus (HPV) infection accounts for APOBEC3B upregulation in cervical and head/neck cancers. However, the responsible mechanisms are unclear for non-viral malignancies such as breast cancer. Here, we demonstrate APOBEC3B upregulation through the PKC-NFκB pathway. PKC activation by the diacylglycerol mimic PMA causes specific and dose-responsive increases in APOBEC3B mRNA, protein, and activity levels, which are strongly suppressed by PKC and NFκB inhibition. Induction correlates with RELB (but not RELA) recruitment to endogenous APOBEC3B implicating non-canonical NFκB signaling. Relevance to tumors is supported by PKC inhibitor-mediated APOBEC3B downregulation in multiple breast cancer cell lines. These data establish the first mechanistic link between APOBEC3B and a common signal transduction pathway, suggesting that existing PKC-NFκB inhibitors could be repurposed to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes such as drug resistance and metastases. Citation Format: McCann J, Leonard B, Starrett G, Kosyakovsky L, Molan A, Burns M, McDougle R, Parker P, Brown W, Harris R. APOBEC3B upregulation by the PKC-NFκB pathway in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-02.