Addressing the benefits of inhibiting APOBEC3-dependent mutagenesis in cancer

Abstract

Mutational signatures associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC)3 cytosine deaminase activity have been found in over half of cancer types, including some therapy-resistant and metastatic tumors. Driver mutations can occur in APOBEC3-favored sequence contexts, suggesting that mutagenesis by APOBEC3 enzymes may drive cancer evolution. The APOBEC3-mediated signatures are often detected in subclonal branches of tumor phylogenies and are acquired in cancer cell lines over long periods of time, indicating that APOBEC3 mutagenesis can be ongoing in cancer. Collectively, these and other observations have led to the proposal that APOBEC3 mutagenesis represents a disease-modifying process that could be inhibited to limit tumor heterogeneity, metastasis and drug resistance. However, critical aspects of APOBEC3 biology in cancer and in healthy tissues have not been clearly defined, limiting well-grounded predictions regarding the benefits of inhibiting APOBEC3 mutagenesis in different settings in cancer. We discuss the relevant mechanistic gaps and strategies to address them to investigate whether inhibiting APOBEC3 mutagenesis may confer clinical benefits in cancer.

Figure 1.. APOBEC3 mutagenesis is predicted to be a disease-modifying process in cancer that may be exploited therapeutically.

A) APOBEC3 enzymes convert cytosine to uracil by deamination of cytosine bases in single-stranded DNA. Depending on the subsequent uracil-processing, different types of mutations can arise. Note, translesion synthesis (TLS) is predicted to give rise to multiple mutation types (C>A, C>G, C>T) at cytosine bases. UNG (uracil DNA glycosylase). B) (1) APOBEC3 mutagenesis is speculated to drive cancer cell evolution and associated phenotypes, including tumor heterogeneity, therapeutic resistance and metastases. (2) Inhibition of APOBEC3 mutagenesis alongside the standards of care may limit phenotypes associated with cancer cell evolution. The extent to which such phenotypes would be limited upon APOBEC3 inhibition in individual cancers likely depends in part on the strength of other mutational processes that contribute to cancer evolution. (3) If mutations driving resistance to a standard of care have already been acquired in a cancer before the start of the treatment, APOBEC3 inhibition alongside the standard of care may diminish evolution of tumor heterogeneity, but is not predicted to eliminate resistance to the relevant therapy.

Figure 2.. Surrogate readouts of active mutagenesis by individual APOBEC3 enzymes.

The degree to which assays of APOBEC3 expression (mRNA and protein) and deaminase activity upon DNA probes (e.g. probe cleavage assays) or upon endogenous RNA (e.g. 3D-PCR of commonly targeted transcripts) reflect active mutagenesis upon a genome is not clear. Mutations in cancer (including mutational signatures, mutations at extended sequence contexts and mutations at hairpin loops) indicate the historic exposure of a genome to APOBEC3 mutagenesis, but such readouts do not inform on whether APOBEC3 mutagenesis is active in a given tumor at the time of sampling. Pol, polymerase; A3A, APOBEC3A; A3B, APOBEC3B; RTCN/YTCN: R, purine base; Y, pyrimidine; N, any base.

Figure 3.. Addressing impact of APOBEC3 enzymes on cancer evolution.

APOBEC3 mutators can be identified by knockouts (KO) of individual enzymes in human cell lines, which actively acquire APOBEC3-associated mutations, from cancer types commonly presenting with APOBEC3-associated signatures (percentages of cancers presenting with APOBEC3-associated signatures in examplar cancer types are indicated). To detect the impact of APOBEC3 deletions on mutation acquisition, genomes of single-cell derived parent clones can be compared to genomes of their respective daughters obtained after defined in vitro periods. To investigate the roles of APOBEC3 mutagenesis in cancer cell evolution, identified APOBEC3 mutators can be expressed in murine models, which lack most human APOBEC3 gene orthologs. APOBEC3 expression can be performed upon genetic backgrounds and in tissues in which APOBEC3-associated mutations present in human cancers. Tumors that may result from APOBEC3 activities can be profiled for driver mutations and those can be compared to driver mutations in human cancers. To control for neomorphic effects, experiments in transgenic models can be accompanied by experiments in patient-derived xenografts and human cancer cell lines with active APOBEC3 mutagenesis. Analyses can be performed in cancers and models of various stages of the disease and/or upon therapy treatment to investigate the ability of APOBEC3 mutagenesis to contribute to different stages of cancer cell evolution, such as tumor heterogeneity, metastasis and resistance.