Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Differentiation signals induce APOBEC3A expression via GRHL3 in squamous epithelia and squamous cell carcinoma.

Two APOBEC (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-like) DNA cytosine deaminase enzymes (APOBEC3A and APOBEC3B) generate somatic mutations in cancer, driving tumour development and drug resistance. Here we used single cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires Grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell cycle stage associated with APOBEC-mediated mutagenesis. In contrast, we show that in squamous cell carcinoma tissues, there is expansion of GRHL3 expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings indicate a mechanism for acquisition of APOBEC3A mutagenic activity in tumours.

Mesoscale DNA Features Impact APOBEC3A and APOBEC3B Deaminase Activity and Shape Tumor Mutational Landscapes.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets singlestranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have been fully established, and the specific influence of the DNA sequence on APOBEC3A APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B selectively targets DNA stem-loop structures, and they are distinct from those subjected deamination by APOBEC3A. We develop Oligo-seq, a novel in vitro sequencing-based to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A an APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Mitochondrial double-stranded RNA triggers induction of the antiviral DNA deaminase APOBEC3A and nuclear DNA damage.

APOBEC3A is an antiviral DNA deaminase often induced by virus infection. APOBEC3A is also a source of cancer mutation in viral and nonviral tumor types. It is therefore critical to identify factors responsible for APOBEC3A upregulation. Here, we test the hypothesis that leaked mitochondrial (mt) double-stranded (ds)RNA is recognized as foreign nucleic acid, which triggers innate immune signaling, APOBEC3A upregulation, and DNA damage. Knockdown of an enzyme responsible for degrading mtdsRNA, the exoribonuclease polynucleotide phosphorylase, results in mtdsRNA leakage into the cytosol and induction of APOBEC3A expression. APOBEC3A upregulation by cytoplasmic mtdsRNA requires RIG-I, MAVS, and STAT2 and is likely part of a broader type I interferon response. Importantly, although mtdsRNA-induced APOBEC3A appears cytoplasmic by subcellular fractionation experiments, its induction triggers an overt DNA damage response characterized by elevated nuclear γ-H2AX staining. Thus, mtdsRNA dysregulation may induce APOBEC3A and contribute to observed genomic instability and mutation signatures in cancer.

Therapy-induced APOBEC3A drives evolution of persistent cancer cells.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.

APOBEC3B drives PKR-mediated translation shutdown and protects stress granules in response to viral infection.

Double-stranded RNA produced during viral replication and transcription activates both protein kinase R (PKR) and ribonuclease L (RNase L), which limits viral gene expression and replication through host shutoff of translation. In this study, we find that APOBEC3B forms a complex with PABPC1 to stimulate PKR and counterbalances the PKR-suppressing activity of ADAR1 in response to infection by many types of viruses. This leads to translational blockage and the formation of stress granules. Furthermore, we show that APOBEC3B localizes to stress granules through the interaction with PABPC1. APOBEC3B facilitates the formation of protein-RNA condensates with stress granule assembly factor (G3BP1) by protecting mRNA associated with stress granules from RNAse L-induced RNA cleavage during viral infection. These results not only reveal that APOBEC3B is a key regulator of different steps of the innate immune response throughout viral infection but also highlight an alternative mechanism by which APOBEC3B can impact virus replication without editing viral genomes.

A digital PCR-based protocol to detect and quantify RNA editing events at hotspots.

APOBEC3A, CRISPR programmable RNA base editors, or other enzymes can edit RNA transcripts at specific locations or hotspots. Precise quantification of these RNA-editing events is crucial to determine the activity and efficiency of these enzymes in cells. We have developed a quick method to quantify RNA-editing activity using digital PCR, a sensitive and quantitative technique to detect rare mutations by micro-partitioning bulk PCR reactions. This assay allows rapid absolute quantification of RNA editing events in cell lines or patient samples. For complete details on the use and execution of this protocol, please refer to Jalili et al. (2020) and Oh et al. (2021).

Genotoxic stress and viral infection induce transient expression of APOBEC3A and pro-inflammatory genes through two distinct pathways.

APOBEC3A is a cytidine deaminase driving mutagenesis in tumors. While APOBEC3A-induced mutations are common, APOBEC3A expression is rarely detected in cancer cells. This discrepancy suggests a tightly controlled process to regulate episodic APOBEC3A expression in tumors. In this study, we find that both viral infection and genotoxic stress transiently up-regulate APOBEC3A and pro-inflammatory genes using two distinct mechanisms. First, we demonstrate that STAT2 promotes APOBEC3A expression in response to foreign nucleic acid via a RIG-I, MAVS, IRF3, and IFN-mediated signaling pathway. Second, we show that DNA damage and DNA replication stress trigger a NF-κB (p65/IkBα)-dependent response to induce expression of APOBEC3A and other innate immune genes, independently of DNA or RNA sensing pattern recognition receptors and the IFN-signaling response. These results not only reveal the mechanisms by which tumors could episodically up-regulate APOBEC3A but also highlight an alternative route to stimulate the immune response after DNA damage independently of cGAS/STING or RIG-I/MAVS.

Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor.

The Hippo pathway, which plays a critical role in organ size control and cancer, features numerous WW domain-based protein-protein interactions. However, ~100 WW domains and 2,000 PY motif-containing peptide ligands are found in the human proteome, raising a "WW-PY" binding specificity issue in the Hippo pathway. In this study, we have established the WW domain binding specificity for Hippo pathway components and uncovered a unique amino acid sequence required for it. By using this criterion, we have identified a WW domain-containing protein, STXBP4, as a negative regulator of YAP. Mechanistically, STXBP4 assembles a protein complex comprising α-catenin and a group of Hippo PY motif-containing components/regulators to inhibit YAP, a process that is regulated by actin cytoskeleton tension. Interestingly, STXBP4 is a potential tumor suppressor for human kidney cancer, whose downregulation is correlated with YAP activation in clear cell renal cell carcinoma. Taken together, our study not only elucidates the WW domain binding specificity for the Hippo pathway, but also reveals STXBP4 as a player in actin cytoskeleton tension-mediated Hippo pathway regulation.