RESUMEN
APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.
Asunto(s)
Transformación Celular Neoplásica/genética , Citidina Desaminasa/genética , ADN/genética , Antígenos de Histocompatibilidad Menor/genética , Conformación de Ácido Nucleico , Proteínas/genética , Secuencia de Bases/genética , Línea Celular Tumoral , Escherichia coli/genética , Células HEK293 , Humanos , Mutagénesis , Mutación , Neoplasias/genéticaRESUMEN
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.
Asunto(s)
Citidina Desaminasa/genética , Daño del ADN , Regulación de la Expresión Génica , Inmunidad/genética , Proteínas/genética , Transducción de Señal/fisiología , Línea Celular , Línea Celular Tumoral , Citidina Desaminasa/metabolismo , Interacciones Huésped-Patógeno , Humanos , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células THP-1 , Factor de Transcripción ReIA/metabolismo , Regulación hacia Arriba , Virus/crecimiento & desarrolloRESUMEN
APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.
Asunto(s)
Citidina Desaminasa/genética , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/genética , Proteínas/genética , Edición de ARN , Línea Celular , Línea Celular Tumoral , Citidina Desaminasa/metabolismo , Pruebas de Enzimas/métodos , Células HEK293 , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Proteínas/metabolismo , Interferencia de ARN , Secuenciación del Exoma/métodosRESUMEN
Cancer drivers require statistical modeling to distinguish them from passenger events, which accumulate during tumorigenesis but provide no fitness advantage to cancer cells. The discovery of driver genes and mutations relies on the assumption that exact positional recurrence is unlikely by chance; thus, the precise sharing of mutations across patients identifies drivers. Examining the mutation landscape in cancer genomes, we found that many recurrent cancer mutations previously designated as drivers are likely passengers. Our integrated bioinformatic and biochemical analyses revealed that these passenger hotspot mutations arise from the preference of APOBEC3A, a cytidine deaminase, for DNA stem-loops. Conversely, recurrent APOBEC-signature mutations not in stem-loops are enriched in well-characterized driver genes and may predict new drivers. This demonstrates that mesoscale genomic features need to be integrated into computational models aimed at identifying mutations linked to diseases.