RESUMEN

Activation-induced cytidine deaminase (AID) is responsible for the initiation of somatic hypermutation (SHM) and class-switch recombination (CSR), which result in antibody affinity maturation and isotype switching, thus producing pathogen-specific antibodies. Chromatin dynamics and accessibility play a significant role in determining AID expression and its targeting. Chromatin remodelers contribute to the accessibility of the chromatin structure, thereby influencing the targeting of AID to Ig genes. Epigenetic modifications, including DNA methylation, histone modifications, and miRNA expression, profoundly impact the regulation of AID and chromatin remodelers targeting Ig genes. Additionally, epigenetic modifications lead to chromatin rearrangement and thereby can change AID expression levels and its preferential targeting to Ig genes. This interplay is symbolized as the ACE phenomenon encapsulates three interconnected aspects: AID, Chromatin remodelers, and Epigenetic modifications. This review emphasizes the importance of understanding the intricate relationship between these aspects to unlock the therapeutic potential of these molecular processes and molecules.

Asunto(s)

Diversidad de Anticuerpos , Ensamble y Desensamble de Cromatina , Citidina Desaminasa , Epigénesis Genética , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Humanos , Animales , Diversidad de Anticuerpos/genética , Cambio de Clase de Inmunoglobulina/genética , Metilación de ADN , Hipermutación Somática de Inmunoglobulina , Cromatina/metabolismo , Cromatina/genética , Regulación de la Expresión Génica

RESUMEN

The combination of cisplatin and pemetrexed remains the gold standard chemotherapy for malignant pleural mesothelioma (MPM), although resistance and poor response pose a significant challenge. Cytidine deaminase (CDA) is a key enzyme in the nucleotide salvage pathway and is involved in the adaptive stress response to chemotherapy. The cytidine analog capecitabine and its metabolite 5'-deoxy-5-fluorocytidine (5'-DFCR) are converted via CDA to 5-fluorouracil, which affects DNA and RNA metabolism. This study investigated a schedule-dependent treatment strategy, proposing that initial chemotherapy induces CDA expression, sensitizing cells to subsequent capecitabine treatment. Basal CDA protein expression was low in different mesothelioma cell lines but increased in the corresponding xenografts. Standard chemotherapy increased CDA protein levels in MPM cells in vitro and in vivo in a schedule-dependent manner. This was associated with epithelial-to-mesenchymal transition and with HIF-1alpha expression at the transcriptional level. In addition, pretreatment with cisplatin and pemetrexed in combination sensitized MPM xenografts to capecitabine. Analysis of a tissue microarray (TMA) consisting of samples from 98 human MPM patients revealed that most human MPM samples had negative CDA expression. While survival curves based on CDA expression in matched samples clearly separated, significance was not reached due to the limited sample size. In non-matched samples, CDA expression before but not after neoadjuvant therapy was significantly associated with worse overall survival. In conclusion, chemotherapy increases CDA expression in xenografts, which is consistent with our in vitro results in MPM and lung cancer. A subset of matched patient samples showed increased CDA expression after therapy, suggesting that a schedule-dependent treatment strategy based on chemotherapy and capecitabine may benefit a selected MPM patient population.

Asunto(s)

Capecitabina , Citidina Desaminasa , Mesotelioma Maligno , Pemetrexed , Neoplasias Pleurales , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Capecitabina/farmacología , Animales , Línea Celular Tumoral , Mesotelioma Maligno/tratamiento farmacológico , Mesotelioma Maligno/metabolismo , Mesotelioma Maligno/patología , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Ratones , Pemetrexed/farmacología , Neoplasias Pleurales/tratamiento farmacológico , Neoplasias Pleurales/metabolismo , Neoplasias Pleurales/patología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Mesotelioma/tratamiento farmacológico , Mesotelioma/metabolismo , Mesotelioma/patología , Femenino , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos

RESUMEN

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

Asunto(s)

Citidina Desaminasa , Cambio de Clase de Inmunoglobulina , Estructuras R-Loop , Cambio de Clase de Inmunoglobulina/genética , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/química , Animales , Ratones , Metilación , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Recombinación Genética , ARN/metabolismo , ARN/genética

RESUMEN

APOBEC proteins are cytidine deaminases that restrict the replication of viruses and transposable elements. Several members of the APOBEC3 family, APOBEC3A, APOBEC3B, and APOBEC3H-I, can access the nucleus and cause what is thought to be indiscriminate deamination of the genome, resulting in mutagenesis and genome instability. Although APOBEC3C is also present in the nucleus, the full scope of its deamination target preferences is unknown. By expressing human APOBEC3C in a yeast model system, I have defined the APOBEC3C mutation signature, as well as the preferred genome features of APOBEC3C targets. The APOBEC3C mutation signature is distinct from those of the known cancer genome mutators APOBEC3A and APOBEC3B. APOBEC3C produces DNA strand-coordinated mutation clusters, and APOBEC3C mutations are enriched near the transcription start sites of active genes. Surprisingly, APOBEC3C lacks the bias for the lagging strand of DNA replication that is seen for APOBEC3A and APOBEC3B. The unique preferences of APOBEC3C constitute a mutation profile that will be useful in defining sites of APOBEC3C mutagenesis in human genomes.

Asunto(s)

Citidina Desaminasa , Mutación , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Humanos , Genoma Humano , Replicación del ADN , Proteínas/genética , Proteínas/metabolismo , Mutagénesis , Saccharomyces cerevisiae/genética , Antígenos de Histocompatibilidad Menor

RESUMEN

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Citidina Desaminasa , Daño del ADN , Replicación del ADN , Humanos , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Inestabilidad Genómica , Línea Celular Tumoral , Proteínas

RESUMEN

APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.

Asunto(s)

Desaminasa APOBEC-3G , ADN Complementario , VIH-1 , Mutación , Humanos , Desaminasa APOBEC-3G/genética , Desaminasa APOBEC-3G/metabolismo , ADN Complementario/genética , ADN Viral/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Linfocitos T/virología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Replicación Viral/genética

RESUMEN

Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA, but the quick degradation in water limited the applicability as a potential therapeutic. To improve stability in water, we synthesised derivatives of phosphapyrimidine nucleoside having a CH2 group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide and a negatively charged phosphinic acid derivative had excellent stability in water at pH 7.4, but only the charge-neutral compound inhibited human CDA, similar to previously described 2'-deoxyzebularine (Ki = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, under basic conditions, the charge-neutral phosphinamide was unstable, which prevented the incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative was incorporated into DNA instead of the target 2'-deoxycytidine using an automated DNA synthesiser, but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that the negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.

RESUMEN

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics and also offering a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

RESUMEN

Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that catalyzes the hydrolytic deamination of free cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Our team discovered that CDA deficiency is associated with several aspects of genetic instability, such as increased sister chromatid exchange and ultrafine anaphase bridge frequencies. Based on these results, we sought (1) to determine how CDA deficiency contributes to genetic instability, (2) to explore the possible relationships between CDA deficiency and carcinogenesis, and (3) to develop a new anticancer treatment targeting CDA-deficient tumors. This review summarizes our major findings indicating that CDA deficiency is associated with a genetic instability that does not confer an increased cancer risk. In light of our results and published data, I propose a novel hypothesis that loss of CDA, by reducing basal PARP-1 activity and increasing Tau levels, may reflect an attempt to prevent, slow or reverse the process of carcinogenesis.

Asunto(s)

Carcinogénesis , Citidina Desaminasa , Poli(ADP-Ribosa) Polimerasa-1 , Humanos , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Carcinogénesis/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Animales , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas tau/metabolismo , Proteínas tau/genética , Inestabilidad Genómica

RESUMEN

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Asunto(s)

Citidina Desaminasa , ADN , Humanos , Desaminación , Citidina Desaminasa/metabolismo , ADN/metabolismo , ADN/química , Cinética , Desaminasas APOBEC/metabolismo , Inhibidores Enzimáticos/farmacología

RESUMEN

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

Asunto(s)

Linfocitos B , Citidina Desaminasa , Centro Germinal , Linfoma de Células B , Humanos , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Centro Germinal/inmunología , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Animales , Linfocitos B/inmunología , Hipermutación Somática de Inmunoglobulina , Regulación Neoplásica de la Expresión Génica , Cambio de Clase de Inmunoglobulina

RESUMEN

Plant mitochondrial and chloroplast transcripts are subject to numerous events of specific cytidine-to-uridine (C-to-U) RNA editing to correct genetic information. Key protein factors for this process are specific RNA-binding pentatricopeptide repeat (PPR) proteins, which are encoded in the nucleus and post-translationally imported into the two endosymbiotic organelles. Despite hundreds of C-to-U editing sites in the plant organelles, no comparable editing has been found for nucleo-cytosolic mRNAs raising the question why plant RNA editing is restricted to chloroplasts and mitochondria. Here, we addressed this issue in the model moss Physcomitrium patens, where all PPR-type RNA editing factors comprise specific RNA-binding and cytidine deamination functionalities in single proteins. To explore whether organelle-type RNA editing can principally also take place in the plant cytosol, we expressed PPR56, PPR65 and PPR78, three editing factors recently shown to also function in a bacterial setup, together with cytosolic co-transcribed native targets in Physcomitrium. While we obtained unsatisfying results upon their constitutive expression, we found strong cytosolic RNA editing under hormone-inducible expression. Moreover, RNA-Seq analyses revealed varying numbers of up to more than 900 off-targets in other cytosolic transcripts. We conclude that PPR-mediated C-to-U RNA editing is not per se incompatible with the plant cytosol but that its limited target specificity has restricted its occurrence to the much less complex transcriptomes of mitochondria and chloroplast in the course of evolution.

Asunto(s)

Bryopsida , Cloroplastos , Citosol , Mitocondrias , Edición de ARN , ARN de Planta , Cloroplastos/metabolismo , Cloroplastos/genética , Citosol/metabolismo , Bryopsida/genética , Bryopsida/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Citidina/metabolismo , Citidina/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Regulación de la Expresión Génica de las Plantas , Uridina/metabolismo , Uridina/genética

RESUMEN

The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various in silico approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, "uncharacterized proteins" and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate in silico predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology.

Asunto(s)

Citidina Desaminasa , Ácidos Nucleicos , Animales , Desaminasas APOBEC-1/genética , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , ADN , Citidina

RESUMEN

Phage-assisted evolution has emerged as a powerful technique for improving a protein's function by using mutagenesis and selective pressure. However, mutations typically occur throughout the host's genome and are not limited to the gene-of-interest (GOI): these undesirable genomic mutations can yield host cells that circumvent the system's selective pressure. Our system targets mutations specifically toward the GOI by combining T7 targeted mutagenesis and phage-assisted evolution. This system improves the structure and function of proteins by accumulating favorable mutations that can change its binding affinity, specificity, and activity.

Asunto(s)

Bacteriófagos , Bacteriófagos/genética , Mutación , Mutagénesis , Bacteriófago T7/genética

RESUMEN

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

Asunto(s)

Antimetabolitos Antineoplásicos , Citidina Desaminasa , Desoxicitidina , Resistencia a Antineoplásicos , Enterococcus faecium , Neoplasias de la Vesícula Biliar , Gemcitabina , Humanos , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/uso terapéutico , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Línea Celular Tumoral , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/química , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/metabolismo , Desoxicitidina/química , Enterococcus faecium/enzimología , Enterococcus faecium/genética , Neoplasias de la Vesícula Biliar/tratamiento farmacológico , Neoplasias de la Vesícula Biliar/genética , Neoplasias de la Vesícula Biliar/microbiología , Gemcitabina/metabolismo , Gemcitabina/farmacología , Gemcitabina/uso terapéutico

RESUMEN

Antibody-coding genes accumulate somatic mutations to achieve antibody affinity maturation. Genetic dissection using various mouse models has shown that intrinsic hypermutations occur preferentially and are predisposed in the DNA region encoding antigen-contacting residues. The molecular basis of nonrandom/preferential mutations is a long-sought question in the field. Here, we summarize recent findings on how single-strand (ss)DNA flexibility facilitates activation-induced cytidine deaminase (AID) activity and fine-tunes the mutation rates at a mesoscale within the antibody variable domain exon. We propose that antibody coding sequences are selected based on mutability during the evolution of adaptive immunity and that DNA mechanics play a noncoding role in the genome. The mechanics code may also determine other cellular DNA metabolism processes, which awaits future investigation.

Asunto(s)

Genes de Inmunoglobulinas , Hipermutación Somática de Inmunoglobulina , Animales , Ratones , Hipermutación Somática de Inmunoglobulina/genética , Mutación , ADN , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo

RESUMEN

PURPOSE: Molecular residual disease (MRD) is the main cause of postoperative recurrence of breast cancer. However, the baseline tumor genomic characteristics and therapeutic implications of breast cancer patients with detectable MRD after surgery are still unknown. MATERIALS AND METHODS: In this study, we enrolled 80 patients with breast cancer who underwent next-generation sequencing-based genetic testing of 1,021 cancer-related genes performed on baseline tumor and postoperative plasma, among which 18 patients had detectable MRD after surgery. RESULTS: Baseline clinical characteristics found that patients with higher clinical stages were more likely to have detectable MRD. Analysis of single nucleotide variations and small insertions/deletions in baseline tumors showed that somatic mutations in MAP3K1, ATM, FLT1, GNAS, POLD1, SPEN, and WWP2 were significantly enriched in patients with detectable MRD. Oncogenic signaling pathway analysis revealed that alteration of the Cell cycle pathway was more likely to occur in patients with detectable MRD (p=0.012). Mutational signature analysis showed that defective DNA mismatch repair and activation-induced cytidine deaminase (AID) mediated somatic hypermutation (SHM) were associated with detectable MRD. According to the OncoKB database, 77.8% (14/18) of patients with detectable MRD had U.S. Food and Drug Administration-approved mutational biomarkers and targeted therapy. CONCLUSION: Our study reports genomic characteristics of breast cancer patients with detectable MRD. The cell cycle pathway, defective DNA mismatch repair, and AID-mediated SHM were found to be the possible causes of detectable MRD. We also found the vast majority of patients with detectable MRD have the opportunity to access targeted therapy.

Asunto(s)

Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Hipermutación Somática de Inmunoglobulina , Mutación , Reparación de la Incompatibilidad de ADN , Genómica , Ubiquitina-Proteína Ligasas/genética

RESUMEN

OBJECTIVE: Cancer is caused by abnormal growth and disruption of homeostatic mechanisms. Breast cancer is a major health problem and the second leading cause of death in women. Capecitabine is a prodrug of 5-fluorouracil, which is a non-cytotoxic agent and is used to treat advanced or metastatic breast cancer. Cytidine deaminase (CDA) plays an important role in the activation of capecitabine by acetylating 5-deoxycytidine in the liver, which ultimately leads to the formation of 5-fluorouracil. In this study, we aimed to investigate the relationship between cytidine deaminase polymorphism and capecitabine efficacy in breast cancer patients. METHODS: One hundred breast cancer patients aged 45 to 75 years were included in this study, all of whom received capecitabine as monotherapy. The serum levels of CA15.3, calcium, and estradiol E2 in breast cancer patients were investigated. In addition, the relationship of these markers with cytidine deaminase polymorphism was investigated in order to show the association of cytidine deaminase polymorphism with capecitabine efficacy in breast cancer patients. RESULT: The findings of this study showed that there is a statistically significant relationship between CDA enzyme polymorphisms (rs2072671 and rs532545) with estradiol and CA15.3 serum levels and a non-significant relationship with calcium level. Furthermore, patients who participated were highly polymorphic and heterozygotes genotypes had the highest frequency in both rs2072671 and rs532545 polymorphisms. CONCLUSION: The results obtained from the present study identified different genetic polymorphisms in the gene encoding the CDA enzyme. Overall, our results clearly showed direct evidence for the association of cytidine deaminase polymorphism with the efficacy of capecitabine in breast cancer patients, which could be useful in reducing side effects and improving drug response to capecitabine.

Asunto(s)

Neoplasias de la Mama , Humanos , Femenino , Capecitabina/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/inducido químicamente , Antimetabolitos Antineoplásicos/uso terapéutico , Citidina Desaminasa/genética , Calcio , Polimorfismo Genético/genética , Fluorouracilo/uso terapéutico , Estradiol

RESUMEN

t(8;14) translocation is the hallmark of Burkitt's lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt's lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt's lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.

Asunto(s)

Linfoma de Burkitt , G-Cuádruplex , Humanos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , ADN , Genes myc , Estructuras R-Loop , Translocación Genética

RESUMEN

Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at specific sites in either DNA or RNA by combining a targeting module (such as a catalytically impaired CRISPR-Cas component) and an effector module (most often a deaminase). Deaminase-based molecular tools are already being utilized in a wide spectrum of therapeutic and research applications; however, their medical and biotechnological potential seems to be much greater. Recent reports indicate that the further development of nucleic acid editing systems depends largely on our ability to engineer the substrate specificity and catalytic activity of the editors themselves. In this review, we summarize the current trends and achievements in deaminase engineering. The presented data indicate that the potential of these enzymes has not yet been fully revealed or understood. Several examples show that even relatively minor changes in the structure of deaminases can give them completely new and unique properties.