RESUMO
Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUb D2). Here, we present a 4.1 Å cryo-EM structure of IUb D2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUb D2Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUb D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub -DNA complex. The IUb D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUb D2Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUb D2 complex.
Assuntos
Anemia de Fanconi , Humanos , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Lisina/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Ubiquitinação , DNA/metabolismo , Dano ao DNA , Reparo do DNARESUMO
Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, Fanconi anemia complementation group A (FANCA), is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous FANCA variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic FANCA variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel FANCA variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in FANCA, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes.
Assuntos
Alelos , Proteína do Grupo de Complementação A da Anemia de Fanconi , Insuficiência Ovariana Primária , Humanos , Insuficiência Ovariana Primária/genética , Feminino , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Adulto , Anemia de Fanconi/genética , Anemia de Fanconi/diagnóstico , Irmãos , Heterozigoto , Predisposição Genética para Doença , Linhagem , Mutação/genéticaRESUMO
High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of ovarian cancer and demonstrates 5-year survival of just 40%. One of the major causes of mortality is the development of tumour resistance to platinum-based chemotherapy, which can be modulated by dysregulation of DNA damage repair pathways. We therefore investigated the contribution of the DNA interstrand crosslink repair protein FANCD2 to chemosensitivity in HGSOC. Increased FANCD2 protein expression was observed in some cell line models of platinum resistant HGSOC compared with paired platinum sensitive models. Knockdown of FANCD2 in some cell lines, including the platinum resistant PEO4, led to increased carboplatin sensitivity. Investigation into mechanisms of FANCD2 regulation showed that increased FANCD2 expression in platinum resistant cells coincides with increased expression of mTOR. Treatment with mTOR inhibitors resulted in FANCD2 depletion, suggesting that mTOR can mediate platinum sensitivity via regulation of FANCD2. Tumours from a cohort of HGSOC patients showed varied nuclear and cytoplasmic FANCD2 expression, however this was not significantly associated with clinical characteristics. Knockout of FANCD2 was associated with increased cell migration, which may represent a non-canonical function of cytoplasmic FANCD2. We conclude that upregulation of FANCD2, possibly mediated by mTOR, is a potential mechanism of chemoresistance in HGSOC and modulation of FANCD2 expression can influence platinum sensitivity and other tumour cell characteristics.
Assuntos
Carboplatina , Cistadenocarcinoma Seroso , Resistencia a Medicamentos Antineoplásicos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Neoplasias Ovarianas , Humanos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Feminino , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/metabolismo , Regulação Neoplásica da Expressão Gênica , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Movimento Celular/genética , Gradação de Tumores , Platina/farmacologia , Platina/uso terapêuticoRESUMO
Fanconi Anaemia is an autosomal recessive disorder, which is characterised by progressive pancytopenia, café au lait spots (>50%), bruising, petechie, recurrent infections, short height (50%), and thumb and radial bone anomalies (40%). Herein, is presented a case of a lean emaciated female child, who presented with the chief complaints of fever, loose stools and decreased appetite for one month reported at Sindh Government General Hospital, Karachi, on February, 1, 2023. She had cutaneous findings of hyperpigmentation and café au lait spots and a tri-phalangeal thumb. On investigation, pancytopenia and a low reticulocyte count of 0.7% was also observed. Karyotype and chromosomal breakage test induced by Diepoxybutane confirmed her as a case of Fanconi Anaemia.
Assuntos
Manchas Café com Leite , Anemia de Fanconi , Humanos , Feminino , Anemia de Fanconi/complicações , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Manchas Café com Leite/genética , Quebra Cromossômica , Compostos de EpóxiRESUMO
The long-term outcomes of adults with Fanconi anaemia (FA) have improved with advances in haematopoietic stem cell transplantation (HSCT) and more detailed follow-up and screening guidelines. The phenotype of those who survive to adulthood may differ from the typical presentation of FA. We collected retrospective clinical data on adults with FA who received their care at the Cincinnati Children's Hospital Medical Center. In our final cohort of 52 patients, there were 29 females and 23 males, with median (range) age of 21 (18-37) years. Overall, 42 patients (81%) were alive at last follow-up. In all, 36 adults (69%) had undergone HSCT, including eight who had developed myelodysplasia or acute myeloid leukaemia. Eight (15%) developed squamous cell carcinoma. Endocrine complications were common, including hypothyroidism (42%), diabetes (10%), low body mass index (31%) and low bone mineral density (51%). The majority of adults with FA were employed (52%) or full-time students (13%). A significant subset of patients with FA are surviving into adulthood without requiring HSCT. Endocrine abnormalities and the development of solid tumours complicate adulthood. With improved survival outcomes following HSCT and more aggressive malignancy screening protocols, ongoing longitudinal analysis will be important to further characterise this cohort and the phenotype of untransplanted adults with FA.
Assuntos
Anemia de Fanconi , Transplante de Células-Tronco Hematopoéticas , Síndromes Mielodisplásicas , Masculino , Feminino , Humanos , Anemia de Fanconi/complicações , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/terapia , Estudos Retrospectivos , Transplante de Células-Tronco Hematopoéticas/métodos , Síndromes Mielodisplásicas/complicações , FenótipoRESUMO
Fanconi anaemia (FA) is a rare autosomal recessive condition resulting in changes in the FANC gene family. This report describes a case of Fanconi anaemia in a family with complex biallelic variants. The patient is a 32-year-old female diagnosed with FA on cascade testing during childhood with chromosome breakage studies. On examination she had a fixed deformity of the right thumb and the proximal interphalangeal joint was immobile. Her brother shared this radial abnormality and had FA, requiring a bone marrow transplant. She presented in adulthood seeking further BRCA advice and had next generation sequencing that showed three variants in the FANCA gene. One allele a known pathogenic change, the other had two sequence variants in tandem that have been reported as variants of uncertain significance. There is one other unrelated case of these two variants occurring together in cis, resulting in Fanconi anaemia. This case is an interesting example of three variants in the FANCA gene, one allele with a pathogenic deletion and the other with a single complex allele made up of two missense variants of uncertain significance, likely manifesting with FA. It highlights the utility of different genetic technologies in the interpretation of next generation sequencing.
Assuntos
Anemia de Fanconi , Humanos , Masculino , Feminino , Adulto , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Genômica , Mutação de Sentido Incorreto , MutaçãoRESUMO
The ubiquitin signalling pathway is a large system associated with numerous intracellular mechanisms. However, its function in the liver regeneration process remains unknown. This particular study investigates the intracellular effect mechanisms of the ubiquitin signalling pathway. It also determines the differences in the expression of 88 genes belonging to the ubiquitin pathway using the RT-PCR array method. To conduct this research, three genes-that differed in the expression analysis were selected. Moreover, their proteins were analysed by western blot analysis while using Ki67 immunohistochemical analysis that determines proliferation rates by hour. It was determined that BRCA1 and BARD1, which are effective in DNA repair, play an active role at PH24. Similarly, Ube2t expression, which belongs to the Fanconi anaemia pathway associated with DNA repair, was also found to be high at PH12-72 h. In addition, it was revealed that the expressions of Anapc2, Anapc11, Cdc20 belonging to the APC/CCdc20 complex, which participate in cell cycle regulation, differed at different hours after PH. Expression of Mul1, which is involved in mitochondrial biogenesis and mitophagy mechanisms, peaked at PH12 under the observation. Considering the Mul1 gene expression difference, MUL1-mediated mitophagy and mitochondrial fission mechanism may be associated with liver regeneration. It was also determined that PARKIN-mediated mitophagy mechanisms are not active in 0-72 h of liver regeneration since PARKIN expression did not show a significant change in PH groups.
Assuntos
Regeneração Hepática , Ubiquitina , Animais , Ratos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Transdução de Sinais , Proteínas de Ciclo Celular/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Haematopoietic cell transplantation (HCT) is the only curative treatment for haematological complications in patients with Fanconi anaemia (FA). Haploidentical (haplo-) HCT is a promising alternative for FA. We aimed to analyse the outcomes of unmanipulated haplo-HCT in patients with FA with radiation-free conditioning. A total of 56 patients from 11 centres between 2013 and 2021 in China were retrospectively analysed. The mean (SD) cumulative incidence was 96.4% (0.08%) for 30-day neutrophil engraftment and 85.5% (0.24%) for 100-day platelet engraftment. With a median (range) follow-up of 2.4 (0.2-5.8) years, favourable mean (SD) overall survival of 80.9% (5.5%) and event-free survival of 79.3% (5.6%) were achieved. The mean (SD) incidences of acute graft-versus-host disease (aGvHD) Grade II-IV and Grade III-IV were 55.4% (0.45%) and 42.9 (0.45%) respectively. The mean (SD) cumulative incidence of 3-year chronic graft-versus-host disease (cGvHD) was 34.7% (0.86%) and that of moderate-to-severe cGvHD was 9.0% (0.19%). Our data demonstrate that in unmanipulated haplo-HCT for patients with FA, radiation-free regimens based on fludarabine and low-dose cyclophosphamide ± busulfan achieved favourable engraftment and survival with relatively high incidences of aGvHD and cGvHD. These results prompt the use of low-intensity conditioning without radiation and intensive GvHD prophylaxis when considering unmanipulated haplo-HCT in patients with FA.
Assuntos
Anemia de Fanconi , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Bussulfano/uso terapêutico , Estudos Retrospectivos , Condicionamento Pré-Transplante/efeitos adversos , Condicionamento Pré-Transplante/métodos , Anemia de Fanconi/terapia , Anemia de Fanconi/complicações , Medula Óssea , Recidiva Local de Neoplasia/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Ciclofosfamida/uso terapêutico , Sistema de RegistrosRESUMO
The optimal haploidentical haematopoietic cell transplant approach for Fanconi anaemia (FA) patients is not well established, given the rarity of the disease, the increased sensitivity to DNA-damaging agents and the high risk of severe graft-versus-host disease (GVHD). The report by Xu et al. suggests that excellent engraftment and short-term survival can be achieved in FA patients without irradiation, but their retrospective cohort was plagued by a high rate of severe GVHD. Our commentary explores the outcomes in T-cell replete haploidentical haematopoietic cell transplant and ponders whether elimination of total body irradiation in FA patients is the best method if it limits the ability to safely administer post-transplant cyclophosphamide. Commentary on: Xu et al. Unmanipulated haploidentical haematopoietic cell transplantation with radiation-free conditioning in Fanconi anaemia: A retrospective analysis from the Chinese Blood and Marrow Transplantation Registry Group. Br J Haematol. 2022;199:401-410.
Assuntos
Anemia de Fanconi , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Anemia de Fanconi/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante Haploidêntico , Estudos Retrospectivos , Linfócitos T , Ciclofosfamida/uso terapêutico , Condicionamento Pré-Transplante/métodosRESUMO
Recent advances have facilitated studies of the clonal architecture of the aging haematopoietic system, and provided clues to the mechanisms underlying the origins of hematopoietic malignancy. Much less is known about the clonal composition of haematopoiesis and its impact in bone marrow failure (BMF) disorders, including Fanconi anaemia (FA). Understanding clonality in FA is likely to inform both the marked predisposition to cancer and the rapid erosion of regenerative reserve seen with this disease. This may also hold broader lessons for haematopoietic stem cell biology in other diseases with a clonal restriction. In this review, we focus on the conceptual basis and available tools to study clonality, and highlight insights in somatic mosaicism and malignant evolution in FA in the context of haematopoietic failure and gene therapy.
Assuntos
Evolução Clonal/genética , Anemia de Fanconi/etiologia , Anemia de Fanconi/metabolismo , Variação Genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Mosaicismo , Animais , Biomarcadores , Diferenciação Celular/genética , Rastreamento de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Terapia Combinada , Análise Citogenética , Dano ao DNA , Gerenciamento Clínico , Suscetibilidade a Doenças , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/terapia , Regulação da Expressão Gênica , Terapia Genética , Células-Tronco Hematopoéticas/citologia , Humanos , Imagem Molecular , Transdução de SinaisRESUMO
Fanconi anaemia (FA) is an inherited bone marrow failure syndrome (IBMFS) with a high cancer predisposition rate. Traditional diagnoses are made before age 10 years due to bone marrow failure (BMF) and characteristic birth defects. Up to 10% of published cases were adults at diagnosis. We hypothesized that FA subgroups diagnosed in childhood are distinct from those diagnosed as adults. We classified patients by age at diagnosis of FA as FA-PED (<18 years) or FA-ADULT (≥18 years). The National Cancer Institute IBMFS cohort included 178 FA-PED and 26 FA-ADULT cases. We compared various features; the cumulative incidences of first adverse events (severe BMF leading to haematopoietic cell transplant or death, leukaemia, or solid tumours) were compared using competing-risk analyses. FA-ADULT lacked the 'typical' FA features (birth defects and early-onset BMF or leukaemia), were mainly female, had more patients with FANCA genotype, and had or developed more head and neck squamous-cell carcinoma (HNSCC) and/or gynaecological cancers compared with FA-PED, albeit at similar ages in both subgroups. FA-ADULT is a distinct subgroup that remained unrecognized during childhood. Centres for adult haematology-oncology should consider FA diagnosis in patients with early-onset HNSCC or gynaecological cancer with or without haematologic problems.
Assuntos
Anemia de Fanconi , Neoplasias de Cabeça e Pescoço , Transplante de Células-Tronco Hematopoéticas , Leucemia , Adulto , Transtornos da Insuficiência da Medula Óssea , Criança , Síndrome Congênita de Insuficiência da Medula Óssea , Anemia de Fanconi/complicações , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Feminino , Humanos , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary "Arm" ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.
Assuntos
Anemia de Fanconi , DNA/genética , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Humanos , Proteína 2 Inibidora de Diferenciação/genética , UbiquitinaçãoRESUMO
The C1ORF112 gene initially drew attention when it was found to be strongly co-expressed with several genes previously associated with cancer and implicated in DNA repair and cell cycle regulation, such as RAD51 and the BRCA genes. The molecular functions of C1ORF112 remain poorly understood, yet several studies have uncovered clues as to its potential functions. Here, we review the current knowledge on C1ORF112 biology, its evolutionary history, possible functions, and its potential relevance to cancer. C1ORF112 is conserved throughout eukaryotes, from plants to humans, and is very highly conserved in primates. Protein models suggest that C1ORF112 is an alpha-helical protein. Interestingly, homozygous knockout mice are not viable, suggesting an essential role for C1ORF112 in mammalian development. Gene expression data show that, among human tissues, C1ORF112 is highly expressed in the testes and overexpressed in various cancers when compared to healthy tissues. C1ORF112 has also been shown to have altered levels of expression in some tumours with mutant TP53. Recent screens associate C1ORF112 with DNA replication and reveal possible links to DNA damage repair pathways, including the Fanconi anaemia pathway and homologous recombination. These insights provide important avenues for future research in our efforts to understand the functions and potential disease relevance of C1ORF112.
Assuntos
Evolução Biológica , Dano ao DNA , Reparo do DNA , Replicação do DNA , Fases de Leitura Aberta/genética , Animais , Humanos , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Mapas de Interação de Proteínas , Testículo/metabolismoRESUMO
During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Ciclo Celular , Humanos , Modelos BiológicosRESUMO
A safer and more effective combination strategy designed to enhance the efficacy and minimize the toxicity of cisplatin in osteosarcoma (OS) is urgently needed. Zeylenone (zey), a cyclohexene oxide compound, exerted an obvious inhibitory effect on several cancer cell lines and exhibited little cytotoxicity towards normal cells, enabling zey to play a unique role in combination therapy. Thus, the study aimed to determine whether the combination of zey and cisplatin produces synergistic antitumour effects on OS and to further explore molecular mechanisms. Initially, we found that zey potentiated the anti-osteosarcoma efficacy of cisplatin and exhibited synergistic interactions with cisplatin in vitro, which also were confirmed in vivo by using xenograft model. Mechanistically, zey and cisplatin synergistically induced DNA damage, cell cycle arrest, necrosis, and apoptosis in OS cells. Importantly, zey had a high binding affinity for Hsp90 and reduced the expression of Hsp90, which further induced the suppression of AKT/GSK3ß signalling axis and the degradation of Fanconi anaemia (FA) pathway proteins. Thus, the Hsp90/AKT/GSK3ß and FA pathway are the key to the synergism between zey and cisplatin. Overall, zey shows promise for development as a cisplatin chemosensitizer with clinical utility in restoring cisplatin sensitivity of cancer cells.
Assuntos
Antineoplásicos , Neoplasias Ósseas , Anemia de Fanconi , Osteossarcoma , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Cicloexanos , Dano ao DNA , Dioxanos , Glicogênio Sintase Quinase 3 beta , Humanos , Necrose , Osteossarcoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-aktRESUMO
Ovarian cancer is the most lethal gynaecological cancer, and resistance of platinum-based chemotherapy is the main reason for treatment failure. The aim of the present study was to identify candidate genes involved in ovarian cancer platinum response by analysing genes from homologous recombination and Fanconi anaemia pathways. Associations between these two functional genes were explored in the study, and we performed a random walk algorithm based on reconstructed gene-gene network, including protein-protein interaction and co-expression relations. Following the random walk, all genes were ranked and GSEA analysis showed that the biological functions focused primarily on autophagy, histone modification and gluconeogenesis. Based on three types of seed nodes, the top two genes were utilized as examples. We selected a total of six candidate genes (FANCA, FANCG, POLD1, KDM1A, BLM and BRCA1) for subsequent verification. The validation results of the six candidate genes have significance in three independent ovarian cancer data sets with platinum-resistant and platinum-sensitive information. To explore the correlation between biomarkers and clinical prognostic factors, we performed differential analysis and multivariate clinical subgroup analysis for six candidate genes at both mRNA and protein levels. And each of the six candidate genes and their neighbouring genes with a mutation rate greater than 10% were also analysed by network construction and functional enrichment analysis. In the meanwhile, the survival analysis for platinum-treated patients was performed in the current study. Finally, the RT-qPCR assay was used to determine the performance of candidate genes in ovarian cancer platinum response. Taken together, this research demonstrated that comprehensive bioinformatics methods could help to understand the molecular mechanism of platinum response and provide new strategies for overcoming platinum resistance in ovarian cancer treatment.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Anemia de Fanconi/genética , Recombinação Homóloga/genética , Neoplasias Ovarianas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Linhagem Celular Tumoral , DNA Polimerase III/genética , Intervalo Livre de Doença , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/genética , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Platina/administração & dosagem , Platina/efeitos adversos , RecQ Helicases/genética , Fatores de RiscoRESUMO
The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , DNA Helicases/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Marcação de Genes/métodos , Proteínas de Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , Recombinação Homóloga , MutaçãoRESUMO
Progressive cytopenia is a serious complication among paediatric patients with inherited bone marrow failure syndromes (IBMFS). Androgens have been used to improve blood counts in different bone marrow failure conditions. Little is known about efficacy and toxicity with new androgens (i.e., danazol) in different types of IBMFS. We identified 29 patients from the Canadian Inherited Marrow Failure Registry, who received oxymetholone or danazol. Sixteen (55%) had haematological response including patients with unclassified IBMFS (45%). Danazol showed a better toxicity profile and similar efficacy compared to oxymetholone. Androgens are an effective and safe option to ameliorate bone marrow failure in IBMFS.
Assuntos
Androgênios/uso terapêutico , Transtornos da Insuficiência da Medula Óssea/tratamento farmacológico , Adolescente , Adulto , Androgênios/efeitos adversos , Transtornos da Insuficiência da Medula Óssea/sangue , Transtornos da Insuficiência da Medula Óssea/genética , Transtornos da Insuficiência da Medula Óssea/terapia , Canadá/epidemiologia , Linhagem da Célula , Criança , Pré-Escolar , Terapia Combinada , Danazol/efeitos adversos , Danazol/uso terapêutico , Progressão da Doença , Substituição de Medicamentos , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Oximetolona/efeitos adversos , Oximetolona/uso terapêutico , Pancitopenia/tratamento farmacológico , Pancitopenia/etiologia , Sistema de Registros , Trombocitopenia/tratamento farmacológico , Trombocitopenia/etiologia , Resultado do Tratamento , Virilismo/induzido quimicamenteRESUMO
AIMS: Fanconi anaemia (FA) is a rare disorder characterized by progressive bone marrow failure that requires haematopoietic cell transplantation (HCT). Busulfan is used in conditioning regimens prior to HCT. Doses used in non-FA patients cause life-threatening toxicities in FA patients and data on busulfan pharmacokinetics (PK) in this population are limited. This study characterized busulfan PK in paediatric FA patients using population PK modelling and evaluated the effect of body composition on steady-state concentrations (Css ). METHODS: A total of 200 busulfan plasma concentrations in 29 FA patients from a recent study (Clinicaltrials.gov; NCT01082133) were available for population PK modelling. The effect of different body size-scaled doses and body compositions on Css was investigated using population PK modelling. RESULTS: Fat free mass (FFM) was identified as the best size descriptor in a two-compartment busulfan PK model in FA patients. Conventional dosing, based on an amount of busulfan per kilogram of total body mass, resulted in higher Css in FA patients with higher body mass index (BMI). A newly proposed FFM-based dosing strategy would eliminate the observed trend of higher concentrations in high BMI patients, and achieve consistent Css across a wide BMI spectrum. CONCLUSIONS: This is the first study to describe the population PK of busulfan in paediatric FA patients. The proposed model will facilitate PK model-informed precision dosing. FFM-based dosing is expected to improve the probability of achieving target Css , particularly in obese patients, while minimizing the risk of overdosing.
Assuntos
Alquilantes , Bussulfano , Anemia de Fanconi , Transplante de Células-Tronco Hematopoéticas , Alquilantes/farmacocinética , Composição Corporal , Bussulfano/farmacocinética , Criança , Anemia de Fanconi/terapia , Feminino , Humanos , Masculino , Condicionamento Pré-TransplanteRESUMO
Diffuse large B cell lymphoma (DLBCL) is traced to a mature B malignance carrying abnormal activation-induced cytidine deaminase (AID) expression. AID activity initially focuses on deamination of cytidine to uracil to generate somatic hypermutation and class-switch recombination of the immunoglobulin (Ig), but recently it has been implicated in DNA demethylation of genes required for B cell development and proliferation in the germinal centre (GC). However, whether AID activity on mutation or demethylation of genes involves oncogenesis of DLBCL has not been well characterized. Our data demonstrate that the proto-oncogene Fanconi anaemia complementation group A (FANCA) is highly expressed in DLBCL patients and cell lines, respectively. AID recruits demethylation enzyme ten eleven translocation family member (TET2) to bind the FANCA promoter. As a result, FANCA is demethylated and its expression increases in DLBCL. On the basis of our findings, we have developed a new therapeutic strategy to significantly inhibit DLBCL cell growth by combination of the proteasome inhibitor bortezomib with AID and TET2 depletion. These findings support a novel mechanism that AID has a crucial role in active demethylation for oncogene activation in DLBCL.