RESUMO
Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B-/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B-/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. ⢠Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. ⢠ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. ⢠The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. ⢠The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Quinases , Humanos , Proteínas Quinases/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Doxorrubicina/farmacologia , Dano ao DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Aneuploidy is a very rare and tissue-specific event in normal conditions, occurring in a low number of brain and liver cells. Its frequency increases in age-related disorders and is one of the hallmarks of cancer. Aneuploidy has been associated with defects in the spindle assembly checkpoint (SAC). However, the relationship between chromosome number alterations, SAC genes and tumor susceptibility remains unclear. Here, we provide a comprehensive review of SAC gene alterations at genomic and transcriptional level across human cancers and discuss the oncogenic and tumor suppressor functions of aneuploidy. SAC genes are rarely mutated but frequently overexpressed, with a negative prognostic impact on different tumor types. Both increased and decreased SAC gene expression show oncogenic potential in mice. SAC gene upregulation may drive aneuploidization and tumorigenesis through mitotic delay, coupled with additional oncogenic functions outside mitosis. The genomic background and environmental conditions influence the fate of aneuploid cells. Aneuploidy reduces cellular fitness. It induces growth and contact inhibition, mitotic and proteotoxic stress, cell senescence and production of reactive oxygen species. However, aneuploidy confers an evolutionary flexibility by favoring genome and chromosome instability (CIN), cellular adaptation, stem cell-like properties and immune escape. These properties represent the driving force of aneuploid cancers, especially under conditions of stress and pharmacological pressure, and are currently under investigation as potential therapeutic targets. Indeed, promising results have been obtained from synthetic lethal combinations exploiting CIN, mitotic defects, and aneuploidy-tolerating mechanisms as cancer vulnerability.
Assuntos
Aneuploidia , Instabilidade Cromossômica/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Fuso Acromático/genética , Animais , Carcinogênese/genética , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Modelos Genéticos , Neoplasias/patologiaRESUMO
BACKGROUND: Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. METHODS: To understand the molecular bases of aneuploid acute myeloid leukemia (A-AML), this study examined the genomic profile in 42 A-AML cases and 35 euploid acute myeloid leukemia (E-AML) cases. RESULTS: A-AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E-AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A-AML, which was associated with a 3-gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A-AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E-AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. CONCLUSIONS: These findings indicate that aneuploidy-related and leukemia-specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Genômica/métodos , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aneuploidia , Ciclo Celular , Bandeamento Cromossômico , Feminino , Dosagem de Genes , Regulação Leucêmica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteólise , Sequenciamento do Exoma , Adulto JovemRESUMO
BACKGROUND: Detecting somatic mutations in whole exome sequencing data of cancer samples has become a popular approach for profiling cancer development, progression and chemotherapy resistance. Several studies have proposed software packages, filters and parametrizations. However, many research groups reported low concordance among different methods. We aimed to develop a pipeline which detects a wide range of single nucleotide mutations with high validation rates. We combined two standard tools - Genome Analysis Toolkit (GATK) and MuTect - to create the GATK-LODN method. As proof of principle, we applied our pipeline to exome sequencing data of hematological (Acute Myeloid and Acute Lymphoblastic Leukemias) and solid (Gastrointestinal Stromal Tumor and Lung Adenocarcinoma) tumors. We performed experiments on simulated data to test the sensitivity and specificity of our pipeline. RESULTS: The software MuTect presented the highest validation rate (90 %) for mutation detection, but limited number of somatic mutations detected. The GATK detected a high number of mutations but with low specificity. The GATK-LODN increased the performance of the GATK variant detection (from 5 of 14 to 3 of 4 confirmed variants), while preserving mutations not detected by MuTect. However, GATK-LODN filtered more variants in the hematological samples than in the solid tumors. Experiments in simulated data demonstrated that GATK-LODN increased both specificity and sensitivity of GATK results. CONCLUSION: We presented a pipeline that detects a wide range of somatic single nucleotide variants, with good validation rates, from exome sequencing data of cancer samples. We also showed the advantage of combining standard algorithms to create the GATK-LODN method, that increased specificity and sensitivity of GATK results. This pipeline can be helpful in discovery studies aimed to profile the somatic mutational landscape of cancer genomes.
Assuntos
Exoma , Genômica/métodos , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Algoritmos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Sensibilidade e Especificidade , SoftwareRESUMO
Background: COVID-19, whose causative pathogen is the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), was declared a pandemic in March 2020. The gastrointestinal tract is one of the targets of this virus, and mounting evidence suggests that gastrointestinal symptoms may contribute to disease severity. The gut-lung axis is involved in the immune response to SARS-CoV-2; therefore, we investigated whether COVID-19 patients' bacterial and fungal gut microbiome composition was linked to disease clinical outcome. Methods: In May 2020, we collected stool samples and patient records from 24 hospitalized patients with laboratory-confirmed SARS-CoV-2 infection. Fungal and bacterial gut microbiome was characterized by amplicon sequencing on the MiSeq, Illumina's integrated next generation sequencing instrument. A cohort of 201 age- and sex-matched healthy volunteers from the project PRJNA661289 was used as a control group for the bacterial gut microbiota analysis. Results: We observed that female COVID-19 patients had a lower gut bacterial microbiota richness than male patients, which was consistent with a different latency in hospital admittance time between the two groups. Both sexes in the COVID-19 patient study group displayed multiple positive associations with opportunistic bacterial pathogens such as Enterococcus, Streptococcus, and Actinomyces. Of note, the Candida genus dominated the gut mycobiota of COVID-19 patients, and adult patients showed a higher intestinal fungal diversity than elderly patients. We found that Saccharomycetales unassigned fungal genera were positively associated with bacterial short-chain fatty acid (SCFA) producers and negatively associated with the proinflammatory genus Bilophila in COVID-19 patients, and we observed that none of the patients who harbored it were admitted to the high-intensity unit. Conclusions: COVID-19 was associated with opportunistic bacterial pathogens, and Candida was the dominant fungal taxon in the intestine. Together, we found an association between commensal SCFA-producers and a fungal genus that was present in the intestines of patients who did not experience the most severe outcome of the disease. We believe that this taxon could have played a role in the disease outcome, and that further studies should be conducted to understand the role of fungi in gastrointestinal and health protection.
Assuntos
COVID-19 , Microbiota , Adulto , Humanos , Masculino , Feminino , Idoso , SARS-CoV-2 , Bactérias/genética , Candida , Gravidade do PacienteRESUMO
The members of the Poly(ADP-ribose) polymerase (PARP) superfamily are involved in several biological processes and, in particular, in the DNA damage response (DDR). The most studied members, PARP1, PARP2 and PARP3, act as sensors of DNA damages, in order to activate different intracellular repair pathways, including single-strand repair, homologous recombination, conventional and alternative non-homologous end joining. This review recapitulates the functional role of PARPs in the DDR pathways, also in relationship with the cell cycle phases, which drives our knowledge of the mechanisms of action of PARP inhibitors (PARPi), encompassing inhibition of single-strand breaks and base excision repair, PARP trapping and sensitization to antileukemia immune responses. Several studies have demonstrated a preclinical activity of the current available PARPi, olaparib, rucaparib, niraparib, veliparib and talazoparib, as single agent and/or in combination with cytotoxic, hypomethylating or targeted drugs in acute leukemia, thus encouraging the development of clinical trials. We here summarize the most recent preclinical and clinical findings and discuss the synthetic lethal interactions of PARPi in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite the low frequency of genomic alterations of PARP and other DDR-related genes in acute leukemia, selective vulnerabilities have been reported in several disease subgroups, along with a "BRCAness phenotype." AML carrying the RUNX1-RUNX1T1 or PML-RARA fusion genes or mutations in signaling genes (FLT3-ITD in combination with TET2 or TET2 and DNMT3A deficiency), cohesin complex members (STAG2), TP53 and BCOR as co-occurring lesions, IDH1/2 and ALL cases expressing the TCF3-HLF chimera or TET1 was highly sensitive to PARPi in preclinical studies. These data, along with the warning coming from the observation of cases of therapy-related myeloid malignancies among patients receiving PARPi for solid tumors treatment, indicate that PARPi represents a promising strategy in a personalized medicine setting. The characterization of the clonal and subclonal genetic background and of the DDR functionality is crucial to select acute leukemia patients that will likely benefit of PARPi-based therapeutic regimens.
Assuntos
Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Animais , Antineoplásicos/farmacologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Terapia de Alvo Molecular , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Acute myeloid leukemia (AML) is a complex hematological malignancy characterized by genetic and clinical heterogeneity and high mortality. Despite the recent introduction of novel pharmaceutical agents in hemato-oncology, few advancements have been made in AML for decades. In the last years, the therapeutic options have rapidly changed, with the approval of innovative compounds that provide new opportunities, together with new challenges for clinicians: among them, on 1 September, 2017 the Food and Drug Administration granted approval for Gemtuzumab Ozogamicin (GO) in combination with daunorubicin and cytarabine for the treatment of adult patients affected by newly diagnosed CD33+ AML. Benefits of GO-based regimens were also reported in the pre- and post-transplantation settings. Moreover, several biomarkers of GO response have been suggested, including expression of CD33 and multidrug resistance genes, cytogenetic and molecular profiles, minimal residual disease and stemness signatures. Among them, elevated CD33 expression on blast cells and non-adverse cytogenetic or molecular risk represent largely validated predictors of good response.
RESUMO
In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML cell line and primary cells, but not in TP53-mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated TP53-wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 TP53-wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in TP53-wt AML.
RESUMO
Adrenomedullin (ADM) is a hypotensive and vasodilator peptide belonging to the calcitonin gene-related peptide family. It is secreted in vitro by endothelial cells and vascular smooth muscle cells, and is significantly upregulated by a number of stimuli. Moreover, ADM participates in the regulation of hematopoietic compartment, solid tumors and leukemias, such as acute myeloid leukemia (AML). To better characterize ADM involvement in AML pathogenesis, we investigated its expression during human hematopoiesis and in leukemic subsets, based on a morphological, cytogenetic and molecular characterization and in T cells from AML patients. In hematopoietic stem/progenitor cells and T lymphocytes from healthy subjects, ADM transcript was barely detectable. It was expressed at low levels by megakaryocytes and erythroblasts, while higher levels were measured in neutrophils, monocytes and plasma cells. Moreover, cells populating the hematopoietic niche, including mesenchymal stem cells, showed to express ADM. ADM was overexpressed in AML cells versus normal CD34+ cells and in the subset of leukemia compared with hematopoietic stem cells. In parallel, we detected a significant variation of ADM expression among cytogenetic subgroups, measuring the highest levels in inv(16)/t(16;16) or complex karyotype AML. According to the mutational status of AML-related genes, the analysis showed a lower expression of ADM in FLT3-ITD, NPM1-mutated AML and FLT3-ITD/NPM1-mutated cases compared with wild-type ones. Moreover, ADM expression had a negative impact on overall survival within the favorable risk class, while showing a potential positive impact within the subgroup receiving a not-intensive treatment. The expression of 135 genes involved in leukemogenesis, regulation of cell proliferation, ferroptosis, protection from apoptosis, HIF-1α signaling, JAK-STAT pathway, immune and inflammatory responses was correlated with ADM levels in the bone marrow cells of at least two AML cohorts. Moreover, ADM was upregulated in CD4+ T and CD8+ T cells from AML patients compared with healthy controls and some ADM co-expressed genes participate in a signature of immune tolerance that characterizes CD4+ T cells from leukemic patients. Overall, our study shows that ADM expression in AML associates with a stem cell phenotype, inflammatory signatures and genes related to immunosuppression, all factors that contribute to therapy resistance and disease relapse.
RESUMO
Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Dano ao DNA/genética , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatina/genética , Feminino , Genômica/métodos , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Prognóstico , Adulto Jovem , CoesinasRESUMO
Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.
Assuntos
DNA Metiltransferase 3A/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Genótipo , Humanos , Leucemia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Neoplásicas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
After being in the therapeutic wilderness for several decades, acute myeloid leukemia has been recently thrust into the limelight with a series of drug approvals. Technical refinements in production, genetic manipulation and chemical modification of monoclonal antibodies led to growing interest in antibodies-based treatment strategies. Much of the focus of these efforts in acute myeloid leukemia has been on CD33 as a target. On September 2, 2017, the U.S. Food and Drug Administration approved gemtuzumab ozogamicin for treatment of relapsed or refractory CD33+ acute myeloid leukemia. This signals a new chapter in the long and unusual story of gemtuzumab ozogamicin, which was the first antibody-drug conjugate approved for human use by the Food and Drug Administration. In this review we have analyzed the history of this drug which, among several mishaps, is experiencing a second youth and still represents a field to be further explored.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Gemtuzumab/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Idoso , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Arsenicais/uso terapêutico , Calicheamicinas/metabolismo , Cloretos/uso terapêutico , Ensaios Clínicos como Assunto , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Daunorrubicina/efeitos adversos , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Gemtuzumab/efeitos adversos , Gemtuzumab/farmacocinética , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Camundongos , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Recidiva , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Tretinoína/uso terapêuticoRESUMO
Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795 ∗ ; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.
RESUMO
Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.
RESUMO
Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix®) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p = 0.002), ELN high risk (HR) (p < 0.001), lower white blood cell (WBC) count (p = 0.040), TP53 loss, and/or mutations (p < 0.001) while FLT3 (p = 0.025), and NPM1 (p = 0.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p < 0.001) compared with HR patients (p = 0.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e., TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair, and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, and 17. CBA. FISH showed that chromothripsis is associated with marker, derivative, and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology.
Assuntos
Cromotripsia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Bandeamento Cromossômico , Feminino , Predisposição Genética para Doença , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Mutação , Nucleofosmina , Polimorfismo de Nucleotídeo Único , Prognóstico , Modelos de Riscos Proporcionais , Cromossomos em Anel , Resultado do Tratamento , Adulto JovemAssuntos
Reparo do DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Deleção de Genes , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Adulto JovemRESUMO
Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work.