RESUMO
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by complex molecular and cytogenetic abnormalities. Pro-oxidant cellular redox status is a common hallmark of AML cells, providing a rationale for redox-based anticancer strategy. We previously discovered that auranofin (AUF), initially used for the treatment of rheumatoid arthritis and repositioned for its anticancer activity, can synergize with a pharmacological concentration of vitamin C (VC) against breast cancer cell line models. In this study, we observed that this drug combination synergistically and efficiently killed cells of leukaemic cell lines established from different myeloid subtypes. In addition to an induced elevation of reactive oxygen species and ATP depletion, a rapid dephosphorylation of 4E-BP1 and p70S6K, together with a strong inhibition of protein synthesis were early events in response to AUF/VC treatment, suggesting their implication in AUF/VC-induced cytotoxicity. Importantly, a study on 22 primary AML specimens from various AML subtypes showed that AUF/VC combinations at pharmacologically achievable concentrations were effective to eradicate primary leukaemic CD34+ cells from the majority of these samples, while being less toxic to normal cord blood CD34+ cells. Our findings indicate that targeting the redox vulnerability of AML with AUF/VC combinations could present a potential anti-AML therapeutic approach.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Mutação , Síndromes Mielodisplásicas , Niclosamida , Proteína Supressora de Tumor p53 , Humanos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Proteína Supressora de Tumor p53/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologiaRESUMO
A low allele burden (i.e., <20%) of the CALR driver mutation is found in 10.8% of CALR-mutated MPNs, mostly in essential thrombocythemia, and correlates with a milder phenotype and a more indolent evolution compared to patients with an allele burden ≥20%.
Assuntos
Trombocitemia Essencial , Humanos , Alelos , Calreticulina/genética , Janus Quinase 2/genética , Mutação , Fenótipo , Trombocitemia Essencial/genéticaRESUMO
Classical myeloproliferative neoplasms (MPNs) are characterized by the proliferation of myeloid cells and the risk of transformation into myelofibrosis or acute myeloid leukemia (AML) and TP53 mutations in MPN patients are linked to AML. However, JAK2V617F has been reported to impact the TP53 response to DNA damage, suggesting potential overlapping role of TP53 inactivation in MPN. We established a mouse model showing that JAK2V617F/Vav-Cre/Trp53-/- mice displayed a similar phenotype to JAK2V617F/Vav-Cre mice, but their proliferation was outcompeted in competitive grafts. RNA-Seq revealed that half of the genes affected by JAK2V617F were affected by p53-inactivation, including the interferon pathway. To validate this finding, mice were repopulated with a mixture of wild-type and JAK2V617F (or JAK2V617F/Vav-Cre/Trp53-/-) cells and treated with pegylated interferonα. JAK2V617F-reconstituted mice entered complete hematological remission, while JAK2V617F/Vav-Cre /Trp53-/--reconstituted mice did not, confirming that p53 loss induced interferon-α resistance. KEGG and Gene Ontology analyses of common deregulated genes showed that these genes were mainly implicated in cytokine response, proliferation, and leukemia evolution, illustrating that in this mouse model, the development of MPN is not affected by TP53 inactivation. Taken together, our results show that many genetic modifications induced by JAK2V617F are influenced by TP53, the MPN phenotype may not be. Trp53 loss alone is insufficient to induce rapid leukemic transformation in steady-state hematopoiesis in JAK2V617F MPN, and Trp53 loss may contribute to interferon resistance in MPN.
Assuntos
Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Humanos , Camundongos , Animais , Proteína Supressora de Tumor p53/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Mutação , Interferon-alfa/farmacologia , GenômicaRESUMO
Comparative clinical characteristics, molecular landscape and prognosis scoring for primary (PMF) and secondary myelofibrosis (SMF).
Assuntos
Mielofibrose Primária , Humanos , Mielofibrose Primária/genética , PrognósticoRESUMO
Current recommended risk scores to predict thrombotic events associated with myeloproliferative neoplasms (MPN) do not discriminate between arterial and venous thrombosis despite their different physiopathology. To define novel stratification systems, we delineated a comprehensive landscape of MPN associated thrombosis across a large long-term follow-up MPN cohort. Prior arterial thrombosis, age >60 years, cardiovascular risk factors and presence of TET2 or DNMT3A mutations were independently associated with arterial thrombosis in multivariable analysis. ARTS, an ARterial Thrombosis Score, based on these four factors, defined low- (0.37% patients-year) and high-risk (1.19% patients-year) patients. ARTS performance was superior to the two-tiered conventional risk stratification in our training cohort, across all MPN subtypes, as well as in two external validation cohorts. Prior venous thrombosis and presence of a JAK2V617F mutation with a variant allelic frequency ≥50% were independently associated with venous thrombosis. The discrimination potential of VETS, a VEnous Thrombosis Score based on these two factors, was poor, similar to the two-tiered conventional risk stratification. Our study pinpoints arterial and venous thrombosis clinico-molecular differences and proposes an arterial risk score for more accurate patients' stratification. Further improvement of venous risk scores, accounting for additional factors and considering venous thrombosis as a heterogeneous entity is warranted.
Assuntos
Transtornos Mieloproliferativos , Neoplasias , Trombose , Trombose Venosa , Humanos , Pessoa de Meia-Idade , Neoplasias/complicações , Trombose Venosa/genética , Trombose/genética , Trombose/complicações , Mutação , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/genética , Fatores de Risco , Janus Quinase 2/genética , Medição de RiscoRESUMO
Myeloproliferative neoplasms are characterized by the acquisition at the hematopoietic stem cell level of driver mutations targeting the JAK/STAT pathway. In addition, they also often exhibit additional mutations targeting various pathways such as intracellular signalling, epigenetics, mRNA splicing or transcription. The natural history of myeloproliferative neoplasms is usually marked by a chronic phase of variable duration depending on the disease subtype, which can be followed by an accelerated phase or transformation towards more aggressive diseases such as myelofibrosis or acute leukemia. Besides, recent studies revealed important new information about the rates and mechanisms of sequential acquisition and selection of mutations in hematopoietic cells of myeloproliferative neoplasms. Better understanding of these events has been made possible in large part with the help of novel techniques that are now available to precisely decipher at the single cell level both the clonal architecture and the mutation-induced cell modifications. In this review, we will summarize the most recent knowledge about the mechanisms leading to clonal selection, how clonal architecture complexity can explain disease heterogeneity, and the impact of clonal evolution on clinical evolution.
Assuntos
Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Humanos , Janus Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição STAT/metabolismo , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Mutação , Evolução ClonalRESUMO
The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
The mechanisms of transformation of chronic myeloproliferative neoplasms (MPN) to leukemia are largely unknown, but TP53 mutations acquisition is considered a key event in this process. p53 is a main tumor suppressor, but mutations in this protein per se do not confer a proliferative advantage to the cells, and a selection process is needed for the expansion of mutant clones. MDM2 inhibitors may rescue normal p53 from degradation and have been evaluated in a variety of cancers with promising results. However, the impact of these drugs on TP53-mutated cells is underexplored. We report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients' cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context, we used a DNA single-cell approach to determine the clonal architecture of TP53-mutated cells. We observed that TP53 mutations are late events in MPN, mainly occurring in the driver clone, whereas clonal evolution frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone. At the single-cell level, the presence of additional mutations does not influence the selection of TP53 mutant cells by MDM2 inhibitor treatment. Also, we describe an in vitro test allowing to predict the emergence of TP53 mutated clones. Altogether, this is the first demonstration that a drug treatment can directly favor the emergence of TP53-mutated subclones in MPN.
Assuntos
Antineoplásicos , Transtornos Mieloproliferativos , Células Clonais/metabolismo , Humanos , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Análise de Célula Única , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.
Assuntos
Eritropoetina/sangue , Janus Quinase 2/genética , Mutação , Policitemia Vera/sangue , Policitemia Vera/genética , Alelos , Substituição de Aminoácidos , Tomada de Decisão Clínica , Gerenciamento Clínico , Índices de Eritrócitos , Volume de Eritrócitos , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Volume Plasmático , Policitemia Vera/diagnóstico , Policitemia Vera/epidemiologia , Sensibilidade e EspecificidadeAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Quinuclidinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azacitidina/farmacologia , Humanos , Quinuclidinas/farmacologia , Resultado do Tratamento , Células Tumorais CultivadasAssuntos
Leucemia Mieloide Aguda/genética , Transtornos Mieloproliferativos/genética , Subunidade p45 do Fator de Transcrição NF-E2/genética , Adulto , Feminino , Humanos , Leucemia Mieloide Aguda/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação , Transtornos Mieloproliferativos/epidemiologia , Análise de SobrevidaRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) represents the malignant expansion of immature T cells blocked in their differentiation. T-ALL is still associated with a poor prognosis, mainly related to occurrence of relapse or refractory disease. A critical medical need therefore exists for new therapies to improve the disease prognosis. Adenylate kinase 2 (AK2) is a mitochondrial kinase involved in adenine nucleotide homeostasis recently reported as essential in normal T-cell development, as defective AK2 signaling pathway results in a severe combined immunodeficiency with a complete absence of T-cell differentiation. In this study, we show that AK2 is constitutively expressed in T-ALL to varying levels, irrespective of the stage of maturation arrest or the underlying oncogenetic features. T-ALL cell lines and patient T-ALL-derived xenografts present addiction to AK2, whereas B-cell precursor ALL cells do not. Indeed, AK2 knockdown leads to early and massive apoptosis of T-ALL cells that could not be rescued by the cytosolic isoform AK1. Mechanistically, AK2 depletion results in mitochondrial dysfunction marked by early mitochondrial depolarization and reactive oxygen species production, together with the depletion of antiapoptotic molecules (BCL-2 and BCL-XL). Finally, T-ALL exposure to a BCL-2 inhibitor (ABT-199 [venetoclax]) significantly enhances the cytotoxic effects of AK2 depletion. We also show that AK2 depletion disrupts the oxidative phosphorylation pathway. Combined with pharmaceutical inhibition of glycolysis, AK2 silencing prevents T-ALL metabolic adaptation, resulting in dramatic apoptosis. Altogether, we pinpoint AK2 as a genuine and promising therapeutic target in T-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Imunodeficiência Combinada Severa , Adenilato Quinase , Humanos , Mitocôndrias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genéticaRESUMO
Interferon α (IFNα) is used to treat JAK2V617F-driven myeloproliferative neoplasms (MPNs) but rarely clears the disease. We investigated the IFNα mechanism of action focusing on PML, an interferon target and key senescence gene whose targeting by arsenic trioxide (ATO) drives eradication of acute promyelocytic leukemia. ATO sharply potentiated IFNα-induced growth suppression of JAK2V617F patient or mouse hematopoietic progenitors, which required PML and was associated with features of senescence. In a mouse MPN model, combining ATO with IFNα enhanced and accelerated responses, eradicating MPN in most mice by targeting disease-initiating cells. These results predict potent clinical efficacy of the IFNα+ATO combination in patients and identify PML as a major effector of therapy, even in malignancies with an intact PML gene.
Assuntos
Trióxido de Arsênio/farmacologia , Interferon-alfa/farmacologia , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Proteína da Leucemia Promielocítica/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Mieloproliferativos/metabolismoRESUMO
Myeloproliferative neoplasms (MPNs) are the most frequent underlying causes of splanchnic vein thromboses (SVTs). MPN patients with SVTs (MPN-SVT) often have a unique presentation including younger age, female predominance, and low Janus kinase 2 (JAK2) mutation allele burden. This study aimed at identifying risk factors for adverse hematologic outcomes in MPN-SVT patients. We performed a retrospective study of a fully characterized cohort of MPN-SVT patients. The primary outcome was the incidence of evolution to myelofibrosis, acute leukemia, or death. Eighty patients were included in the testing cohort. Median follow-up was 11 years. Most of the patients were women with a mean age of 42 years and a diagnosis of polycythemia vera. The primary outcome was met in 13% of the patients and was associated with a JAK2V617F allele burden ≥50% (odds ratio [OR], 14.7) and presence of additional mutations in genes affecting chromatin/spliceosome (OR, 9). We identified high-risk patients (29% of the cohort) as those harboring at least 1 molecular risk factor: JAK2-mutant allele burden ≥50%, presence of chromatin/spliceosome/TP53 mutation. High-risk patients had worse event-free survival (81% vs 100%; P = .001) and overall survival at 10 years (89% vs 100%; P = .01) than low-risk patients. These results were confirmed in an independent validation cohort of 30 MPN-SVT patients. In conclusion, molecular profiling identified MPN-SVT patients with dismal outcome. In this high-risk population, a disease-modifying therapy should be taken into consideration to minimize the probability of transformation.
Assuntos
Transtornos Mieloproliferativos , Policitemia Vera , Mielofibrose Primária , Trombose Venosa , Adulto , Feminino , Humanos , Masculino , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Estudos RetrospectivosRESUMO
INTRODUCTION: The pathogenesis of myeloproliferative neoplasms (MPNs) is closely related to the acquisition of specific molecular alterations in JAK2, MPL, or CALR genes, the presence of which represent major diagnostic criteria in the WHO classification. The CALR exon 9 insertions and deletions are very heterogeneous, and their detection mainly relies on polymerase chain reaction (PCR) fragment length analysis. METHODS: We report on the rare nonclassical profiles that we observed among the 1382 patients analyzed by PCR fragment length analysis. In difficult cases, we tested germline DNA and performed NGS analysis. RESULTS: We faced some troubling results because of the presence of several unexpected peaks. Our investigations showed that they resulted from a mix of isolated or double somatic mutations combined with germline alterations which could be misleading for a correct diagnosis. The precise interpretation of such difficult cases is mandatory as a misinterpretation could lead to the prescription of cytoreductive drugs to nondiseased persons or to an absence of treatment in true MPN patients. CONCLUSION: Our observations showed that every mutation should be verified by direct Sanger sequencing, and we show that sometimes it may be necessary to study germline DNA and to complement with NGS analysis to precisely interpret the molecular alterations.