RESUMO
The V617F mutation in the JH2 domain of Janus kinase 2 (JAK2) is an oncogenic driver in several myeloproliferative neoplasms (MPNs), including essential thrombocythemia, myelofibrosis, and polycythemia vera (PV). Other mutations in JAK2 have been identified in MPNs, most notably exon 12 mutations in PV. Here, we describe a novel recurrent mutation characterized by a common 4-amino-acid deletion and variable 1-amino-acid insertion (Leu583-Ala586DelInsSer/Gln/Pro) within the JH2 domain of JAK2. All 4 affected patients had eosinophilia, and both patients with Leu583-Ala586DelInsSer fulfilled diagnostic criteria of both PV and chronic eosinophilic leukemia (CEL). Computational and functional studies revealed that Leu583-Ala586DelInsSer (herein referred to as JAK2ex13InDel) deregulates JAK2 through a mechanism similar to JAK2V617F, activates signal transducer and activator of transcription 5 and extracellular signal-regulated kinase, and transforms parental Ba/F3 cells to growth factor independence. In contrast to JAK2V617F, JAK2ex13InDel does not require an exogenous homodimeric type 1 cytokine receptor to transform Ba/F3 cells and is capable of activating ß common chain family cytokine receptor (interleukin-3 receptor [IL-3R], IL-5R, and granulocyte-macrophage colony stimulating factor receptor) signaling in the absence of ligand, with the maximum effect observed for IL-5R, consistent with the clinical phenotype of eosinophilia. Recognizing this new PV/CEL-overlap MPN has significant clinical implications, as both PV and CEL patients are at high risk for thrombosis, and concomitant cytoreduction of red cells, neutrophils, and eosinophils may be required for prevention of thromboembolic events. Targeted next-generation sequencing for genes recurrently mutated in myeloid malignancies in patients with unexplained eosinophilia may reveal additional cases of Leu583-Ala586DelInsSer/Gln/Pro, allowing for complete characterization of this unique MPN.
Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/patologia , Síndrome Hipereosinofílica/patologia , Mutação INDEL , Janus Quinase 2/genética , Leucemia/patologia , Policitemia Vera/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Evolução Clonal , Feminino , Humanos , Síndrome Hipereosinofílica/genética , Síndrome Hipereosinofílica/metabolismo , Janus Quinase 2/metabolismo , Leucemia/genética , Leucemia/metabolismo , Masculino , Camundongos , Oncogenes , Policitemia Vera/genética , Policitemia Vera/metabolismoRESUMO
Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3+, CD14+, and CD34+ cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 107 (range, 1.62 × 105-2.52 × 108), and the median yield of CD34+ cells was 1.40 × 106 (range, 3.60 × 105-9.90 × 106). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.
Assuntos
Cabeça do Fêmur/citologia , Células-Tronco Hematopoéticas/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/metabolismo , Artroplastia de Quadril , Estudos de Casos e Controles , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-IdadeRESUMO
Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1(G2032R) mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.
Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Modelos Moleculares , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Piridinas/farmacologia , Quinase do Linfoma Anaplásico , Crizotinibe , Descoberta de Drogas/métodos , Humanos , Immunoblotting , Técnicas In Vitro , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/química , Pirazóis , Quinolinas , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/químicaRESUMO
UNLABELLED: Avian leukosis virus (ALV) induces tumors by integrating its proviral DNA into the chicken genome and altering the expression of nearby genes via strong promoter and enhancer elements. Viral integration sites that contribute to oncogenesis are selected in tumor cells. Deep-sequencing analysis of B-cell lymphoma DNA confirmed that the telomerase reverse transcriptase (TERT) gene promoter is a common ALV integration target. Twenty-six unique proviral integration sites were mapped between 46 and 3,552 nucleotides (nt) upstream of the TERT transcription start site, predominantly in the opposite transcriptional orientation to TERT Transcriptome-sequencing (RNA-seq) analysis of normal bursa revealed a transcribed region upstream of TERT in the opposite orientation, suggesting the TERT promoter is bidirectional. This transcript appears to be an uncharacterized antisense RNA. We have previously shown that TERT expression is upregulated in tumors with integrations in the TERT promoter region. We now report that the viral promoter drives the expression of a chimeric transcript containing viral sequences spliced to exons 4 through 7 of this antisense RNA. Clonal expansion of cells with ALV integrations driving overexpression of the TERT antisense RNA suggest it may have a role in tumorigenesis. IMPORTANCE: The data suggest that ALV integrations in the TERT promoter region drive the overexpression of a novel antisense RNA and contribute to the development of lymphomas.
Assuntos
Vírus da Leucose Aviária/genética , Leucose Aviária/genética , Leucose Aviária/virologia , Linfoma de Células B/genética , Linfoma de Células B/virologia , RNA Antissenso/genética , Telomerase/genética , Animais , Transformação Celular Neoplásica/genética , Galinhas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição/fisiologia , Transcriptoma/genética , Regulação para Cima/genética , Integração Viral/genéticaRESUMO
The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting â¼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.
Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , RNA Interferente Pequeno/genética , Transporte Ativo do Núcleo Celular/genética , Benzamidas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Humanos , Hidrazinas/farmacologia , Mesilato de Imatinib , Células K562 , Carioferinas/antagonistas & inibidores , Carioferinas/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais , Triazóis/farmacologia , Ensaio Tumoral de Célula-Tronco , Proteína ran de Ligação ao GTP/antagonistas & inibidores , Proteína ran de Ligação ao GTP/genética , Proteína Exportina 1RESUMO
We tested the hypothesis with a sample of community mental health clients (N = 132) that Hispanic clients would report significantly greater post-traumatic stress symptoms than African-American or white clients when controlling for gender, psychiatric symptoms of SMI, and subjective distress from six of the most commonly reported trauma in the SMI literature. Results supported our main hypothesis: being self-identified as Hispanic was significantly associated with greater post-traumatic stress symptoms. Subjective distress from having been sexually abused along with being "Hispanic" were the only two significant variables left in the equation. Limitations of this study include its modest sample size.
Assuntos
Negro ou Afro-Americano/psicologia , Hispânico ou Latino/psicologia , Transtornos de Estresse Pós-Traumáticos/etnologia , População Branca/psicologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Fatores de Risco , Índice de Gravidade de Doença , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Transtornos de Estresse Pós-Traumáticos/psicologia , Estados Unidos/epidemiologiaRESUMO
Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27(CK-)) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27(T187A)) or nuclear retention (p27(S10A)) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization.
Assuntos
Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Citoplasma/metabolismo , Proteínas de Fusão bcr-abl/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Animais , Células Cultivadas , Genes Supressores de Tumor , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Oncogênicas/metabolismo , Transporte Proteico/genéticaRESUMO
BACKGROUND: Resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL) is frequently caused by mutations in the BCR-ABL kinase domain. Ponatinib (AP24534) is a potent oral tyrosine kinase inhibitor that blocks native and mutated BCR-ABL, including the gatekeeper mutant T315I, which is uniformly resistant to tyrosine kinase inhibitors. METHODS: In this phase 1 dose-escalation study, we enrolled 81 patients with resistant hematologic cancers, including 60 with CML and 5 with Ph-positive ALL. Ponatinib was administered once daily at doses ranging from 2 to 60 mg. Median follow-up was 56 weeks (range, 2 to 140). RESULTS: Dose-limiting toxic effects included elevated lipase or amylase levels and pancreatitis. Common adverse events were rash, myelosuppression, and constitutional symptoms. Among Ph-positive patients, 91% had received two or more approved tyrosine kinase inhibitors, and 51% had received all three approved tyrosine kinase inhibitors. Of 43 patients with chronic-phase CML, 98% had a complete hematologic response, 72% had a major cytogenetic response, and 44% had a major molecular response. Of 12 patients who had chronic-phase CML with the T315I mutation, 100% had a complete hematologic response and 92% had a major cytogenetic response. Of 13 patients with chronic-phase CML without detectable mutations, 100% had a complete hematologic response and 62% had a major cytogenetic response. Responses among patients with chronic-phase CML were durable. Of 22 patients with accelerated-phase or blast-phase CML or Ph-positive ALL, 36% had a major hematologic response and 32% had a major cytogenetic response. CONCLUSIONS: Ponatinib was highly active in heavily pretreated patients with Ph-positive leukemias with resistance to tyrosine kinase inhibitors, including patients with the BCR-ABL T315I mutation, other mutations, or no mutations. (Funded by Ariad Pharmaceuticals and others; ClinicalTrials.gov number, NCT00660920.).
Assuntos
Antineoplásicos/administração & dosagem , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridazinas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Amilases/sangue , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Seguimentos , Proteínas de Fusão bcr-abl/genética , Humanos , Imidazóis/efeitos adversos , Imidazóis/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Lipase/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Pancreatite/induzido quimicamente , Proteínas Tirosina Quinases/genética , Piridazinas/efeitos adversos , Piridazinas/química , Relação Estrutura-AtividadeRESUMO
BCR-ABL mutations result in clinical resistance to ABL tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). Although in vitro 50% inhibitory concentration (IC(50)) values for specific mutations have been suggested to guide TKI choice in the clinic, the quantitative relationship between IC(50) and clinical response has never been demonstrated. We used Hill's equation for in vitro response of Ba/F3 cells transduced with various BCR-ABL mutants to determine IC(50) and the slope of the dose-response curve. We found that slope variability between mutants tracked with in vitro TKI resistance, provides particular additional interpretive value in cases where in vitro IC(50) and clinical response are disparate. Moreover, unlike IC(50) alone, higher inhibitory potential at peak concentration (IPP), which integrates IC(50), slope, and peak concentration (Cmax), correlated with improved complete cytogenetic response (CCyR) rates in CML patients treated with dasatinib. Our findings suggest a metric integrating in vitro and clinical data may provide an improved tool for BCR-ABL mutation-guided TKI selection.
Assuntos
Benzamidas/farmacologia , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Biomarcadores Farmacológicos/análise , Linhagem Celular Tumoral , Dasatinibe , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Concentração Inibidora 50 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Modelos Estatísticos , MutaçãoRESUMO
BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação de Sentido Incorreto/genética , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Benzamidas , Clonagem Molecular , Análise Mutacional de DNA/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/química , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Mesilato de Imatinib , Estrutura Terciária de ProteínaRESUMO
Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.
Assuntos
Leucemia Experimental/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas Tirosina Quinases/genética , Alelos , Animais , Apoptose/efeitos dos fármacos , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Janus Quinase 2 , Janus Quinase 3 , Células K562 , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , TYK2 QuinaseRESUMO
We examined associations among six forms of common lifetime traumatic/adverse events and lifetime suicide attempts while controlling for gender, psychiatric symptoms, self-injury, and substance use in 371 community mental health clients with severe mental illness. Most clients (88.1%) reported at least one traumatic event, and more than half had attempted suicide at least once. Regression revealed that three factors were significantly associated with lifetime suicide attempts: lifetime self-injurious behaviors, lifetime physical abuse, and alcohol use. Having been physically abused appears to be uniquely associated with lifetime suicide attempts when other key risk factors are controlled. Limitations include the cross-sectional design.
Assuntos
Acontecimentos que Mudam a Vida , Transtornos Mentais/psicologia , Tentativa de Suicídio/estatística & dados numéricos , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/psicologia , Serviços Comunitários de Saúde Mental/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Comportamento Autodestrutivo/complicações , Comportamento Autodestrutivo/psicologia , Fatores Sexuais , Tentativa de Suicídio/psicologia , Violência/psicologiaRESUMO
BCR-ABL1 compound mutations can lead to resistance to ABL1 inhibitors in chronic myeloid leukemia (CML), which could be targeted by combining the ATP-site inhibitor ponatinib and the allosteric inhibitor asciminib. Here, we report the clinical validation of this approach in a CML patient, providing a basis for combination therapy to overcome such resistance.
RESUMO
Chronic myeloid leukemia is effectively treated with imatinib, but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain mutations. The additional approved ABL tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib, along with investigational TKIs such as ponatinib (AP24534) and DCC-2036, support the possibility that mutation-mediated resistance in chronic myeloid leukemia can be fully controlled; however, the molecular events underlying resistance in patients lacking BCR-ABL point mutations are largely unknown. We previously reported on an insertion/truncation mutant, BCR-ABL(35INS), in which structural integrity of the kinase domain is compromised and all ABL sequence beyond the kinase domain is eliminated. Although we speculated that BCR-ABL(35INS) is kinase-inactive, recent reports propose this mutant contributes to ABL TKI resistance. We present cell-based and biochemical evidence establishing that BCR-ABL(35INS) is kinase-inactive and does not contribute to TKI resistance, and we find that detection of BCR-ABL(35INS) does not consistently track with or explain resistance in clinical samples from chronic myeloid leukemia patients.
Assuntos
Genes abl , Mutação INDEL , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Adulto , Idoso , Sequência de Bases , Benzamidas , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Masculino , Pessoa de Meia-Idade , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Adulto JovemRESUMO
Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.
Assuntos
Transformação Celular Neoplásica/metabolismo , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Substituição de Aminoácidos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/sangue , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/tratamento farmacológico , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mutantes/metabolismo , Células Progenitoras Mieloides/metabolismo , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/genéticaAssuntos
Proteínas Angiogênicas/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Pré-Escolar , Evolução Fatal , Rearranjo Gênico , Humanos , Masculino , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The Abl inhibitor imatinib is a highly effective therapy for patients with chronic myeloid leukemia. Relapses are relatively uncommon in newly diagnosed patients, but are the rule in patients with more advanced disease. Mutations in the BCR-ABL gene are the most common cause of relapse. Working from the imatinib chemical structure, a higher-affinity family member, AMN107, was designed. AMN107 is approximately 20-fold more potent than imatinib, and this translates into improved inhibitory activity against most of the common BCR-ABL mutations. The implications of these results, and the potential role this and other novel ABL inhibitors may have in treating patients with CML, are discussed.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Benzamidas , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Modelos Químicos , Mutação , Piperazinas/química , RecidivaRESUMO
The current study examines correlations among trauma, high risk behaviors, subjective distress from both trauma and high risk behaviors, and substance use in community mental health clients diagnosed with a severe mental illness, and tests the following key hypothesis: clients with major mood disorders (major depression, bipolar I) will show higher rates of posttraumatic stress disorder (PTSD) symptoms than clients with either schizophrenia or schizoaffective disorder when trauma, high risk behaviors, subjective distress, substance use and gender are controlled. Linear regression demonstrated that only major depression and bipolar disorder varied significantly with PTSD symptoms when controlling for other key factors.
Assuntos
Acontecimentos que Mudam a Vida , Transtornos Mentais/psicologia , Transtornos de Estresse Pós-Traumáticos/psicologia , Estresse Psicológico/psicologia , Transtornos Relacionados ao Uso de Substâncias/psicologia , Transtorno Bipolar/epidemiologia , Transtorno Bipolar/psicologia , Transtorno Depressivo Maior/epidemiologia , Transtorno Depressivo Maior/psicologia , Feminino , Humanos , Masculino , Transtornos Mentais/epidemiologia , Pessoa de Meia-Idade , New England/epidemiologia , Transtornos Psicóticos/epidemiologia , Transtornos Psicóticos/psicologia , Análise de Regressão , Assunção de Riscos , Esquizofrenia/epidemiologia , Psicologia do Esquizofrênico , Fatores Sexuais , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Estresse Psicológico/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Inquéritos e QuestionáriosRESUMO
Targeted therapy has vastly improved outcomes in certain types of cancer. Extension of this paradigm across a broad spectrum of malignancies will require an efficient method to determine the molecular vulnerabilities of cancerous cells. Improvements in sequencing technology will soon enable high-throughput sequencing of entire genomes of cancer patients; however, determining the relevance of identified sequence variants will require complementary functional analyses. Here, we report an RNAi-assisted protein target identification (RAPID) technology that individually assesses targeting of each member of the tyrosine kinase gene family. We demonstrate that RAPID screening of primary leukemia cells from 30 patients identifies targets that are critical to survival of the malignant cells from 10 of these individuals. We identify known, activating mutations in JAK2 and K-RAS, as well as patient-specific sensitivity to down-regulation of FLT1, CSF1R, PDGFR, ROR1, EPHA4/5, JAK1/3, LMTK3, LYN, FYN, PTK2B, and N-RAS. We also describe a previously undescribed, somatic, activating mutation in the thrombopoietin receptor that is sensitive to down-stream pharmacologic inhibition. Hence, the RAPID technique can quickly identify molecular vulnerabilities in malignant cells. Combination of this technique with whole-genome sequencing will represent an ideal tool for oncogenic target identification such that specific therapies can be matched with individual patients.
Assuntos
Leucemia/genética , Leucemia/terapia , Interferência de RNA , Alelos , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia/metabolismo , Mutação/genética , Receptores de Trombopoetina/genética , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. RESULTS: The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. CONCLUSION: The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.