RESUMO
The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common ß-chain (ßc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade ßc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Endocitose , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Regulação para Baixo , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Membrana/genética , Camundongos , Transcriptoma , Células Tumorais CultivadasRESUMO
We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.
Assuntos
Leucemia Mieloide Aguda , Sirtuínas , Apoptose , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Lisina/metabolismo , Mitocôndrias/genética , Fosforilação Oxidativa , Sirtuínas/genéticaRESUMO
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-IdadeAssuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Hidrazinas/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Triazóis/farmacologia , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Mesilato de Imatinib/farmacologia , Camundongos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the cornerstone of treatment in chronic myeloid leukemia. Although there are now four TKIs approved for use in the front-line setting, acquired TKI resistance via secondary kinase domain mutations remains a problem for patients. K0706 is a novel BCR-ABL1 TKI currently under clinical investigation with structural elements similar to those of ponatinib and dasatinib. In this article, we functionally characterize the anti-leukemic activity of K0706 using cell proliferation assays in conjunction with drug resistance screening. We provide details from molecular modeling to support our in vitro findings and additionally describe our limited clinical experience with this drug in two patients treated on trial. We demonstrate that although K0706 retains efficacy against a large spectrum of clinically relevant mutations, it does not appear to have activity against BCR-ABL1T315I. Early trial experience suggests excellent tolerability, which may positively affect the place of K0706 within the ever-expanding chronic myeloid leukemia treatment paradigm.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Cromossomo Filadélfia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , CamundongosRESUMO
BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Niacinamida/análogos & derivados , Pirazóis/farmacologia , Piridazinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Imidazóis/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular/métodos , Mutação , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Cultura Primária de Células , Pirazóis/uso terapêutico , Piridazinas/uso terapêuticoRESUMO
Drug-free macromolecular therapeutics (DFMT) is a new paradigm for the treatment of B cell malignancies. Apoptosis is initiated by the biorecognition of complementary oligonucleotide motifs at the cell surface resulting in crosslinking of CD20 receptors. DMFT is composed from two nanoconjugates: 1) bispecific engager, Fab'-MORF1 (anti-CD20 Fab' fragment conjugated with morpholino oligonucleotide), and 2) a crosslinking (effector) component P-(MORF2)X (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple copies of complementary morpholino oligonucleotide). We evaluated this concept in 44 samples isolated from patients diagnosed with various subtypes of B cell malignancies. Apoptosis was observed in 65.9% of the samples tested. Pretreatment of cells with gemcitabine (GEM) or polymer-gemcitabine conjugate (2P-GEM) enhanced CD20 expression levels thus increasing apoptosis induced by DFMT. These positive results demonstrated that DFMT has remarkable therapeutic potential in various subtypes of B cell malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Linfoma de Células B/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD20 , Ciclo Celular/efeitos dos fármacos , Desoxicitidina/uso terapêutico , Feminino , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Pessoa de Meia-Idade , Nanomedicina/métodos , Adulto Jovem , GencitabinaRESUMO
PURPOSE: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. EXPERIMENTAL DESIGN: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). RESULTS: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. CONCLUSIONS: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Mielofibrose Primária/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Biomarcadores , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Biologia Computacional/métodos , Citoplasma/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Camundongos , Terapia de Alvo Molecular , Mutação , Transtornos Mieloproliferativos/etiologia , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/etiologia , Fatores de Transcrição STAT/metabolismo , TranscriptomaRESUMO
Tumor necrosis factor alpha (TNF) is increased in myelofibrosis (MF) and promotes survival of malignant over normal cells. The mechanisms altering TNF responsiveness in MF cells are unknown. We show that the proportion of marrow (BM) cells expressing TNF is increased in MF compared to controls, with the largest differential in primitive cells. Blockade of TNF receptor 2 (TNFR2), but not TNFR1, selectively inhibited colony formation by MF CD34+ and mouse JAK2V617F progenitor cells. Microarray of mouse MPN revealed reduced expression of X-linked inhibitor of apoptosis (Xiap) and mitogen-activated protein kinase 8 (Mapk8) in JAK2V617F relative to JAK2WT cells, which were normalized by TNFR2 but not TNFR1 blockade. XIAP and MAPK8 were also reduced in MF CD34+ cells compared to normal BM, and their ectopic expression induced apoptosis. Unlike XIAP, expression of cellular IAP (cIAP) protein was increased in MF CD34+ cells. Consistent with cIAP's role in NF-κB activation, TNF-induced NF-κB activity was higher in MF vs. normal BM CD34+ cells. This suggests that JAK2V617F reprograms TNF response toward survival by downregulating XIAP and MAPK8 through TNFR2. Our results reveal an unexpected pro-apoptotic role for XIAP in MF and identify TNFR2 as a key mediator of TNF-induced clonal expansion.
Assuntos
Comunicação Autócrina/fisiologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antígenos CD/metabolismo , Apoptose/fisiologia , Humanos , Janus Quinase 2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismoRESUMO
Squamous cell carcinoma (SCC) of the lung is the second most common subtype of lung cancer. With limited treatment options, the 5-year survival rate of SCC is only 15%. Although genomic alterations in SCC have been characterized, identifying the alterations that drive SCC is critical for improving treatment strategies. Mouse models of SCC are currently limited. Using lentiviral delivery of Sox2 specifically to the mouse lung, we tested the ability of Sox2 to promote tumorigenesis in multiple tumor suppressor backgrounds. Expression of Sox2, frequently amplified in human SCC, specifically cooperates with loss of Lkb1 to promote squamous lung tumors. Mouse tumors exhibit characteristic histopathology and biomarker expression similar to human SCC. They also mimic human SCCs by activation of therapeutically relevant pathways including STAT and mTOR. This model may be utilized to test the contribution of additional driver alterations in SCC, as well as for preclinical drug discovery.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição STAT/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The tropical bont tick (TBT) was first identified in St. Kitts in 1978. Initially, infestation was observed on cattle that developed a severe dermatitis. Over a period of seven years, the TBT spread to most areas of the island, affecting cattle, sheep, and goats. The ruminant livestock population declined from, 6,000, 9,000, and 7,000 cattle, sheep, and goats, respectively, in 1984 to an estimated 400, 800, and 1,000, cattle, sheep, and goats in 1990. A project to control the tropical bont tick on St. Kitts was implemented in 1983. This effort was based on the use of plunge dips located in communal grazing areas. In October of 1995, the Caribbean Amblyomma Programme was initiated on St. Kitts to eradicate the TBT from the Caribbean. In 1996, there were 416 animal owners with 2,000, 4,300, and 4,000 cattle, sheep, and goats, respectively. Cases of dermatophilosis, declined from 657 in 1995 to 153 in 1996. During the period 1997 to 1998 treatment with Flumethrin continued. The Department of Agriculture initiated activities for treatment compliance, monitoring, and TBT surveillance. The animal population increased to 3,000, 6,000, and 4,500 cattle, sheep, and goats, respectively, and with 810 animal owners participating. The number of cases of dermatophilosis declined to 42. In 1998, the TBT was confined to three foci. The remainder of the island was declared provisionally tick free.