RESUMEN
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.
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Proteínas de Ciclo Celular/metabolismo , Endocitosis , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Citocinas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Regulación hacia Abajo , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de la Membrana/genética , Ratones , Transcriptoma , Células Tumorales CultivadasRESUMEN
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.
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Transporte Activo de Núcleo Celular , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , ARN Interferente Pequeño/genética , Transporte Activo de Núcleo Celular/genética , Benzamidas/farmacología , Línea Celular Tumoral , Supervivencia Celular , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Técnicas de Silenciamiento del Gen , Biblioteca de Genes , Humanos , Hidrazinas/farmacología , Mesilato de Imatinib , Células K562 , Carioferinas/antagonistas & inhibidores , Carioferinas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Triazoles/farmacología , Ensayo de Tumor de Célula Madre , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genética , Proteína Exportina 1RESUMEN
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.
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Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación Missense/genética , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Benzamidas , Clonación Molecular , Análisis Mutacional de ADN/métodos , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/química , Regulación Leucémica de la Expresión Génica/genética , Humanos , Mesilato de Imatinib , Estructura Terciaria de ProteínaRESUMEN
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.
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Genes abl , Mutación INDEL , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Adulto , Anciano , Secuencia de Bases , Benzamidas , Línea Celular Tumoral , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/genética , Femenino , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Masculino , Persona de Mediana Edad , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Adulto JovenAsunto(s)
Proteínas Angiogénicas/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Preescolar , Resultado Fatal , Reordenamiento Génico , Humanos , Masculino , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
BACKGROUND: Better understanding of vessel biology and vascular pathophysiology is needed to improve understanding of cerebrovascular disorders. Tissue from diseased vessels can offer the best data. Rabbit models can be effective for studying intracranial vessels, filling gaps resulting from difficulties acquiring human tissue. Spatially-resolved transcriptomics (SRT) in particular hold promise for studying such models as they build on RNA sequencing methods, augmenting such data with histopathology. METHODS: Rabbit brains with intact arteries were flash frozen, cryosectioned, and stained with H&E to confirm adequate inclusion of intracranial vessels before proceeding with tissue optimization and gene expression analysis using the Visium SRT platform. SRT results were analyzed with k-means clustering analysis, and differential gene expression was examined, comparing arteries to veins. RESULTS: Cryosections were successfully mounted on Visium proprietary slides. Quality control thresholds were met. Optimum permeabilization was determined to be 24â min for the tissue optimization step. In analysis of SRT data, k-means clustering distinguished vascular tissue from parenchyma. When comparing gene expression traits, the most differentially expressed genes were those found in smooth muscle cells. These genes were more commonly expressed in arteries compared to veins. CONCLUSIONS: Intracranial vessels from model rabbits can be processed and analyzed with the Visium SRT platform. Face validity is found in the ability of SRT data to distinguish vessels from parenchymal tissue and differential expression analysis accurately distinguishing arteries from veins. SRT should be considered for future animal model investigations into cerebrovascular diseases.
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Trastornos Cerebrovasculares , Transcriptoma , Animales , Humanos , Conejos , Músculo Liso Vascular/metabolismo , Arterias , Venas , Perfilación de la Expresión GénicaRESUMEN
Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Humanos , Conejos , Accidente Cerebrovascular/complicaciones , Presión Sanguínea/fisiología , Encéfalo/patología , Isquemia Encefálica/patología , Modelos Animales de EnfermedadRESUMEN
OBJECTIVE: Alternative contrast agents for MRI are needed for individuals who may respond adversely to gadolinium, and need an intravascular agent for specific indications. One potential contrast agent is intracellular methemoglobin, a paramagnetic molecule that is normally present in small amounts in red blood cells. An animal model was used to determine whether methemoglobin modulation with intravenous sodium nitrite transiently changes the T1 relaxation of blood. METHODS: Four adult New Zealand white rabbits were treated with 30 mg intravenous sodium nitrite. 3D TOF and 3D MPRAGE images were acquired before (baseline) and after methemoglobin modulation. T1 of blood was measured with 2D ss EPl acquisitions with inversion recovery preparation performed at two-minute intervals up to 30 min. T1 maps were calculated by fitting the signal recovery curve within major blood vessels. RESULTS: Baseline T1 was 1758 ± 53 ms in carotid arteries and 1716 ± 41 ms in jugular veins. Sodium nitrite significantly changed intravascular T1 relaxation. The mean minimum value of T1 was 1126 ± 28 ms in carotid arteries 8 to 10 min after the injection of sodium nitrite. The mean minimum value of T1 was 1171 ± 52 ms in jugular veins 10 to 14 min after the injection of sodium nitrite. Arterial and venous T1 recovered to baseline after a period of 30 min. CONCLUSION: Methemoglobin modulation produces intravascular contrast on T1-weighted MRI in vivo. Additional studies are needed to safely optimize methemoglobin modulation and sequence parameters for maximal tissue contrast.
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Medios de Contraste , Metahemoglobina , Conejos , Animales , Nitrito de Sodio , Imagen por Resonancia Magnética/métodos , Imagenología TridimensionalRESUMEN
BACKGROUND: The study of chronic pain and its treatments requires a robust animal model with objective and quantifiable metrics. Porcine neuropathic pain models have been assessed with peripheral pain recordings and behavioral responses, but thus far central nervous system electrophysiology has not been investigated. This work aimed to record non-invasive, somatosensory-evoked potentials (SEPs) via electroencephalography in order to quantitatively assess chronic neuropathic pain induced in a porcine model. NEW METHOD: Peripheral neuritis trauma (PNT) was induced unilaterally in the common peroneal nerve of domestic farm pigs, with the contralateral leg serving as the control for each animal. SEPs were generated by stimulation of the peripheral nerves distal to the PNT and were recorded non-invasively using transcranial electroencephalography (EEG). The P30 wave of the SEP was analyzed for latency changes. RESULTS: P30 SEPs were successfully recorded with non-invasive EEG. PNT resulted in significantly longer P30 SEP latencies (p < 0.01 [n = 8]) with a median latency increase of 14.3 [IQR 5.0 - 17.5] ms. Histological results confirmed perineural inflammatory response and nerve damage around the PNT nerves. COMPARISON WITH EXISTING METHOD(S): Control P30 SEPs were similar in latency and amplitude to those previously recorded invasively in healthy pigs. Non-invasive recordings have numerous advantages over invasive measures. CONCLUSIONS: P30 SEP latency can serve as a quantifiable neurological measure that reflects central nervous system processing in a porcine model of chronic pain. Advancing the development of a porcine chronic pain model will facilitate the translation of experimental therapies into human clinical trials.
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Dolor Crónico , Neuralgia , Humanos , Porcinos , Animales , Potenciales Evocados Somatosensoriales/fisiología , Electroencefalografía , Sistema Nervioso Central , Neuralgia/diagnóstico , Estimulación Eléctrica , Nervio MedianoRESUMEN
INTRODUCTION: Vessel wall magnetic resonance imaging can improve the evaluation of intracranial atherosclerotic disease. However, pathological validation is needed to improve vessel wall magnetic resonance imaging techniques. Human pathology samples are not practical for such analysis, so an animal model is therefore needed. MATERIALS AND METHODS: Watanabe heritable hyperlipidemic rabbits and apolipoprotein E knockout rabbits were evaluated against New Zealand white wild-type rabbits. Evaluation of intracranial arteries was performed with vessel wall magnetic resonance imaging and pathological analysis, rating the presence and severity of disease in each segment. Two-tailed t-tests were performed to compare disease occurrence and severity prevalence among rabbit subtypes. Sensitivity and specificity were calculated to assess the diagnostic accuracy of vessel wall magnetic resonance imaging. RESULTS: Seventeen rabbits (five Watanabe heritable hyperlipidemic, four apolipoprotein E knockout and eight New Zealand white) were analysed for a total of 51 artery segments. Eleven segments (five Watanabe heritable hyperlipidemic and six apolipoprotein E knockout) demonstrated intracranial atherosclerotic disease on pathology. Disease model animals had lesions more frequently than New Zealand white animals (P<0.001). The sensitivity and specificity of vessel wall magnetic resonance imaging for the detection of intracranial atherosclerotic disease were 68.8% and 95.2%, respectively. When excluding mild cases to assess vessel wall magnetic resonance imaging accuracy for detecting moderate to severe intracranial atherosclerotic disease lesions, sensitivity improved to 100% with unchanged specificity. CONCLUSION: Intracranial atherosclerotic disease can be reliably produced and detected using 3T vessel wall magnetic resonance imaging-compatible Watanabe heritable hyperlipidemic and ApoE rabbit models. Further analysis is needed to characterize better the development and progression of the disease to correlate tissue-validated animal findings with those in human vessel wall magnetic resonance imaging studies.
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Arteriosclerosis Intracraneal/diagnóstico por imagen , Angiografía por Resonancia Magnética/métodos , Animales , Modelos Animales de Enfermedad , Arteriosclerosis Intracraneal/patología , Conejos , Sensibilidad y EspecificidadRESUMEN
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.
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Leucemia Mieloide Aguda , Sirtuinas , Apoptosis , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Lisina/metabolismo , Mitocondrias/genética , Fosforilación Oxidativa , Sirtuinas/genéticaRESUMEN
Intracranial atherosclerotic disease (ICAD) is the most common cause of ischemic stroke. Poor understanding of the disease due to limited human data leads to imprecise treatment. Apolipoprotein E knockout (ApoE-KO) rabbits were compared to an existing model, the Watanabe heritable hyperlipidemic (WHHL) rabbit, and wild-type New Zealand white (NZW) rabbit controls. Intracranial artery samples were assessed on histopathology for the presence of ICAD. Logistic and ordinal regression analyses were performed to assess for disease presence and severity, respectively. Eighteen rabbits and 54 artery segments were analyzed. Univariate logistic analysis confirmed the presence of ICAD in model rabbits (P < .001), while no difference was found between WHHL and ApoE-KO rabbits (P = .178). In multivariate analysis, only classification as a model vs wild-type animal (P < .001) was associated with the presence of ICAD. Univariate ordinal regression analysis demonstrated an association between ICAD severity and model animals (P = .001), with no difference was noted between WHHL and ApoE-KO rabbits (P = .528). In multivariate ordinal regression analysis, only classification as a model retained significance (P < .001). ICAD can be reliably produced in ApoE-KO rabbits, developing the disease comparably to the older WHHL model. Further analysis is warranted to optimize accelerated development of ICAD in ApoE-KO rabbits to more efficiently study this disease.
RESUMEN
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.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Imidazoles/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Niacinamida/análogos & derivados , Pirazoles/farmacología , Piridazinas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Imidazoles/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Terapia Molecular Dirigida/métodos , Mutación , Niacinamida/farmacología , Niacinamida/uso terapéutico , Cultivo Primario de Células , Pirazoles/uso terapéutico , Piridazinas/uso terapéuticoRESUMEN
PURPOSE: Rearranged ROS1 is a crizotinib-sensitive oncogenic driver in lung cancer. The development of acquired resistance, however, poses a serious clinical challenge. Consequently, experimental and clinical validation of resistance mechanisms and potential second-line therapies is essential. EXPERIMENTAL DESIGN: We report the discovery of a novel, solvent-front ROS1(D2033N) mutation in a patient with CD74-ROS1-rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib resistance of CD74-ROS1(D2033N) was functionally evaluated using cell-based assays and structural modeling. RESULTS: In biochemical and cell-based assays, the CD74-ROS1(D2033N) mutant demonstrated significantly decreased sensitivity to crizotinib. Molecular dynamics simulation revealed compromised crizotinib binding due to drastic changes in the electrostatic interaction between the D2033 residue and crizotinib and reorientation of neighboring residues. In contrast, cabozantinib binding was unaffected by the D2033N substitution, and inhibitory potency against the mutant was retained. Notably, cabozantinib treatment resulted in a rapid clinical and near-complete radiographic response in this patient. CONCLUSIONS: These results provide the first example of successful therapeutic intervention with targeted therapy to overcome crizotinib resistance in a ROS1-rearranged cancer. Clin Cancer Res; 22(10); 2351-8. ©2015 AACR.
Asunto(s)
Anilidas/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Antígenos de Diferenciación de Linfocitos B/genética , Línea Celular Tumoral , Ensayos Clínicos Fase II como Asunto , Crizotinib , Resistencia a Antineoplásicos/genética , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Persona de Mediana Edad , Mutación/genética , Oncogenes/efectos de los fármacos , Oncogenes/genéticaRESUMEN
Activation of Janus kinase 2 (JAK2), frequently as a result of the JAK2(V617F) mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and myelofibrosis, and it is thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2-selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34(+) progenitor cells from patients with JAK2(V617F)-positive MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction-transplantation model of JAK2(V617F) MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high-dose group and were maintained well below the levels in vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red blood cell parameters. After 42 days of treatment, the proportion of JAK2(V617F)-positive cells in hematopoietic tissues was identical or slightly increased compared with controls. Plasma concentrations of interleukin 6, interleukin 15, and tumor necrosis factor α were elevated in MPN mice and reduced in the high-dose treatment group, whereas other cytokines were unchanged. Inhibitor activity after dosing was confirmed in a cell culture assay using the plasma of dosed mice and phosphorylated signal transducer and activator of transcription 5 flow cytometry. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2(V617F)-driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control.
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Antineoplásicos/uso terapéutico , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Janus Quinasa 2/antagonistas & inhibidores , Terapia Molecular Dirigida , Trastornos Mieloproliferativos/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Antineoplásicos/farmacología , Médula Ósea/patología , Citocinas/sangre , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Janus Quinasa 2/genética , Ratones , Ratones Endogámicos BALB C , Mutación Missense , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Proteínas de Neoplasias/genética , Mutación Puntual , Inhibidores de Proteínas Quinasas/farmacología , Quimera por Radiación , Bazo/patologíaRESUMEN
Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen-deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 µM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.
Asunto(s)
Ácidos Aminosalicílicos/farmacología , Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica , Leucocitos Mononucleares/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Factor de Transcripción STAT3/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Ácidos Aminosalicílicos/síntesis química , Ácidos Aminosalicílicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Línea Celular Tumoral , Dasatinib , Descubrimiento de Drogas , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/metabolismo , Genes Reporteros , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Luciferasas/genética , Luciferasas/metabolismo , Simulación del Acoplamiento Molecular , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Pirimidinas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Sulfonamidas/síntesis química , Sulfonamidas/química , Tiazoles/farmacologíaRESUMEN
Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Imidazoles/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Piridazinas/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Dominio Catalítico , Proteínas de Fusión bcr-abl/química , Humanos , Imidazoles/química , Imidazoles/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Simulación de Dinámica Molecular , Mutación Missense , Cromosoma Filadelfia , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridazinas/química , Piridazinas/uso terapéutico , Insuficiencia del TratamientoRESUMEN
The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Apoptosis/efectos de los fármacos , Benzamidas/farmacocinética , Benzamidas/farmacología , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Piperazinas/farmacocinética , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Tyrosine kinase inhibitor (TKI) therapy targeting the BCR-ABL1 kinase is effective against chronic myeloid leukaemia (CML), but is not curative for most patients. Minimal residual disease (MRD) is thought to reside in TKI-insensitive leukaemia stem cells (LSCs) that are not fully addicted to BCR-ABL1. Recent conceptual advances in both CML biology and therapeutic intervention have increased the potential for the elimination of CML cells, including LSCs, through simultaneous inhibition of BCR-ABL1 and other newly identified, crucial targets.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Terapia Molecular Dirigida/métodos , Proteínas Tirosina Quinasas/metabolismoRESUMEN
We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.