RESUMEN
Myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid cells, disease-related mutations in certain driver-genes including JAK2, CALR, and MPL, and a substantial risk to progress to secondary acute myeloid leukemia (sAML). Although behaving as stem cell neoplasms, little is known about disease-initiating stem cells in MPN. We established the phenotype of putative CD34+ /CD38- stem cells and CD34+ /CD38+ progenitor cells in MPN. A total of 111 patients with MPN suffering from polycythemia vera, essential thrombocythemia, or primary myelofibrosis (PMF) were examined. In almost all patients tested, CD34+ /CD38- stem cells expressed CD33, CD44, CD47, CD52, CD97, CD99, CD105, CD117, CD123, CD133, CD184, CD243, and CD274 (PD-L1). In patients with PMF, MPN stem cells often expressed CD25 and sometimes also CD26 in an aberrant manner. MPN stem cells did not exhibit substantial amounts of CD90, CD273 (PD-L2), CD279 (PD-1), CD366 (TIM-3), CD371 (CLL-1), or IL-1RAP. The phenotype of CD34+ /CD38- stem cells did not change profoundly during progression to sAML. The disease-initiating capacity of putative MPN stem cells was confirmed in NSGS mice. Whereas CD34+ /CD38- MPN cells engrafted in NSGS mice, no substantial engraftment was produced by CD34+ /CD38+ or CD34- cells. The JAK2-targeting drug fedratinib and the BRD4 degrader dBET6 induced apoptosis and suppressed proliferation in MPN stem cells. Together, MPN stem cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules, and other clinically relevant target antigens. Phenotypic characterization of neoplastic stem cells in MPN and sAML should facilitate their enrichment and the development of stem cell-eradicating (curative) therapies.
Asunto(s)
Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Policitemia Vera , Animales , Ratones , Calreticulina/genética , Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/genética , Células Madre Neoplásicas , Proteínas Nucleares/genética , Fenotipo , Policitemia Vera/genética , Factores de Transcripción/genética , HumanosRESUMEN
In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream-effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34+ /CD38- leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22, and KCL22T315I . The BRD4-targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1-resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast-induced TKI resistance of CML LSC in a co-culture system and to block interferon-gamma-induced upregulation of the checkpoint antigen PD-L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Proteínas Nucleares , Animales , Crisis Blástica/tratamiento farmacológico , Proteínas de Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Ratones , Proteínas Nucleares/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-myc , Células Madre , Factores de Transcripción/genéticaRESUMEN
Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.
Asunto(s)
Azepinas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Triazoles/farmacología , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Genes myc/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Mastocytosis is a term used to denote a group of rare diseases characterized by an abnormal accumulation of neoplastic mast cells in various tissues and organs. In most patients with systemic mastocytosis, the neoplastic cells carry activating mutations in KIT Progress in mastocytosis research has long been hindered by the lack of suitable in vitro models, such as permanent human mast cell lines. In fact, only a few human mast cell lines are available to date: HMC-1, LAD1/2, LUVA, ROSA and MCPV-1. The HMC-1 and LAD1/2 cell lines were derived from patients with mast cell leukemia. By contrast, the more recently established LUVA, ROSA and MCPV-1 cell lines were derived from CD34+ cells of non-mastocytosis donors. While some of these cell lines (LAD1/2, LUVA, ROSAKIT WT and MCPV-1) do not harbor KIT mutations, HMC-1 and ROSAKIT D816V cells exhibit activating KIT mutations found in mastocytosis and have thus been used to study disease pathogenesis. In addition, these cell lines are increasingly employed to validate new therapeutic targets and to screen for effects of new targeted drugs. Recently, the ROSAKIT D816V subclone has been successfully used to generate a unique in vivo model of advanced mastocytosis by injection into immunocompromised mice. Such a model may allow in vivo validation of data obtained in vitro with targeted drugs directed against mastocytosis. In this review, we discuss the major characteristics of all available human mast cell lines, with particular emphasis on the use of HMC-1 and ROSAKIT D816V cells in preclinical therapeutic research in mastocytosis.
Asunto(s)
Línea Celular Tumoral , Mastocitos , Mastocitosis Sistémica , Modelos Biológicos , Animales , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/patología , Humanos , Mastocitos/metabolismo , Mastocitos/patología , Mastocitosis Sistémica/genética , Mastocitosis Sistémica/metabolismo , Mastocitosis Sistémica/patologíaRESUMEN
Systemic mastocytosis is a complex disease defined by abnormal growth and accumulation of neoplastic mast cells in various organs. Most patients exhibit a D816V-mutated variant of KIT, which confers resistance against imatinib. Clinical problems in systemic mastocytosis arise from mediator-related symptoms and/or organ destruction caused by malignant expansion of neoplastic mast cells and/or other myeloid cells in various organ systems. DCC-2618 is a spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. We found that DCC-2618 inhibits the proliferation and survival of various human mast cell lines (HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC50 <1 µM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. Furthermore, DCC-2618 was found to suppress the proliferation of endothelial cells, suggesting additional drug effects on systemic mastocytosis-related angiogenesis. Finally, DCC-2618 was found to downregulate IgE-mediated histamine release from basophils and tryptase release from mast cells. Together, DCC-2618 inhibits growth, survival and activation of multiple cell types relevant to advanced systemic mastocytosis. Whether DCC-2618 is effective in vivo in patients with advanced systemic mastocytosis is currently under investigation in clinical trials.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Mastocitos/patología , Mastocitosis Sistémica/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Mastocitosis Sistémica/tratamiento farmacológico , Mastocitosis Sistémica/metabolismo , Persona de Mediana Edad , Mutación , Células Tumorales CultivadasRESUMEN
Mast cell leukemia (MCL) is a rare, life-threatening malignancy defined by a substantial increase in neoplastic mast cells (MCs) in bone marrow (BM) smears, drug-resistance, and a poor prognosis. In most patients, the survival time is less than 1 year. However, exceptional cases may present with a less malignant course. We report on a 49-year-old female patient with MCL diagnosed in 2013. In February 2013, first symptoms, including flushing, headache, and diarrhea, were recorded. In addition, mild anemia was detected. The disease was characterized by a massive increase in well-granulated, mature, and often spindle-shaped MCs (80 %) in BM smears. The serum tryptase level amounted to 332 ng/mL. Like in most other MCL patients, no skin lesions were detected. However, unlike in other patients, tryptase levels remained stable, and no other signs or symptoms of MCL-induced organ damage were found. Sequencing studies revealed an isolated S476I point mutation in KIT but no mutation in codon 816. The patient received histamine receptor blockers but refused cytoreductive therapy. After 9 months, still no progression or organ damage was detected. However, progression with transformation to acute MCL occurred after 12 months. We propose that the chronic type of MCL with stable conditions, absence of organ damage, and a mature MC morphology is recognized as a distinct entity that should be distinguished from the acute variant of MCL.
Asunto(s)
Leucemia de Mastocitos/genética , Mastocitos/metabolismo , Mutación Missense , Proteínas Proto-Oncogénicas c-kit/genética , Examen de la Médula Ósea , Proliferación Celular/efectos de los fármacos , Enfermedad Crónica , Femenino , Humanos , Leucemia de Mastocitos/patología , Mastocitos/efectos de los fármacos , Mastocitos/patología , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are advanced hematopoietic neoplasms with poor prognosis. In these patients, neoplastic mast cells (MCs) are resistant against various drugs. We examined the effects of 2 demethylating agents, 5-azacytidine and decitabine on growth and survival of neoplastic MCs and the MC line HMC-1. Two HMC-1 subclones were used, HMC-1.1 lacking KIT D816V and HMC-1.2 exhibiting KIT D816V. Both agents induced apoptosis in HMC-1.1 and HMC-1.2 cells. Decitabine, but not 5-azacytidine, also produced a G(2)/M cell-cycle arrest in HMC-1 cells. Drug-induced apoptosis was accompanied by cleavage of caspase-8 and caspase-3 as well as FAS-demethylation and FAS-re-expression in neoplastic MCs. Furthermore, both demethylating agents were found to synergize with the FAS-ligand in inducing apoptosis in neoplastic MCs. Correspondingly, siRNA against FAS was found to block drug-induced expression of FAS and drug-induced apoptosis in HMC-1 cells. Neither 5-azacytidine nor decitabine induced substantial apoptosis or growth arrest in normal MCs or normal bone marrow cells. Together, 5-azacytidine and decitabine exert growth-inhibitory and proapoptotic effects in neoplastic MCs. These effects are mediated through "FAS-re-expression" and are augmented by the FAS-ligand. Whether epigenetic drugs produce antineoplastic effects in vivo in patients with ASM and MCL remains to be determined.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacología , Leucemia de Mastocitos/patología , Mastocitos/efectos de los fármacos , Mastocitosis Sistémica/patología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptor fas/metabolismo , Adulto , Anciano , Secuencia de Bases , Línea Celular Tumoral/efectos de los fármacos , Islas de CpG , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Decitabina , Sinergismo Farmacológico , Proteína Ligando Fas/fisiología , Femenino , Humanos , Masculino , Mastocitos/patología , Metilación/efectos de los fármacos , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Mutación Puntual , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-kit/genética , ARN Interferente Pequeño/farmacología , Receptor fas/antagonistas & inhibidores , Receptor fas/genéticaRESUMEN
A major problem in SARS-CoV-2-infected patients is the massive tissue inflammation in certain target organs, including the lungs. Mast cells (MC), basophils (BA), and eosinophils (EO) are key effector cells in inflammatory processes. These cells have recently been implicated in the pathogenesis of SARS-CoV-2 infections. We explored coronavirus receptor (CoV-R) expression profiles in primary human MC, BA, and EO, and in related cell lines (HMC-1, ROSA, MCPV-1, KU812, and EOL-1). As determined using flow cytometry, primary MC, BA, and EO, and their corresponding cell lines, displayed the CoV-R CD13 and CD147. Primary skin MC and BA, as well as EOL-1 cells, also displayed CD26, whereas primary EO and the MC and BA cell lines failed to express CD26. As assessed using qPCR, most cell lines expressed transcripts for CD13, CD147, and ABL2, whereas ACE2 mRNA was not detectable, and CD26 mRNA was only identified in EOL-1 cells. We also screened for drug effects on CoV-R expression. However, dexamethasone, vitamin D, and hydroxychloroquine did not exert substantial effects on the expression of CD13, CD26, or CD147 in the cells. Together, MC, BA, and EO express distinct CoV-R profiles. Whether these receptors mediate virus-cell interactions and thereby virus-induced inflammation remains unknown at present.
Asunto(s)
Dipeptidil Peptidasa 4 , Mastocitos , Humanos , Dipeptidil Peptidasa 4/genética , Receptores de Coronavirus , Basófilos , Eosinófilos , InflamaciónRESUMEN
Patients with advanced systemic mastocytosis, including mast cell leukemia, have a poor prognosis. In these patients, neoplastic mast cells usually harbor the KIT mutant D816V that confers resistance against tyrosine kinase inhibitors. We examined the effects of the multi-kinase blocker ponatinib on neoplastic mast cells and investigated whether ponatinib acts synergistically with other antineoplastic drugs. Ponatinib was found to inhibit the kinase activity of KIT G560V and KIT D816V in the human mast cell leukemia cell line HMC-1. In addition, ponatinib was found to block Lyn- and STAT5 activity in neoplastic mast cells. Ponatinib induced growth inhibition and apoptosis in HMC-1.1 cells (KIT G560V(+)) and HMC-1.2 cells (KIT G560V(+)/KIT D816V(+)) as well as in primary neoplastic mast cells. The effects of ponatinib were dose-dependent, but higher IC50-values were obtained in HMC-1 cells harboring KIT D816V than in those lacking KIT D816V. In drug combination experiments, ponatinib was found to synergize with midostaurin in producing growth inhibition and apoptosis in HMC-1 cells and primary neoplastic mast cells. The ponatinib+midostaurin combination induced substantial inhibition of KIT-, Lyn-, and STAT5 activity, but did not suppress Btk. We then applied a Btk short interfering RNA and found that Btk knockdown sensitizes HMC-1 cells against ponatinib. Finally, we were able to show that ponatinib synergizes with the Btk-targeting drug dasatinib to produce growth inhibition in HMC-1 cells. In conclusion, ponatinib exerts major growth-inhibitory effects on neoplastic mast cells in advanced systemic mastocytosis and synergizes with midostaurin and dasatinib in inducing growth arrest in neoplastic mast cells.
Asunto(s)
Inhibidores de Crecimiento/administración & dosificación , Imidazoles/administración & dosificación , Mastocitosis Sistémica/tratamiento farmacológico , Mastocitosis Sistémica/genética , Proteínas Proto-Oncogénicas c-kit/genética , Piridazinas/administración & dosificación , Estaurosporina/análogos & derivados , Adulto , Anciano , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Masculino , Mastocitosis Sistémica/diagnóstico , Persona de Mediana Edad , Estaurosporina/administración & dosificaciónRESUMEN
INTRODUCTION: The Vienna Cancer Stem Cell Club (VCSCC) was launched by a group of scientists in Vienna in 2002. AREAS COVERED: Major aims of the VCSCC are to support research on cancer stem cells (CSC) in hematopoietic malignancies and to translate CSC-related markers and targets into clinical application. A primary focus of research in the VCSCC is the leukemic stem cell (LSC). Between 2013 and 2021, members of the VCSCC established a special research program on myeloproliferative neoplasms and since 2008, members of the VCSCC run the Ludwig Boltzmann Institute for Hematology and Oncology. In all these years, the VCSCC provided a robust intellectual platform for translational hematology and LSC research in Vienna. Furthermore, the VCSCC interacts with several national and international study groups and societies in the field. Representatives of the VCSCC also organized a number of international meetings and conferences on neoplastic stem cells, including LSC, in the past 15 years, and contributed to the definition and classification of CSC/LSC and related pre-malignant and malignant conditions. EXPERT OPINION: The VCSCC will continue to advance the field and to develop LSC-detecting and LSC-eradicating concepts through which diagnosis, prognostication, and therapy of blood cancer patients should improve.
Asunto(s)
Neoplasias Hematológicas , Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/patología , Neoplasias Hematológicas/patología , PredicciónRESUMEN
Systemic mastocytosis (SM) is a hematopoietic neoplasm with a complex pathology and a variable clinical course. Clinical symptoms result from organ infiltration by mast cells (MC) and the effects of pro-inflammatory mediators released during MC activation. In SM, growth and survival of MC are triggered by various oncogenic mutant-forms of the tyrosine kinase KIT. The most prevalent variant, D816V, confers resistance against various KIT-targeting drugs, including imatinib. We examined the effects of two novel promising KIT D816V-targeting drugs, avapritinib and nintedanib, on growth, survival, and activation of neoplastic MC and compared their activity profiles with that of midostaurin. Avapritinib was found to suppress growth of HMC-1.1 cells (KIT V560G) and HMC-1.2 cells (KIT V560G + KIT D816V) with comparable IC50 values (0.1-0.25 µM). In addition, avapritinib was found to inhibit the proliferation of ROSAKIT WT cells, (IC50: 0.1-0.25 µM), ROSAKIT D816V cells (IC50: 1-5 µM), and ROSAKIT K509I cells (IC50: 0.1-0.25 µM). Nintedanib exerted even stronger growth-inhibitory effects in these cells (IC50 in HMC-1.1: 0.001-0.01 µM; HMC-1.2: 0.25-0.5 µM; ROSAKIT WT: 0.01-0.1 µM; ROSAKIT D816V: 0.5-1 µM; ROSAKIT K509I: 0.01-0.1 µM). Avapritinib and nintedanib also suppressed the growth of primary neoplastic cells in most patients with SM examined (avapritinib IC50: 0.5-5 µM; nintedanib IC50: 0.1-5 µM). Growth-inhibitory effects of avapritinib and nintedanib were accompanied by signs of apoptosis and decreased surface expression of the transferrin receptor CD71 in neoplastic MC. Finally, we were able to show that avapritinib counteracts IgE-dependent histamine secretion in basophils and MC in patients with SM. These effects of avapritinib may explain the rapid clinical improvement seen during treatment with this KIT inhibitor in patients with SM. In conclusion, avapritinib and nintedanib are new potent inhibitors of growth and survival of neoplastic MC expressing various KIT mutant forms, including D816V, V560G, and K509I, which favors the clinical development and application of these new drugs in advanced SM. Avapritinib is of particular interest as it also blocks mediator secretion in neoplastic MC.
RESUMEN
Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression, or by BH3-mimetics such as obatoclax, may be an attractive therapy concept in SM.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Regulación Neoplásica de la Expresión Génica , Mastocitos/metabolismo , Mastocitosis Sistémica/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Northern Blotting , Western Blotting , Ácidos Borónicos/farmacología , Bortezomib , Citometría de Flujo , Expresión Génica , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Mastocitos/efectos de los fármacos , Mastocitos/patología , Mastocitosis Sistémica/tratamiento farmacológico , Mastocitosis Sistémica/genética , Proteínas de la Membrana/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/genética , Pirazinas/farmacología , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , TransfecciónRESUMEN
BACKGROUND: In advanced systemic mastocytosis the response of neoplastic mast cells to conventional drugs is poor and the prognosis is bad. Current research is, therefore, attempting to identify novel drug targets in neoplastic mast cells. Polo-like kinase-1 is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias and solid tumors. DESIGN AND METHODS: In the present study, we analyzed the expression and function of Polo-like kinase-1 in neoplastic mast cells in systemic mastocytosis. RESULTS: As determined by immunostaining, primary neoplastic mast cells as well as the human mast cell leukemia cell line HMC-1 displayed phosphorylated Polo-like kinase-1. In addition, neoplastic mast cells expressed Polo-like kinase-1 mRNA. Polo-like kinase-1-specific small interfering RNA induced apoptosis in neoplastic mast cells, whereas no effect was seen with a control small interfering RNA. BI 2536, a drug targeting Polo-like kinase-1, was found to inhibit proliferation in HMC-1 cells in a dose-dependent manner. BI 2536 also inhibited the growth of primary neoplastic mast cells and cells of the canine mastocytoma cell line C2. The growth-inhibitory effects of BI 2536 on neoplastic mast cells were found to be associated with mitotic arrest and subsequent apoptosis. Finally, BI 2536 was found to synergize with the KIT-targeting kinase inhibitor midostaurin (PKC412) in inhibiting the growth of neoplastic mast cells. In control experiments, BI 2536 did not induce apoptosis in normal cultured mast cells. CONCLUSIONS: Collectively, our data show that Polo-like kinase-1 is a potential therapeutic target in neoplastic mast cells. Targeting Polo-like kinase-1 may be an attractive pharmacological concept in the management of advanced systemic mastocytosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Mastocitos/metabolismo , Mastocitosis Sistémica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Células HL-60 , Humanos , Inmunohistoquímica , Células K562 , Mastocitos/efectos de los fármacos , Mastocitos/patología , Mastocitosis Sistémica/genética , Mastocitosis Sistémica/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pteridinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismo , Timidina/farmacocinética , Células Tumorales Cultivadas , Células U937 , Quinasa Tipo Polo 1RESUMEN
Chronic myelomonocytic leukemia (CMML) is a stem cell-derived neoplasm characterized by dysplasia, uncontrolled expansion of monocytes, and substantial risk to transform to secondary acute myeloid leukemia (sAML). So far, little is known about CMML-initiating cells. We found that leukemic stem cells (LSC) in CMML reside in a CD34+/CD38- fraction of the malignant clone. Whereas CD34+/CD38- cells engrafted NSGS mice with overt CMML, no CMML was produced by CD34+/CD38+ progenitors or the bulk of CD34- monocytes. CMML LSC invariably expressed CD33, CD117, CD123 and CD133. In a subset of patients, CMML LSC also displayed CD52, IL-1RAP and/or CLL-1. CMML LSC did not express CD25 or CD26. However, in sAML following CMML, the LSC also expressed CD25 and high levels of CD114, CD123 and IL-1RAP. No correlations between LSC phenotypes, CMML-variant, mutation-profiles, or clinical course were identified. Pre-incubation of CMML LSC with gemtuzumab-ozogamicin or venetoclax resulted in decreased growth and impaired engraftment in NSGS mice. Together, CMML LSC are CD34+/CD38- cells that express a distinct profile of surface markers and target-antigens. During progression to sAML, LSC acquire or upregulate certain cytokine receptors, including CD25, CD114 and CD123. Characterization of CMML LSC should facilitate their enrichment and the development of LSC-eradicating therapies.
Asunto(s)
Antígenos CD34/inmunología , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Crónica/complicaciones , Células Madre Neoplásicas/patología , Fenotipo , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD34/metabolismo , Apoptosis , Estudios de Casos y Controles , Proliferación Celular , Femenino , Humanos , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.
RESUMEN
PURPOSE: Since tear film stability can be affected by fluorescein, the Dry Eye Workshop (DEWSII) recommended non-invasive measurement of tear breakup time (NIBUT). The aim of this study was to investigate the agreement and repeatability of four different instruments in the measurement of NIBUT. METHODS: 72 participants (mean 24.2 ± 3.6 years) were recruited for this multi-centre, cross-sectional study. NIBUT was measured three times from one eye using each of the instruments in randomized order on two separate sessions during a day, separated by at least 2 h. NIBUT was performed at three sites (Switzerland, Germany and UK) using three subjective instruments, Tearscope Plus (Keeler, Windsor, UK) (TS), Polaris (bon Optic, Lübeck, Germany) (POL), EasyTear Viewplus (Easytear, Rovereto, Italy) (ET) and the objective Keratograph 5 M (Oculus Optikgeräte GmbH, Wetzlar, Germany) (KER). As the latter instrument only analyses for 24 s, all data was capped at this value. RESULTS: NIBUT measurements (average of both sessions) between the four instruments were not statistically significantly different: TS (median 10.4, range 2.0-24.0 s), POL (10.1, 1.0-24.0 s), ET (10.6, 1.0-24.0 s) and KER (11.1, 2.6-24.0 s) (p = 0.949). The objective KER measures were on average (1.2 s ± 9.6 s, 95 % confidence interval) greater than the subjective evaluations of NIBUT with the other instruments (mean difference 0.4 s ± 7.7 s, 95 % confidence interval), resulting in a higher limits of agreement. The slope was -0.08 to 0.11 indicating no bias in the difference between instruments with the magnitude of the NIBUT. Repeated measurements from the two sessions were not significantly different for TS (p = 0.584), POL (p = 0.549), ET (p = 0.701) or KER (p = 0.261). CONCLUSIONS: The four instruments evaluated for their measurement of tear stability were reasonably repeatable and give similar average results.
Asunto(s)
Síndromes de Ojo Seco , Lágrimas , Estudios Transversales , Síndromes de Ojo Seco/diagnóstico , Fluoresceína , Humanos , Pruebas de VisiónRESUMEN
OBJECTIVE: Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of clonality of eosinophils, marked eosinophilia, and organ damage. In many patients, the transforming mutation FIP1L1-PDGFRalpha and the related CHIC2 deletion are found. The respective oncoprotein, FIP1L1-PDGFRalpha, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRalpha in most patients. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils. MATERIALS AND METHODS: We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRalpha. RESULTS: The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5 to 1 nM, which was found to be in the same range when compared to IC50 values produced with imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC50: 1-10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and in a TUNEL assay. In Western blot experiments, dasatinib completely blocked the phosphorylation of FIP1L1-PDGFRalpha in EOL-1 cells. CONCLUSIONS: Dasatinib inhibits the growth of leukemic eosinophils through targeting of the disease-related oncoprotein FIP1L1-PDGFRalpha. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL.
Asunto(s)
División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Eosinófilos/fisiología , Síndrome Hipereosinofílico/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacología , Factores de Escisión y Poliadenilación de ARNm/fisiología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Dasatinib , Eosinófilos/efectos de los fármacos , Eosinófilos/enzimología , Citometría de Flujo , Humanos , Eliminación de Secuencia , Timidina/metabolismo , Factores de Escisión y Poliadenilación de ARNm/efectos de los fármacosRESUMEN
OBJECTIVE: Advanced mast cell (MC) neoplasms are usually resistant to conventional therapy. Therefore, current research focuses on new targets in neoplastic MC and development of respective targeted drugs. Mastocytomas in dogs often behave as aggressive tumors. We report that heat-shock protein 32 (Hsp32), also known as heme oxygenase-1, is a survival-enhancing molecule and new target in canine mastocytoma cells. MATERIALS AND METHODS: As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, immunocytochemistry, and Western blotting, primary neoplastic dog MC, and the canine mastocytoma-derived cell line C2 expressed Hsp32 mRNA and the Hsp32 protein in a constitutive manner. RESULTS: The KIT-targeting drug midostaurin inhibited expression of Hsp32, as well as survival in C2 cells. Confirming the functional role of Hsp32, the inhibitory effect of midostaurin on C2 cells was markedly reduced by the Hsp32-inductor hemin. Two pharmacologic Hsp32-inhibitors, styrene maleic-acid micelle-encapsulated ZnPP (SMA-ZnPP) and pegylated zinc-protoporphyrin (PEG-ZnPP) were applied. Both drugs were found to inhibit proliferation of C2 cells as well as growth of primary neoplastic canine MC. The growth-inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose- and time-dependent (IC(50): 1-10 muM) and found to be associated with induction of apoptosis. CONCLUSIONS: Hsp32 is an important survival factor and interesting new target in neoplastic canine MC. Trials with Hsp32-targeted drugs are now warranted to define the clinical efficacy of these drugs.
Asunto(s)
Apoptosis/efectos de los fármacos , Hemo-Oxigenasa 1/antagonistas & inhibidores , Mastocitoma/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Perros , Hemo-Oxigenasa 1/análisis , Hemo-Oxigenasa 1/genética , Mastocitoma/enzimología , Mastocitoma/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/análisisRESUMEN
The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE-dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI-1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC50 values ranging between 0.1 and 1 µM in primary MCT cells and between 1 and 3 µM in C2 and NI-1 cells. In C2 cells, the combination "ibrutinib + midostaurin" produced synergistic growth-inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE-dependent histamine release in primary MCT cells, with IC50 values ranging from 0.05 to 0.1 µM in NI-1 cells, and from 0.05 to 1 µM in primary MCT cells. In summary, ibrutinib exerts anti-proliferative effects in canine neoplastic MCs and counteracts IgE-dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Enfermedades de los Perros/tratamiento farmacológico , Histamina/metabolismo , Mastocitoma/veterinaria , Pirazoles/farmacología , Pirimidinas/farmacología , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa/genética , Agammaglobulinemia Tirosina Quinasa/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Perros , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inmunoglobulina E/farmacología , Mastocitoma/tratamiento farmacológico , Piperidinas , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismoRESUMEN
In humans, advanced mast cell (MC) neoplasms are rare malignancies with a poor prognosis. Only a few preclinical models are available, and current treatment options are limited. In dogs, MC neoplasms are the most frequent malignant skin tumours. Unlike low-grade MC neoplasms, high-grade MC disorders usually have a poor prognosis with short survival. In both species, neoplastic MCs display activating KIT mutations, which are considered to contribute to disease evolution. Therefore, tyrosine kinase inhibitors against KIT have been developed. Unfortunately, clinical responses are unpredictable and often transient, which remains a clinical challenge in both species. Therefore, current efforts focus on the development of new improved treatment strategies. The field of comparative oncology may assist in these efforts and accelerate human and canine research regarding diagnosis, prognostication, and novel therapies. In this article, we review the current status of comparative oncology approaches and perspectives in the field of MC neoplasms.