RESUMEN
Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.
Asunto(s)
Leucemia Mieloide Aguda , Diferenciación Celular , Estudios de Cohortes , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Receptores de Superficie Celular/genética , TranscriptomaAsunto(s)
Aurora Quinasa A , Leucemia-Linfoma Linfoblástico de Células Precursoras , Aurora Quinasa A/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Humanos , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocación GenéticaRESUMEN
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.
Asunto(s)
Médula Ósea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Linfocitos T/metabolismo , Microambiente Tumoral/fisiología , Médula Ósea/inmunología , Antígeno CTLA-4/metabolismo , Proliferación Celular , Citocinas/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T/inmunologíaRESUMEN
To identify new therapeutic targets in acute myeloid leukemia (AML), we performed small-molecule and small-interfering RNA (siRNA) screens of primary AML patient samples. In 23% of samples, we found sensitivity to inhibition of colony-stimulating factor 1 (CSF1) receptor (CSF1R), a receptor tyrosine kinase responsible for survival, proliferation, and differentiation of myeloid-lineage cells. Sensitivity to CSF1R inhibitor GW-2580 was found preferentially in de novo and favorable-risk patients, and resistance to GW-2580 was associated with reduced overall survival. Using flow cytometry, we discovered that CSF1R is not expressed on the majority of leukemic blasts but instead on a subpopulation of supportive cells. Comparison of CSF1R-expressing cells in AML vs healthy donors by mass cytometry revealed expression of unique cell-surface markers. The quantity of CSF1R-expressing cells correlated with GW-2580 sensitivity. Exposure of primary AML patient samples to a panel of recombinant cytokines revealed that CSF1R inhibitor sensitivity correlated with a growth response to CSF1R ligand, CSF1, and other cytokines, including hepatocyte growth factor (HGF). The addition of CSF1 increased the secretion of HGF and other cytokines in conditioned media from AML patient samples, whereas adding GW-2580 reduced their secretion. In untreated cells, HGF levels correlated significantly with GW-2580 sensitivity. Finally, recombinant HGF and HS-5-conditioned media rescued cell viability after GW-2580 treatment in AML patient samples. Our results suggest that CSF1R-expressing cells support the bulk leukemia population through the secretion of HGF and other cytokines. This study identifies CSF1R as a novel therapeutic target of AML and provides a mechanism of paracrine cytokine/growth factor signaling in this disease.
Asunto(s)
Anisoles/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Comunicación Paracrina/efectos de los fármacos , Pirimidinas/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Microambiente Tumoral/efectos de los fármacos , Antineoplásicos/farmacología , Diferenciación Celular , Supervivencia Celular , Medios de Cultivo Condicionados/farmacología , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Pronóstico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Genómica , Leucemia Mieloide Aguda/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Conjuntos de Datos como Asunto , Exoma/genética , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Terapia Molecular Dirigida , Proteínas Nucleares/genética , Nucleofosmina , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Análisis de Secuencia de ARN , Factores de Empalme Serina-Arginina/genéticaRESUMEN
The protein tyrosine phosphatase PTPN11 is implicated in the pathogenesis of juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and other malignancies. Activating mutations in PTPN11 increase downstream proliferative signaling and cell survival. We investigated the signaling upstream of PTPN11 in JMML and AML cells and found that PTPN11 was activated by the nonreceptor tyrosine/serine/threonine kinase TNK2 and that PTPN11-mutant JMML and AML cells were sensitive to TNK2 inhibition. In cultured human cell-based assays, PTPN11 and TNK2 interacted directly, enabling TNK2 to phosphorylate PTPN11, which subsequently dephosphorylated TNK2 in a negative feedback loop. Mutations in PTPN11 did not affect this physical interaction but increased the basal activity of PTPN11 such that TNK2-mediated activation was additive. Consequently, coexpression of TNK2 and mutant PTPN11 synergistically increased mitogen-activated protein kinase (MAPK) signaling and enhanced colony formation in bone marrow cells from mice. Chemical inhibition of TNK2 blocked MAPK signaling and colony formation in vitro and decreased disease burden in a patient with PTPN11-mutant JMML who was treated with the multikinase (including TNK2) inhibitor dasatinib. Together, these data suggest that TNK2 is a promising therapeutic target for PTPN11-mutant leukemias.
Asunto(s)
Dasatinib/farmacología , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Juvenil/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Mutaciones Letales Sintéticas , Animales , Niño , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Juvenil/tratamiento farmacológico , Leucemia Mielomonocítica Juvenil/enzimología , Leucemia Mielomonocítica Juvenil/genética , Masculino , Ratones , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Tasa de Supervivencia , Ensayo de Tumor de Célula MadreRESUMEN
In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.
RESUMEN
Although small molecule inhibitors of B-cell receptor-associated kinases have revolutionized therapy in chronic lymphocytic leukemia (CLL), responses are incomplete. Pro-survival signaling emanating from the microenvironment may foster therapeutic resistance of the malignant B cells resident in the protective lymphoid niches. B-cell activating factor (BAFF) is critical to the survival of both healthy and neoplastic B cells. However, the pro-survival pathways triggered by BAFF have not been fully characterized. Here we show that BAFF elicited resistance to spontaneous and drug-induced apoptosis in stromal co-cultures, induced activation of both canonical and non-canonical NFκB signaling pathways, and triggered B-cell receptor signaling in CLL cells, independently of IGHV mutational status. SYK, a proximal kinase in the B-cell receptor signaling cascade, acted via STAT3 to bolster transcription of the anti-apoptotic protein Mcl-1, thereby contributing to apoptosis resistance in BAFF-stimulated cells. SYK inhibitor entospletinib downregulated Mcl-1, abrogating BAFF-mediated cell survival. BAFF-B-cell receptor crosstalk in neoplastic B cells was mediated by SYK interaction with TRAF2/TRAF3 complex. Thus, SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and B-cell receptor, thereby disrupting the pro-survival microenvironment signaling in chronic lymphocytic leukemia.
Asunto(s)
Factor Activador de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Quinasa Syk/antagonistas & inhibidores , Animales , Células CHO , Línea Celular Tumoral , Supervivencia Celular/genética , Análisis por Conglomerados , Cricetulus , Perfilación de la Expresión Génica , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Inmunohistoquímica , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Modelos Biológicos , FN-kappa B/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNFAsunto(s)
Autofagia/efectos de los fármacos , Hidroxicloroquina/farmacología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Recurrencia , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
PURPOSE: Identify AML patients most likely to respond to CPX-351, a nano-scale liposome formulation containing cytarabine and daunorubicin co-encapsulated at a 5:1 molar ratio. METHODS: We examined the ex vivo cytotoxic activity of CPX-351 against leukemic cells isolated from 53 AML patients and an additional 127 samples including acute lymphoblastic leukemia, myelodysplastic syndrome/myeloproliferative neoplasms, or chronic lymphocytic leukemia/lymphoma. We assessed activity with respect to common molecular lesions and used flow cytometry to assess CPX-351 cellular uptake. RESULTS: AML specimen sensitivity to CPX-351 was similar across conventional risk groups. FLT3-ITD cases were five-fold more sensitive to CPX-351. CPX-351 was active across other indications with nearly all cases exhibiting IC50 values markedly lower than reported 72-h plasma drug concentration in patients receiving CPX-351. The range and distribution of CPX-351 IC50 values were comparable for AML, CLL, and ALL, whereas MDS/MPN cases were less sensitive. CPX-351 uptake analysis revealed a correlation between uptake of CPX-351 and cytotoxic potency. CONCLUSIONS: Our findings are consistent with clinical data, in which CPX-351 activity is retained in high-risk AML patients. Ex vivo analysis of cytotoxic potency may provide a means to identify specific AML subsets, such as FLT3-ITD, that benefit most from CPX-351 and warrant additional clinical evaluation.
Asunto(s)
Crisis Blástica/tratamiento farmacológico , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Liposomas/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Antibióticos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Humanos , Concentración 50 Inhibidora , Mutación , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) remains a challenge. Although the addition of targeted tyrosine kinase inhibitors (TKIs) to standard cytotoxic therapy has greatly improved upfront treatment, treatment-related morbidity and mortality remain high. TKI monotherapy provides only temporary responses and renders patients susceptible to the development of TKI resistance. Thus, identifying agents that could enhance the activity of TKIs is urgently needed. Recently, a selective inhibitor of B cell lymphoma 2 (BCL-2), ABT-199 (venetoclax), has shown impressive activity against hematologic malignancies. We demonstrate that the combination of TKIs with venetoclax is highly synergistic in vitro, decreasing cell viability and inducing apoptosis in Ph(+)ALL. Furthermore, the multikinase inhibitors dasatinib and ponatinib appear to have the added advantage of inducing Lck/Yes novel tyrosine kinase (LYN)-mediated proapoptotic BCL-2-like protein 11 (BIM) expression and inhibiting up-regulation of antiapoptotic myeloid cell leukemia 1 (MCL-1), thereby potentially overcoming the development of venetoclax resistance. Evaluation of the dasatinib-venetoclax combination for the treatment of primary Ph(+)ALL patient samples in xenografted immunodeficient mice confirmed the tolerability of this drug combination and demonstrated its superior antileukemic efficacy compared to either agent alone. These data suggest that the combination of dasatinib and venetoclax has the potential to improve the treatment of Ph(+)ALL and should be further evaluated for patient care.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Familia-src Quinasas/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Línea Celular Tumoral , Dasatinib/administración & dosificación , Humanos , Imidazoles/administración & dosificación , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Piridazinas/administración & dosificación , Factor de Transcripción STAT5/metabolismo , Investigación Biomédica Traslacional , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in the majority of chronic neutrophilic leukemia (CNL) and a smaller percentage of atypical chronic myeloid leukemia (aCML) cases. Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. In this study, we assess the importance of the CSF3R T640N mutation as a marker of CNL/aCML and potential therapeutic target. EXPERIMENTAL DESIGN: Sanger sequencing of leukemia samples was performed to identify CSF3R mutations in CNL and aCML. The oncogenicity of the CSF3R T640N mutation relative to the T618I mutation was assessed by cytokine independent growth assays and by mouse bone marrow transplant. Receptor dimerization and O-glycosylation of the mutants was assessed by Western blot, and JAK inhibitor sensitivity was assessed by colony assay. RESULTS: Here, we identify a CSF3R T640N mutation in two patients with CNL/aCML, one of whom was originally diagnosed with MDS and acquired the T640N mutation upon evolution of disease to aCML. The T640N mutation is oncogenic in cellular transformation assays and an in vivo mouse bone marrow transplantation model. It exhibits many similar phenotypic features to T618I, including ligand independence and altered patterns of O-glycosylation--despite the transmembrane location of T640 preventing access by GalNAc transferase enzymes. Cells transformed by the T640N mutation are sensitive to JAK kinase inhibition to a similar degree as cells transformed by CSF3R T618I. CONCLUSIONS: Because of its similarities to CSF3R T618I, the T640N mutation likely has diagnostic and therapeutic relevance in CNL/aCML.
Asunto(s)
Codón , Quinasas Janus/antagonistas & inhibidores , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Receptores del Factor Estimulante de Colonias/genética , Sustitución de Aminoácidos , Animales , Médula Ósea/patología , Trasplante de Médula Ósea , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Femenino , Glicosilación , Humanos , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia/metabolismo , Leucemia/mortalidad , Leucemia/patología , Ligandos , Masculino , Ratones , Persona de Mediana Edad , Receptores del Factor Estimulante de Colonias/metabolismoAsunto(s)
Antineoplásicos/uso terapéutico , Exosomas/patología , Leucemia Mieloide Aguda/patología , Células Madre Mesenquimatosas/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Adolescente , Adulto , Anciano , Niño , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adulto JovenRESUMEN
PURPOSE: Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma. EXPERIMENTAL DESIGN: A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells. RESULTS: The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events. CONCLUSION: Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma.
Asunto(s)
Acetamidas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azepinas/farmacología , Bortezomib/farmacología , Mieloma Múltiple/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Acetamidas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis/efectos de los fármacos , Azepinas/administración & dosificación , Bortezomib/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Mieloma Múltiple/patologíaRESUMEN
Genome sequencing is revealing a vast mutational landscape in leukemia, offering new opportunities for treatment with targeted therapy. Here, we identify two patients with acute myelogenous leukemia and B-cell acute lymphoblastic leukemia whose tumors harbor point mutations in the ALK kinase. The mutations reside in the extracellular domain of ALK and are potently transforming in cytokine-independent cellular assays and primary mouse bone marrow colony formation studies. Strikingly, both mutations conferred sensitivity to ALK kinase inhibitors, including the FDA-approved drug crizotinib. On the basis of our results, we propose that tumors harboring ALK mutations may be therapeutically tractable for personalized treatment of certain aggressive leukemias with ALK inhibitors.
Asunto(s)
Leucemia Mieloide Aguda/genética , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Crizotinib , Resistencia a Antineoplásicos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Mutación , Medicina de Precisión , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidoresRESUMEN
BACKGROUND: Novel-targeted therapies are in rapid development for the treatment of acute lymphoblastic leukemia (ALL) to overcome resistance and decrease toxicity. Survivin, a member of the inhibitor of apoptosis gene family and chromosome passenger complex, is critical in a variety of human cancers, including ALL. A well-established suppressor of survivin has been the small molecule, YM155. Reports are identifying other mechanisms of action for YM155. Therefore, we sought to investigate the mode of action and role of YM155 for therapeutic use in the context of ALL. METHODS: Primary ALL samples and ALL cell lines were interrogated with YM155 to identify drug sensitivity. Ph(+)ALL harboring the BCR-ABL1 oncogene were tested for any interaction with YM155 and the multi-kinase inhibitor dasatinib. Representative ALL cell lines were tested to identify the response to YM155 using standard biochemical assays as well as RNA expression and phosphorylation arrays. RESULTS: ALL samples exhibited significant sensitivity to YM155, and an additive response was observed with dasatinib in the setting of Ph(+)ALL. ALL cells were more sensitive to YM155 during S phase during DNA replication. YM155 activates the DNA damage pathway leading to phosphorylation of Chk2 and H2AX. Interestingly, screening of primary patient samples identified unique and exquisite YM155 sensitivity in some but not all ALL specimens. CONCLUSION: These results are the first to have screened a large number of primary patient leukemic samples to identify individual variations of response to YM155. Our studies further support that YM155 in ALL induces DNA damage leading to S phase arrest. Finally, only subsets of ALL have exquisite sensitivity to YM155 presumably through both suppression of survivin expression and activation of the DNA damage pathway underscoring its potential for therapeutic development.
Asunto(s)
Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , Imidazoles/farmacología , Naftoquinonas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Humanos , Immunoblotting , Concentración 50 InhibidoraRESUMEN
Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.
Asunto(s)
Basófilos/patología , Diferenciación Celular/efectos de los fármacos , Eosinófilos/patología , Factor de Transcripción Ikaros/antagonistas & inhibidores , Leucemia Mieloide de Fase Crónica/patología , Factor de Transcripción STAT5/metabolismo , Animales , Antígenos CD34/metabolismo , Apoptosis/efectos de los fármacos , Basófilos/efectos de los fármacos , Basófilos/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Progresión de la Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Citometría de Flujo , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Técnicas para Inmunoenzimas , Leucemia Mieloide de Fase Crónica/genética , Leucemia Mieloide de Fase Crónica/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/genéticaRESUMEN
Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27(CK-)) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27(T187A)) or nuclear retention (p27(S10A)) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization.
Asunto(s)
Transformación Celular Neoplásica/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Citoplasma/metabolismo , Proteínas de Fusión bcr-abl/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Animales , Células Cultivadas , Genes Supresores de Tumor , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Oncogénicas/metabolismo , Transporte de Proteínas/genéticaRESUMEN
Agents that target B-cell receptor (BCR) signaling in lymphoid malignancies including idelalisib (GS-1101) and fostamatinib which inhibit the delta isoform of PI3 kinase (PI3Kd) and spleen tyrosine kinase (Syk) respectively have shown significant clinical activity. By disrupting B-cell signaling pathways, idelalisib treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high risk disease remain challenges. Targeting the BCR signaling pathway with simultaneous inhibition of PI3Kd and Syk has not yet been reported. We evaluated the pre-clinical activity of idelalisib combined with the novel and selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow Chronic Lymphocytic Leukemia (CLL) samples. Both PI3Kd and Syk inhibition reduced CLL survival and in combination induced synergistic growth inhibition and further disrupted chemokine signaling at nanomolar concentrations including in bone marrow derived and poor risk samples. Simultaneous targeting of these kinases may significantly increase clinical activity.