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1.
Leukemia ; 38(7): 1581-1591, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38811818

RESUMEN

Internal tandem duplication mutations of FLT3 (FLT3/ITD) confer poor prognosis in AML. FLT3 tyrosine kinase inhibitors (TKIs) alone have limited and transient clinical efficacy thus calling for new targets for more effective combination therapy. In a loss-of-function RNAi screen, we identified NOTCH4 as one such potential target whose inhibition proved cytotoxic to AML cells, and also sensitized them to FLT3 inhibition. Further investigation found increased NOTCH4 expression in FLT3/ITD AML cell lines and primary patient samples. Inhibition of NOTCH4 by shRNA knockdown, CRISPR-Cas9-based knockout or γ-secretase inhibitors synergized with FLT3 TKIs to kill FLT3/ITD AML cells in vitro. NOTCH4 inhibition sensitized TKI-resistant FLT3/ITD cells to FLT3 TKI inhibition. The combination reduced phospho-ERK and phospho-AKT, indicating inhibition of MAPK and PI3K/AKT signaling pathways. It also led to changes in expression of genes involved in regulating cell cycling, DNA repair and transcription. A patient-derived xenograft model showed that the combination reduced both the level of leukemic involvement of primary human FLT3/ITD AML cells and their ability to engraft secondary recipients. In summary, these results demonstrate that NOTCH4 inhibition synergizes with FLT3 TKIs to eliminate FLT3/ITD AML cells, providing a new therapeutic target for AML with FLT3/ITD mutations.


Asunto(s)
Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Receptor Notch4 , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms , Humanos , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ratones , Receptor Notch4/genética , Mutación , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Transducción de Señal/efectos de los fármacos
2.
CRISPR J ; 4(2): 169-177, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33876959

RESUMEN

Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.


Asunto(s)
Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Edición Génica , Mutación , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN , Roturas del ADN de Doble Cadena , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN
3.
Cancer Discov ; 10(10): 1500-1513, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32606137

RESUMEN

The cell of origin of oncogenic transformation is a determinant of therapeutic sensitivity, but the mechanisms governing cell-of-origin-driven differences in therapeutic response have not been delineated. Leukemias initiating in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and highly express the transcription factor MECOM (EVI1) compared with leukemias derived from myeloid progenitors. Here, we compared leukemias initiated in either HSCs or myeloid progenitors to reveal a novel function for EVI1 in modulating p53 protein abundance and activity. HSC-derived leukemias exhibit decreased apoptotic priming, attenuated p53 transcriptional output, and resistance to lysine-specific demethylase 1 (LSD1) inhibitors in addition to classical genotoxic stresses. p53 loss of function in Evi1 lo progenitor-derived leukemias induces resistance to LSD1 inhibition, and EVI1hi leukemias are sensitized to LSD1 inhibition by venetoclax. Our findings demonstrate a role for EVI1 in p53 wild-type cancers in reducing p53 function and provide a strategy to circumvent drug resistance in chemoresistant EVI1 hi acute myeloid leukemia. SIGNIFICANCE: We demonstrate that the cell of origin of leukemia initiation influences p53 activity and dictates therapeutic sensitivity to pharmacologic LSD1 inhibitors via the transcription factor EVI1. We show that drug resistance could be overcome in HSC-derived leukemias by combining LSD1 inhibition with venetoclax.See related commentary by Gu et al., p. 1445.This article is highlighted in the In This Issue feature, p. 1426.


Asunto(s)
Regulación Leucémica de la Expresión Génica/genética , Histona Demetilasas/antagonistas & inhibidores , Leucemia/fisiopatología , Apoptosis , Humanos , Factores de Transcripción
4.
iScience ; 23(3): 100941, 2020 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-32169821

RESUMEN

Repair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically been centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lysine methyltransferase, Setd2, and its modification of lysine-36 trimethylation (H3K36me3), in the processing and joining of DNA ends during V(D)J recombination. Loss leads to mis-repair of Rag-induced DNA DSBs, especially when combined with loss of Atm kinase activity. Furthermore, loss reduces immune repertoire and a severe block in lymphogenesis as well as causes post-mitotic neuronal apoptosis. Together, these studies are suggestive of an important role of Setd2/H3K36me3 in these two mammalian developmental processes that are influenced by double-stranded break repair.

5.
Cancer Discov ; 10(2): 214-231, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31771968

RESUMEN

Spleen tyrosine kinase (SYK) is a nonmutated therapeutic target in acute myeloid leukemia (AML). Attempts to exploit SYK therapeutically in AML have shown promising results in combination with chemotherapy, likely reflecting induced mechanisms of resistance to single-agent treatment in vivo. We conducted a genome-scale open reading frame (ORF) resistance screen and identified activation of the RAS-MAPK-ERK pathway as one major mechanism of resistance to SYK inhibitors. This finding was validated in AML cell lines with innate and acquired resistance to SYK inhibitors. Furthermore, patients with AML with select mutations activating these pathways displayed early resistance to SYK inhibition. To circumvent SYK inhibitor therapy resistance in AML, we demonstrate that a MEK and SYK inhibitor combination is synergistic in vitro and in vivo. Our data provide justification for use of ORF screening to identify resistance mechanisms to kinase inhibitor therapy in AML lacking distinct mutations and to direct novel combination-based strategies to abrogate these. SIGNIFICANCE: The integration of functional genomic screening with the study of mechanisms of intrinsic and acquired resistance in model systems and human patients identified resistance to SYK inhibitors through MAPK signaling in AML. The dual targeting of SYK and the MAPK pathway offers a combinatorial strategy to overcome this resistance.This article is highlighted in the In This Issue feature, p. 161.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasa Syk/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Benzamidas/farmacología , Benzamidas/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Difenilamina/análogos & derivados , Difenilamina/farmacología , Difenilamina/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Indazoles/farmacología , Indazoles/uso terapéutico , Leucemia Mieloide Aguda/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Sistemas de Lectura Abierta/genética , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirazinas/farmacología , Pirazinas/uso terapéutico , Quinasa Syk/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Blood Adv ; 2(19): 2478-2490, 2018 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-30266823

RESUMEN

Infant B-cell acute lymphoblastic leukemias (B-ALLs) that harbor MLL-AF4 rearrangements are associated with a poor prognosis. One important obstacle to progress for this patient population is the lack of immunocompetent models that faithfully recapitulate the short latency and aggressiveness of this disease. Recent whole-genome sequencing of MLL-AF4 B-ALL samples revealed a high frequency of activating RAS mutations; however, single-agent targeting of downstream effectors of the RAS pathway in these mutated MLL-r B-ALLs has demonstrated limited and nondurable antileukemic effects. Here, we demonstrate that the expression of activating mutant N-Ras G12D cooperates with Mll-Af4 to generate a highly aggressive serially transplantable B-ALL in mice. We used our novel mouse model to test the sensitivity of Mll-Af4/N-Ras G12D leukemia to small molecule inhibitors and found potent and synergistic preclinical efficacy of dual targeting of the Mek and Atr pathways in mouse- and patient-derived xenografts with both mutations in vivo, suggesting this combination as an attractive therapeutic opportunity that might be used to treat patients with these mutations. Our studies indicate that this mouse model of Mll-Af4/N-Ras B-ALL is a powerful tool to explore the molecular and genetic pathogenesis of this disease subtype, as well as a preclinical discovery platform for novel therapeutic strategies.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Genes ras , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Activación Transcripcional , Animales , Apoptosis/genética , Ciclo Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Vectores Genéticos/genética , Humanos , Ratones , Ratones Transgénicos , Mutación , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Inhibidores de Proteínas Quinasas/farmacología , Retroviridae/genética , Transducción de Señal
7.
Blood ; 130(24): 2631-2641, 2017 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-29018079

RESUMEN

Mutations in SETD2, encoding the histone 3 lysine 36 trimethyltransferase, are enriched in relapsed acute lymphoblastic leukemia and MLL-rearranged acute leukemia. We investigated the impact of SETD2 mutations on chemotherapy sensitivity in isogenic leukemia cell lines and in murine leukemia generated from a conditional knockout of Setd2. SETD2 mutations led to resistance to DNA-damaging agents, cytarabine, 6-thioguanine, doxorubicin, and etoposide, but not to a non-DNA damaging agent, l-asparaginase. H3K36me3 localizes components of the DNA damage response (DDR) pathway and SETD2 mutation impaired DDR, blunting apoptosis induced by cytotoxic chemotherapy. Consistent with local recruitment of DDR, genomic regions with higher H3K36me3 had a lower mutation rate, which was increased with SETD2 mutation. Heterozygous conditional inactivation of Setd2 in a murine model decreased the latency of MLL-AF9-induced leukemia and caused resistance to cytarabine treatment in vivo, whereas homozygous loss delayed leukemia formation. Treatment with JIB-04, an inhibitor of the H3K9/36me3 demethylase KDM4A, restored H3K36me3 levels and sensitivity to cytarabine. These findings establish SETD2 alteration as a mechanism of resistance to DNA-damaging chemotherapy, consistent with a local loss of DDR, and identify a potential therapeutic strategy to target SETD2-mutant leukemias.


Asunto(s)
Daño del ADN , Resistencia a Antineoplásicos/genética , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Experimental/genética , Aminopiridinas/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Citarabina/farmacología , Células HEK293 , N-Metiltransferasa de Histona-Lisina/deficiencia , Histonas/metabolismo , Humanos , Hidrazonas/farmacología , Leucemia Experimental/tratamiento farmacológico , Lisina/metabolismo , Metilación/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Supervivencia
8.
J Exp Med ; 214(8): 2453-2470, 2017 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-28733386

RESUMEN

The microRNA-99 (miR-99) family comprises a group of broadly conserved microRNAs that are highly expressed in hematopoietic stem cells (HSCs) and acute myeloid leukemia stem cells (LSCs) compared with their differentiated progeny. Herein, we show that miR-99 regulates self-renewal in both HSCs and LSCs. miR-99 maintains HSC long-term reconstitution activity by inhibiting differentiation and cell cycle entry. Moreover, miR-99 inhibition induced LSC differentiation and depletion in an MLL-AF9-driven mouse model of AML, leading to reduction in leukemia-initiating activity and improved survival in secondary transplants. Confirming miR-99's role in established AML, miR-99 inhibition induced primary AML patient blasts to undergo differentiation. A forward genetic shRNA library screen revealed Hoxa1 as a critical mediator of miR-99 function in HSC maintenance, and this observation was independently confirmed in both HSCs and LSCs. Together, these studies demonstrate the importance of noncoding RNAs in the regulation of HSC and LSC function and identify miR-99 as a critical regulator of stem cell self-renewal.


Asunto(s)
Autorrenovación de las Células/genética , Autorrenovación de las Células/fisiología , Células Madre Hematopoyéticas/fisiología , MicroARNs/genética , Animales , Ciclo Celular/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Factores de Transcripción/genética
9.
Nature ; 544(7648): 53-58, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-28355185

RESUMEN

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1ß, G-CSF, TGFß and CXCL4) and activation of an inducible Irf8 and ß-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.


Asunto(s)
Autorrenovación de las Células , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Progenitoras de Granulocitos y Macrófagos/patología , Leucemia/patología , Mielopoyesis , Células Madre Neoplásicas/patología , Animales , Reprogramación Celular , Citocinas/metabolismo , Granulocitos/citología , Granulocitos/patología , Factores Reguladores del Interferón/metabolismo , Macrófagos/citología , Macrófagos/patología , Ratones , Imagen Molecular , Nicho de Células Madre/fisiología , beta Catenina/metabolismo
10.
Cancer Cell ; 30(6): 863-878, 2016 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-27889185

RESUMEN

The nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in multiple hematopoietic malignancies. Here, we demonstrate that NUP98 fusion proteins, including NUP98-HOXA9 (NHA9), NUP98-HOXD13 (NHD13), NUP98-NSD1, NUP98-PHF23, and NUP98-TOP1 physically interact with mixed lineage leukemia 1 (MLL1) and the non-specific lethal (NSL) histone-modifying complexes. Chromatin immunoprecipitation sequencing illustrates that NHA9 and MLL1 co-localize on chromatin and are found associated with Hox gene promoter regions. Furthermore, MLL1 is required for the proliferation of NHA9 cells in vitro and in vivo. Inactivation of MLL1 leads to decreased expression of genes bound by NHA9 and MLL1 and reverses a gene expression signature found in NUP98-rearranged human leukemias. Our data reveal a molecular dependency on MLL1 function in NUP98-fusion-driven leukemogenesis.


Asunto(s)
Cromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/genética , Leucemia/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Fusión Oncogénica/metabolismo , Animales , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Proteínas de Complejo Poro Nuclear/metabolismo , Regiones Promotoras Genéticas , Células Tumorales Cultivadas
11.
J Clin Invest ; 126(3): 997-1011, 2016 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-26878175

RESUMEN

Self-renewal is a hallmark of both hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs); therefore, the identification of mechanisms that are required for LSC, but not HSC, function could provide therapeutic opportunities that are more effective and less toxic than current treatments. Here, we employed an in vivo shRNA screen and identified jumonji domain-containing protein JMJD1C as an important driver of MLL-AF9 leukemia. Using a conditional mouse model, we showed that loss of JMJD1C substantially decreased LSC frequency and caused differentiation of MLL-AF9- and homeobox A9-driven (HOXA9-driven) leukemias. We determined that JMJD1C directly interacts with HOXA9 and modulates a HOXA9-controlled gene-expression program. In contrast, loss of JMJD1C led to only minor defects in blood homeostasis and modest effects on HSC self-renewal. Together, these data establish JMJD1C as an important mediator of MLL-AF9- and HOXA9-driven LSC function that is largely dispensable for HSC function.


Asunto(s)
Proteínas de Homeodominio/fisiología , Histona Demetilasas con Dominio de Jumonji/fisiología , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/fisiología , Proteínas de Fusión Oncogénica/fisiología , Animales , Autorrenovación de las Células , Expresión Génica , Células Madre Hematopoyéticas/fisiología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones Noqueados , Trasplante de Neoplasias , Células Tumorales Cultivadas
13.
Cell Stem Cell ; 11(3): 346-58, 2012 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-22958930

RESUMEN

Internal tandem duplication (ITD) mutations within the FMS-like tyrosine kinase-3 (FLT3) render the receptor constitutively active driving proliferation and survival in leukemic blasts. Expression of FLT3-ITD from the endogenous promoter in a murine knockin model results in progenitor expansion and a myeloproliferative neoplasm. In this study, we show that this expansion begins with overproliferation within a compartment of normally quiescent long-term hematopoietic stem cells (LT-HSCs), which become rapidly depleted. This depletion is reversible upon treatment with the small molecule inhibitor Sorafenib, which also ablates the disease. Although the normal LT-HSC has been defined as FLT3(-) by flow cytometric detection, we demonstrate that FLT3 is capable of playing a role within this compartment by examining the effects of constitutively activated FLT3-ITD. This indicates an important link between stem cell quiescence/homeostasis and myeloproliferative disease while also giving novel insight into the emergence of FLT3-ITD mutations in the evolution of leukemic transformation.


Asunto(s)
Neoplasias de la Médula Ósea/patología , Duplicación de Gen , Técnicas de Sustitución del Gen , Células Madre Hematopoyéticas/patología , Homeostasis , Secuencias Repetidas en Tándem/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Neoplasias de la Médula Ósea/genética , Compartimento Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Duplicación de Gen/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/genética , Masculino , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Niacinamida/análogos & derivados , Niacinamida/farmacología , Fenotipo , Compuestos de Fenilurea/farmacología , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células de Población Lateral/efectos de los fármacos , Células de Población Lateral/metabolismo , Células de Población Lateral/patología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Sorafenib , Tirosina Quinasa 3 Similar a fms/metabolismo
14.
Drug Resist Updat ; 12(1-2): 8-16, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19162530

RESUMEN

The success of the small molecule tyrosine kinase receptor inhibitor (TKI) imatinib mesylate (Gleevec) in the treatment of chronic myeloid leukemia (CML) constitutes an eminent paradigm shift advocating the rational design of cancer therapeutics specifically targeting the transformation events that drive tumorigenicity. In acute myeloid leukemias (AMLs), the most frequent identified transforming events are activating mutations in the FLT3 receptor tyrosine kinase that constitutively activate survival and proliferation pathways. FLT3 TKIs that are in various phases of clinical trials are showing some initial promise. However, primary and secondary acquired resistance stands to severely compromise long-term and durable efficacy of these inhibitors as a therapeutic strategy. Here, we discuss the mechanisms of resistance to FLT3 inhibitors and possible strategies to overcome resistance through closer examination of the events of leukemogenesis and design of combination therapy.


Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Inhibidores de Proteínas Quinasas/farmacocinética , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo
15.
Biotechniques ; 37(3): 376-8, 380, 382, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15470891

RESUMEN

Transformation of Escherichia coli plays an important role in recombinant DNA technology. Most current transformation protocols require that the cells be treated to attain a particular physiological state known as "competence," and this makes transformation procedures lengthy and arduous. Here we describe a protocol for transforming log-phase E. coli using dimethyl sulfoxide (DMSO) solutions of poly-(R)-3-hydroxybutyrate (PHB) to facilitate the transfer of plasmid DNA into cells, and certain reagents and temperature shocks to promote DNA uptake. The protocol was optimized using factorial design techniques across variables that included PHB molecular weight and concentration, DMSO concentration, monovalent and divalent salts, glucose, cold and heat shocks, cell density, and pH. Using 10 ng DNA, the optimized protocol produces approximately 1000 colony-forming units (CFUs) from 100 microL early log-phase cell culture or approximately 300 CFU from a 21-24 h single colony, sufficient for many applications. The total volume of the transformation reaction mixture is only 150 microL suggesting that the procedure may be adapted for use in microplates or automated transformation technologies.


Asunto(s)
Escherichia coli/genética , Hidroxibutiratos , Poliésteres , Transformación Bacteriana , ADN Bacteriano , Escherichia coli/metabolismo , Indicadores y Reactivos , Plásmidos , Recombinación Genética
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