RESUMO
Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Morfolinas/administração & dosagem , Niacinamida/análogos & derivados , Compostos de Fenilureia/administração & dosagem , Triazinas/administração & dosagem , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Mutação , Niacinamida/administração & dosagem , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Spleen tyrosine kinase (SYK) was recently identified as a new target in acute myeloid leukemia (AML); however, its mechanistic role in this disease is poorly understood. Based on the known interaction between SYK and mammalian target of rapamycin (mTOR) signaling in lymphoma, we hypothesized that SYK may regulate mTOR signaling in AML. Both small-molecule inhibition of SYK and SYK-directed shRNA suppressed mTOR and its downstream signaling effectors, as well as its upstream activator, AKT. Moreover, the inhibition of multiple nodes of the phosphatidylinositol 3'-kinase (PI3K) signaling pathway enhanced the effects of SYK suppression on AML cell viability and differentiation. Evaluation of the collateral mitogen-activated protein kinase (MAPK) pathway revealed a heterogeneous response to SYK inhibition in AML with downregulation of MEK and extracellular signal-regulated kinase (ERK) phosphorylation in some AML cell lines but a paradoxical increase in MEK/ERK phosphorylation in RAS-mutated AML. These studies reveal SYK as a regulator of mTOR and MAPK signaling in AML and demonstrate that inhibition of PI3K pathway activity enhances the effects of SYK inhibition on AML cell viability and differentiation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Quinase Syk , Serina-Treonina Quinases TOR/genética , Células Tumorais CultivadasRESUMO
While target-based small-molecule discovery has taken centre-stage in the pharmaceutical industry, there are many cancer-promoting proteins not easily addressed with a traditional target-based screening approach. In order to address this problem, as well as to identify modulators of biological states in the absence of knowing the protein target of the state switch, alternative phenotypic screening approaches, such as gene expression-based and high-content imaging, have been developed. With this renewed interest in phenotypic screening, however, comes the challenge of identifying the binding protein target(s) of small-molecule hits. Emerging technologies have the potential to improve the process of target identification. In this review, we discuss the application of genomic (gene expression-based), genetic (short hairpin RNA and open reading frame screening), and proteomic approaches to protein target identification.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/metabolismo , Proteômica , Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
With the sequencing of the human genome and the development of new genomic technologies, biomedical discovery has been transformed. The applications of these new approaches are ever-expanding from disease classification, to identification of new targets, to outcome prediction. A logical next step is the integration of genomic approaches into small molecule discovery. This review will focus on the application of genomics to compound discovery, with an emphasis on the hematological malignancies. It will focus on the use of genomic tools to discover cancer targets and the development and application of both cell-based and in silico gene expression-based approaches to small molecule discovery.
Assuntos
Descoberta de Drogas , Genoma Humano , Genômica , Expressão Gênica , HumanosAssuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/fisiologia , Regulação Leucêmica da Expressão Gênica , Leucemia/genética , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Fatores de Transcrição/fisiologia , Adulto , Sequência de Aminoácidos , Criança , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 12/ultraestrutura , Clonagem Molecular , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/genética , Deleção de Genes , Genes abl , Sequências Hélice-Alça-Hélice/genética , Humanos , Leucemia/patologia , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/patologia , Modelos Genéticos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-ets , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Translocação Genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Polymerase chain reaction-based screening of childhood acute lymphoblastic leukemia (ALL) samples showed that a TEL/AML1 fusion transcript was detected in 27% of all cases, representing the most common known gene rearrangement in childhood cancer. The TEL/AML1 fusion results from a t(12;21)(p13;q22) chromosomal translocation, but was undetectable at the routine cytogenetic level. TEL/AML1-positive patients had exclusively B-lineage ALL, and most patients were between the ages of 2 and 9 years at diagnosis. Only 3/89 (3.4%) adult ALL patients were TEL/AML1-positive. Most importantly, TEL/AML1-positive children had a significantly lower rate of relapse compared with TEL/AML1-negative patients (0/22 v 16/54, P = .004). Co-immunoprecipitation experiments demonstrated that TEL/AML-1 formed homodimers in vitro, and heterodimerized with the normal TEL protein when the two proteins were expressed together. The elucidation of the precise mechanism of transformation by TEL/AML1 and the role of TEL/AML1 testing in the treatment of childhood ALL will require additional studies.
Assuntos
Biomarcadores Tumorais , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 21/genética , Proteínas de Neoplasias/química , Proteínas de Fusão Oncogênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/química , Criança , Pré-Escolar , Cromossomos Humanos Par 12/ultraestrutura , Cromossomos Humanos Par 21/ultraestrutura , Clonagem Molecular , Subunidade alfa 2 de Fator de Ligação ao Core , DNA de Neoplasias/genética , Dimerização , Feminino , Deleção de Genes , Humanos , Lactente , Tábuas de Vida , Masculino , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Proto-Oncogenes , Análise de Sobrevida , Resultado do TratamentoRESUMO
We have shown previously that loss of heterozygosity at chromosome band 12p13 is among the most frequent genetic abnormalities identified in acute lymphoblastic leukemia (ALL) of childhood. Two known genes map within the critically deleted region of 12p: TEL, the gene encoding a new member of the ETS family of transcription factors, which is rearranged in a variety of hematological malignancies; and KIP1, the gene encoding the cyclin-dependent kinase inhibitor p27. Both genes are, therefore, excellent candidate tumor suppressor genes. In this report, we determined the exon organization of the TEL gene and performed mutational analysis of TEL and KIP1 in 33 childhood ALL patients known to have loss of heterozygosity at this locus. No mutations in either TEL or KIP1 were found; this suggest that neither TEL nor KIP1 is the critical 12p tumor suppressor gene in childhood ALL.
Assuntos
Proteínas de Ciclo Celular , Cromossomos Humanos Par 12/genética , Éxons/genética , Deleção de Genes , Genes Supressores de Tumor/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Oncogênicas , Mutação Puntual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor , Sequência de Bases , Criança , Inibidor de Quinase Dependente de Ciclina p27 , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas c-etsAssuntos
Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Leucemia Mielomonocítica Crônica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Fatores de Transcrição/genética , Transformação Celular Neoplásica , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 5 , Cromossomos Humanos Par 9 , Subunidade alfa 2 de Fator de Ligação ao Core , Humanos , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-ets , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Translocação Genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
TEL is a new member of the ETS family of transcription factors which is rearranged in a number of hematologic malignancies with translocations involving chromosome band 12p13. In some cases, both TEL alleles are affected, resulting in loss of wild-type TEL function in the leukemic cells. In addition, 5% of children with acute lymphoblastic leukemia (ALL) have 12p12-p13 deletions, suggesting that a tumor suppressor gene resides on 12p. These observations led us to consider whether TEL loss of function may contribute to the pathogenesis of ALL. In this report we show that the TEL gene maps between the polymorphic markers D12S89 and D12S98, and we use these flanking markers to screen paired diagnosis and remission samples from 81 children with ALL for loss of heterozygosity (LOH) at the TEL gene locus. Fifteen percent of informative patients showed TEL LOH which was not evident on cytogenetic analysis. Detailed examination of patients with LOH at this locus showed that the critically deleted region included two candidate tumor suppressor genes: TEL and KIP1, the gene encoding the cyclin-dependent kinase inhibitor p27. These studies show that LOH at the TEL locus is a frequent finding in childhood ALL.
