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1.
Leukemia ; 32(4): 931-940, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28972594

RESUMO

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50-70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27-4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.


Assuntos
Linfócitos B/metabolismo , Biomarcadores Tumorais/genética , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas ras/genética , Adolescente , Animais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos NOD , Taxa de Mutação , Proteínas de Fusão Oncogênica/genética , Prognóstico , Transdução de Sinais/genética
2.
Leukemia ; 31(7): 1491-1501, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-27899802

RESUMO

Children with P2RY8-CRLF2-positive acute lymphoblastic leukemia have an increased relapse risk. Their mutational and transcriptional landscape, as well as the respective patterns at relapse remain largely elusive. We, therefore, performed an integrated analysis of whole-exome and RNA sequencing in 41 major clone fusion-positive cases including 19 matched diagnosis/relapse pairs. We detected a variety of frequently subclonal and highly instable JAK/STAT but also RTK/Ras pathway-activating mutations in 76% of cases at diagnosis and virtually all relapses. Unlike P2RY8-CRLF2 that was lost in 32% of relapses, all other genomic alterations affecting lymphoid development (58%) and cell cycle (39%) remained stable. Only IKZF1 alterations predominated in relapsing cases (P=0.001) and increased from initially 36 to 58% in matched cases. IKZF1's critical role is further corroborated by its specific transcriptional signature comprising stem cell features with signs of impaired lymphoid differentiation, enhanced focal adhesion, activated hypoxia pathway, deregulated cell cycle and increased drug resistance. Our findings support the notion that P2RY8-CRLF2 is dispensable for relapse development and instead highlight the prominent rank of IKZF1 for relapse development by mediating self-renewal and homing to the bone marrow niche. Consequently, reverting aberrant IKAROS signaling or its disparate programs emerges as an attractive potential treatment option in these leukemias.


Assuntos
Fusão Gênica , Genômica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Citocinas/genética , Receptores Purinérgicos P2Y/genética , Transcrição Gênica , Adolescente , Criança , Pré-Escolar , Dosagem de Genes , Genes Supressores de Tumor , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/fisiologia , Lactente , Janus Quinases/fisiologia , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição STAT/fisiologia
3.
Leukemia ; 29(8): 1656-67, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25917266

RESUMO

High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18-30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína de Ligação a CREB/genética , Diploide , Mutação/genética , Recidiva Local de Neoplasia/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adolescente , Estudos de Casos e Controles , Criança , Evolução Clonal , Feminino , Seguimentos , Humanos , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Proteínas Proto-Oncogênicas p21(ras) , Taxa de Sobrevida
4.
Leukemia ; 28(9): 1828-37, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24625531

RESUMO

Still 20% of pediatric acute lymphoblastic leukemia (ALL) patients relapse on or after current treatment strategies. Treatment failure is associated with resistance to prednisolone. We aimed to find new druggable targets that modulate prednisolone resistance. We generated microarray gene expression profiles of 256 pediatric ALL patient samples and identified a 3.4-fold increase in epithelial membrane protein 1 (EMP1) expression in in vitro prednisolone-resistant compared with -sensitive patients (P=0.003). EMP1 silencing in six precursor-B ALL (BCP-ALL) and T-ALL cell lines induced apoptosis and cell-cycle arrest leading to 84.1±4.5% reduction in survival compared with non-silencing control transduced cells (non-silencing control short hairpin, shNSC) (P=0.014). Moreover, EMP1 silencing sensitized to prednisolone up to 18.8-fold (P<0.001). EMP1 silencing also abrogated migration and adhesion to mesenchymal stromal cells (MSCs) by 78.3±9.0 and 29.3±4.1% compared with shNSC (P<0.05). We discovered that EMP1 contributes to MSC-mediated prednisolone resistance. Pathway analysis indicated that EMP1 signals through the Src kinase family. EMP1-high BCP-ALL patients showed a poorer 5-year event-free survival compared with EMP1-low patients (77±2 vs. 89±2%, P=0.003). Multivariate analysis taking along white blood cell count, age, prednisolone resistance and subtype identified EMP1 as an independent predictor for poor outcome in BCP-ALL (P=0.004, hazard ratio: 2.36 (1.31-4.25). This study provides preclinical evidence that EMP1 is an interesting candidate for drug development to optimize treatment of BCP-ALL.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia/tratamento farmacológico , Proteínas de Neoplasias/fisiologia , Prednisolona/farmacologia , Receptores de Superfície Celular/fisiologia , Apoptose , Adesão Celular , Movimento Celular , Proliferação de Células , Células HEK293 , Humanos , Leucemia/mortalidade , Leucemia/patologia , NF-kappa B/fisiologia , Proteínas de Neoplasias/análise , Prognóstico , Receptores de Superfície Celular/análise , Quinases da Família src/fisiologia
6.
Pediatr Blood Cancer ; 61(5): 788-96, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24249312

RESUMO

BACKGROUND: Alterations in the NOTCH1 signaling pathway are found in about 60% of pediatric T-ALL, but its impact on prognosis remains unclear. PROCEDURE: We extended the previously published CoALL cohort (n = 74) to a larger cohort (n = 127) and additionally included 38 Argentine patients from ALL IC-BFM to potentially identify novel mutations and decipher a stronger discriminatory effect on the genotype/phenotype relationship with regard to early treatment response and long-term outcome. RESULTS: Overall, 101 out of 165 (61.2%) T-ALL samples revealed at least one NOTCH1 mutation, 28 of whom had combined NOTCH1 and FBXW7 mutations. Eight T-ALL samples (4.8%) exclusively revealed FBXW7 mutations. Fifty-six T-ALL (33.9%) exhibited a wild-type configuration of either gene. Four novel NOTCH1 mutations were identified localized in the C-terminal PEST domain, in the rarely affected LNR repeat domain and in the ankyrin domain. Novel LNR mutations may contribute to a better understanding of the structure of the NOTCH1 negative regulatory region (NRR) and the R1946 mutation in the ankyrin domain may represent an unusual loss-of-function mutation. CONCLUSIONS: Overall, NOTCH1 pathway mutations did not affect the relapse rate and outcome of the extended T-ALL cohort uniformly treated according to CoALL protocols, although NOTCH1 mutations were associated with good response to induction therapy (P = 0.009). Individually, HD and PEST domain mutations might exert distinct functional effects on cellular homeostasis under treatment NOTCH1 pathway activity with prognostic implications.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Mutação/genética , Recidiva Local de Neoplasia/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Ubiquitina-Proteína Ligases/genética , Criança , Estudos de Coortes , DNA de Neoplasias/genética , Proteína 7 com Repetições F-Box-WD , Genótipo , Humanos , Recidiva Local de Neoplasia/terapia , Fenótipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Taxa de Sobrevida
7.
Leukemia ; 24(2): 298-308, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20016530

RESUMO

In this study, the long-term outcome of 1818 patients treated in five consecutive clinical trials (the cooperative study group for childhood acute lymphoblastic leukaemia (COALL) 82, 85, 89, 92 and 97) from 24 cooperating centres in Germany is reported. The probability of event-free survival (pEFS) improved significantly from the first two trials conducted in the 1980s (COALL 82 and COALL 85) to the three trials conducted in the 1990s (COALL 89, 92 and 97) (P=0.001). Through all COALL studies, age > or =10 years and initial white blood cell count (WBC) > or =50 x 10(9)/l and pro-B immunophenotype were of significant prognostic relevance. A refinement of risk assessment has been achieved by in vitro drug sensitivity testing in COALL 92 and 97. In patients with very sensitive leukaemic cells, therapy could be reduced without loss of efficacy. In COALL 97, a further improvement in risk stratification was gained by the molecular assessment of minimal residual disease (MRD) under treatment, which proved to have a superior prognostic effect when compared with in vitro drug sensitivity testing. Importantly, the gradual reduction in central nervous system (CNS) irradiation led to a decreased incidence of brain tumours as a second malignancy. In general, the prevention of treatment-related late effects will be one of the major issues in future studies. It remains to be shown whether prolonged infusions of anthracyclines, which have been implemented into the COALL studies after equal efficacy compared with short-time infusions was confirmed, will be associated with fewer cardiac late effects. Another way to prevent late effects may be a more refined risk assessment allowing for a reduction in cumulative treatment burden. A great challenge in the future will be to improve the overall treatment results, which very likely can only be achieved by the identification of molecularly defined subgroups to which novel, rational therapeutic strategies can be applied.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Irradiação Craniana , Recidiva Local de Neoplasia/terapia , Neoplasia Residual/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Criança , Pré-Escolar , Terapia Combinada , Feminino , Seguimentos , Humanos , Imunofenotipagem , Lactente , Contagem de Leucócitos , Masculino , Recidiva Local de Neoplasia/diagnóstico , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Prognóstico , Indução de Remissão , Fatores de Risco , Taxa de Sobrevida , Fatores de Tempo , Resultado do Tratamento
8.
Gene Ther ; 14(8): 699-703, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17268534

RESUMO

Juvenile myelomonocytic leukemia (JMML) is a malignant disease of early childhood characterized by a hypersensitivity to granulocyte/macrophage colony-stimulating factor (GM-CSF). Mutations in RAS or PTPN11 are frequently detected in JMML patients. The SH2-containing inositol 5-phosphatase 1 (SHIP-1) is a negative regulator of GM-CSF signaling, and inactivation of SHIP-1 in mice results in a myeloproliferative disease. Here, we report the effects of SHIP-1 expression on GM-CSF-dependent proliferation and colony formation of human hematopoietic cells. After retroviral-mediated transduction of SHIP-1 into CD34+ cells from cord blood of healthy newborns or peripheral blood of JMML patients carrying mutations in KRAS2 or PTPN11, we observed a reduction in GM-CSF-dependent proliferation and colony formation. An enzymatically inactive form of SHIP-1 (D672A) had no effect. These data indicate that SHIP-1 can effectively block GM-CSF hypersensitivity in JMML progenitor cells with mutations in KRAS2 or PTPN11 and may be a useful approach for the treatment of JMML patients.


Assuntos
Terapia Genética/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mielomonocítica Crônica/terapia , Monoéster Fosfórico Hidrolases/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Recém-Nascido , Inositol Polifosfato 5-Fosfatases , Leucemia Mielomonocítica Crônica/imunologia , Mutação , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Proto-Oncogênicas p21(ras) , Transdução Genética/métodos
9.
Genes Dev ; 14(3): 301-12, 2000 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-10673502

RESUMO

Microphthalmia (Mi) is a bHLHZip transcription factor that is essential for melanocyte development and postnatal function. It is thought to regulate both differentiated features of melanocytes such as pigmentation as well as proliferation/survival, based on phenotypes of mutant mouse alleles. Mi activity is controlled by at least two signaling pathways. Melanocyte-stimulating hormone (MSH) promotes transcription of the Mi gene through cAMP elevation, resulting in sustained Mi up-regulation over many hours. c-Kit signaling up-regulates Mi function through MAP kinase phosphorylation of Mi, thereby recruiting the p300 transcriptional coactivator. The current study reveals that c-Kit signaling triggers two phosphorylation events on Mi, which up-regulate transactivation potential yet simultaneously target Mi for ubiquitin-dependent proteolysis. The specific activation/degradation signals derive from MAPK/ERK targeting of serine 73, whereas serine 409 serves as a substrate for p90 Rsk-1. An unphosphorylatable double mutant at these two residues is at once profoundly stable and transcriptionally inert. These c-Kit-induced phosphorylations couple transactivation to proteasome-mediated degradation. c-Kit signaling thus triggers short-lived Mi activation and net Mi degradation, in contrast to the profoundly increased Mi expression after MSH signaling, potentially explaining the functional diversity of this transcription factor in regulating proliferation, survival, and differentiation in melanocytes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Melanócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa , Fatores de Transcrição , Animais , Western Blotting , Células Cultivadas , Cricetinae , Cisteína Endopeptidases/metabolismo , Humanos , Camundongos , Fator de Transcrição Associado à Microftalmia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação , Complexo de Endopeptidases do Proteassoma , Fator de Células-Tronco/metabolismo , Ativação Transcricional , Ubiquitinas/metabolismo
10.
J Biol Chem ; 273(49): 33042-7, 1998 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-9830058

RESUMO

The pituitary peptide alpha-melanocyte-stimulating hormone (alpha-MSH) stimulates melanocytes to up-regulate cAMP, but the downstream targets of cAMP are not well understood mechanistically. One consequence of alpha-MSH stimulation is increased melanization attributable to induction of pigmentation enzymes, including tyrosinase, which catalyzes a rate-limiting step in melanin synthesis. The tyrosinase promoter is a principle target of the melanocyte transcription factor Microphthalmia (Mi), a factor for which deficiency in humans causes Waardenburg syndrome II. We show here that both alpha-MSH and forskolin, a drug that increases cAMP, stimulate a rapid increase in Mi mRNA and protein levels in both melanoma cell lines and primary melanocytes. This up-regulation requires a cAMP-responsive element within the Mi promoter, and the pathway leading to Mi stimulation is subject to tight homeostatic regulation. Although cAMP signaling is ubiquitous, the Mi promoter was seen to be cAMP-responsive in melanocytes but not in nonmelanocytes. Moreover, dominant negative interference with Mi impeded successful alpha-MSH stimulation of tyrosinase. The regulation of Mi expression via alpha-MSH thus provides a direct mechanistic link to pigmentation. In addition, because the human melanocyte and deafness condition Waardenburg syndrome is sometimes caused by haploinsufficiency of Mi, its modulation by alpha-MSH suggests therapeutic strategies targeted at up-regulating the remaining wild type Mi allele.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Transdução de Sinais , Fatores de Transcrição , Síndrome de Waardenburg/genética , alfa-MSH/metabolismo , Animais , Linhagem Celular , Cricetinae , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Fator de Transcrição Associado à Microftalmia , Fosforilação , Regiões Promotoras Genéticas , Síndrome de Waardenburg/metabolismo
11.
J Exp Med ; 187(5): 775-85, 1998 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-9480987

RESUMO

Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Microftalmia/genética , Osteoclastos/fisiologia , Osteopetrose/etiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Expressão Gênica , Sequências Hélice-Alça-Hélice , Humanos , Zíper de Leucina , Camundongos , Fator de Transcrição Associado à Microftalmia , Dados de Sequência Molecular , Osteoblastos/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Mutantes , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
12.
Br J Cancer ; 75(9): 1309-17, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9155051

RESUMO

The 'deleted in colon carcinoma' (DCC) gene has been considered a candidate tumour-suppressor gene that encodes for a transmembrane protein with strong structural similarity to members of the superfamily of neural cell adhesion molecules. It has been mapped to the chromosomal region 18q21.1 and it is implicated in cellular differentiation and developmental processes. In human osteosarcoma allelic loss frequently occurs on the long arm of chromosome 18, suggesting a possible involvement of the DCC gene in the pathogenesis of this tumour entity. In the present study the mRNA and protein expression and rearrangements at the DNA level of the DCC gene were addressed in 25 osteosarcomas and several tumour cell lines, including osteosarcoma- and colon carcinoma-derived cell lines. Using an reverse transcriptase polymerase chain reach in (RT-PCR)-based approach DCC expression was found to be lost or substantially reduced in 14 of 19 high-grade osteosarcomas, in three of six lower grade osteosarcomas and most of the tumour cell lines, in contrast to normally differentiated osteoblasts. Immunohistochemical studies on DCC protein expression of 14 selected tumours correlated well with the RT-PCR-based results. In view of the putative tumour-suppressor characteristics of the DCC gene its loss or reduction of expression could be a specific event in the development or progression of many high-grade osteosarcomas.


Assuntos
Neoplasias Ósseas/genética , Deleção Cromossômica , Cromossomos Humanos Par 18/genética , Regulação Neoplásica da Expressão Gênica , Genes DCC/genética , Osteossarcoma/genética , Proteínas Supressoras de Tumor , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Criança , Receptor DCC , Primers do DNA/química , Citometria de Fluxo , Genes Supressores de Tumor/genética , Humanos , Pessoa de Meia-Idade , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores de Superfície Celular , Células Tumorais Cultivadas
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