A leucemia mieloide crônica (LMC) é uma expansão clonal da célula progenitora hematopoiética, traduzindo-se por hiperplasia mielóide, leucocitose, neutrofilia, basofilia e esplenomegalia. O cromossomo Filadélfia é característico da doença, sendo produto da translocação t(9;22) (q34;q11), resultando na fusão dos genes ABL e BCR. Esta fusão gera um gene híbrido que produz uma proteína com elevada atividade tirosinoquinase que tem um papel central da patogenia da LMC. O mesilato de imatinibe (MI) é um derivado da fenilaminopirimidina que inibe a proteína-tirosina quinase ABL in vitro e in vivo. O MI interage com transportadores de membrana de efluxo, como o ATP binding cassette B1 (ABCB1). Polimorfismos no gene ABCB1 têm sido associados com alteração na sua funcionalidade e podem estar envolvidos na resposta ao tratamento farmacológico. Este estudo tem por objetivo investigar a relação dos polimorfismos C1236T, C3435T e G2677T/A no gene ABCB1 com marcadores de resposta ao tratamento com MI, em indivíduos com LMC, e determinar os fatores de predisposição de resposta ao MI. Foram incluídos 118 pacientes portadores de LMC. Foram constituídos dois grupos: Grupo 1 com 70 pacientes com resposta citogenética completa com a dose padrão de MI (400 mg/dia de MI) por até 18 meses e, Grupo 2 com 48 pacientes sem resposta citogenética completa com a dose inicial de 400 mg/dia de MI ou que perderam esta resposta ao longo do tratamento . Amostras de sangue foram obtidas para: quantificação de BCR-ABL1, extração de DNA genômico e análise citogenética de banda G. As análises dos polimorfismos foram realizadas por PCR-RFLP. A resposta ao tratamento foi avaliada segundo os critérios da European LeukemiaNet. A distribuição da frequência dos genótipos dos polimorfismos C1236T, C3435T e G2677T/A foi similar nos dois gêneros e entre brancos e não brancos. Não houve influência dos polimorfismos estudados no risco de desenvolvimento da LMC e na resposta ao MI. O haplótipo ABCB1 1236CT/2677GT/3435CT (para os polimorfismos C1236T/G2677T/C3435T no gene ABCB1) foi encontrado em 51,7% dos pacientes com resposta molecular maior (P=0,010). Houve tendência a maior frequência de pacientes portadores de genótipos 1236 CT e TT no grupo de respondedores (86,7%) quando foi analisada a resposta molecular completa (p=0,069). O mesmo aconteceu no grupo de não respondedores quando foi considerado o polimorfismo C1236T. Houve tendência a maior frequência de resposta molecular completa em portadores de genótipo 2677 GT+TT+TA nos dois grupos (respondedores P=0,074 e não respondedores P=0,076). Em conclusão, os genótipos e haplótipos para os polimorfismos ABCB1 C1236T, C3435T e G2677T/A estão associados com a resposta molecular em portadores de LMC respondedores ao tratamento com MI. / The chronic myeloid leukemia (CML) is a clonal expansion of the hematopoietic progenitor cell, representing myeloid hyperplasia, leukocytosis, neutrophilia, basophilia and splenomegaly. The Philadelphia chromosome is peculiar on the disease, being the result of the translocation t(9; 22) (q34; q11), leading on the fusion of the ABL and BCR genes. This merger creates a hybrid gene that produces a protein with high activity of tyrosine kinase that is the main pathogenesis of CML. The Imatinib mesylate (IM) is a fenilaminopirimidine derivative which inhibits the ABL protein-tyrosine kinase in vitro and in vivo. The MI interacts with membrane efflux transporters, such as ATP binding cassette B1 (ABCB1). Polymorphisms in the ABCB1 gene have been associated with changes in its functionality and may be involved on the response to drug treatment. This study aims to investigate the relationship of C1236T, C3435T and G2677T / A polymorphisms in ABCB1 gene with response markers for MI treatment in individuals with CML, and to determine the predisposing factors of response to MI. 118 patients with CML were included and divided in two groups. Group 1: 70 patients with complete cytogenetic response and a standard dose of IM (400 mg / day IM) for up to 18 months. Group 2: 48 patients without a complete cytogenetic response and initial dose of 400 mg / day IM or whith response lost throughout the treatment. Blood samples were obtained for: quantification of BCR-ABL1, genomic DNA extraction and band G cytogenetic analysis. The analysis of the polymorphisms were performed by PCR-RFLP. The treatment response was evaluated according to European LeukemiaNet criteria. The frequency distribution of genotypes of C1236T, C3435T and G2677T / A polymorphisms were similar in both sexes and between whites and nonwhites. The polymorphisms studied had no influence on the CML development or MI response. The haplotype ABCB1 1236CT/2677GT/3435CT (for C1236T/G2677T/C3435T polymorphisms in the ABCB1) was found in 51.7% patients with major molecular response (P = 0.010). There was a tendency for higher frequency of patients with 1236 CT and TT genotypes in the responders group (86.7%) when the molecular response was analyzed (p = 0.069). The same happened in the nonresponders group when the C1236T polymorphism was considered. There was a tendency for a higher frequency of complete molecular response in patients with 2677 GT + TT + TA genotype in both groups (responders P = 0.074 and nonresponders P = 0.076). In conclusion, genotypes and haplotypes for ABCB1 C1236T, C3435T and G2677T / A polymorphisms are associated with molecular response in patients with CML that respond to MI treatment.

Orientador: Valdemiro Carlos Sgarbieri / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T14:32:40Z (GMT). No. of bitstreams: 1 Ziegler_FabianeLaFlor_D.pdf: 4176640 bytes, checksum: fccef651838684499cf4cf698073b39e (MD5) Previous issue date: 2010 / Resumo: O objetivo geral da presente pesquisa foi avaliar o efeito de um concentrado de proteínas do soro do leite bovino (WPC) enriquecido com o fator de crescimento e transformação beta (TGF-?) e lactoferrina, em pacientes pediátricos com Leucemia Mieloide Aguda - LMA (Capítulo 2); desenvolver um modelo de mucosite gastrointestinal induzida por quimioterápicos, em ratos Wistar, para posterior avaliação da eficácia das proteínas lácteas na proteção da mucosa (Capítulo 3); avaliar os efeitos pelo WPC e por uma caseína comercial, in vivo e in vitro sobre células leucêmicas humanas transplantadas em camundongos imunodeficientes (NOD/SCID) e em cultura de células (Capítulo 4). Para todos os estudos foi utilizado WPC enriquecido com TGF-??e lactoferrina doado pela empresa Hilmar Cheese Company (Cal, USA). No capítulo 2 realizou-se estudo de intervenção nutricional, prospectivo, duplo cego com placebo controlado, onde foram avaliados 21 pacientes, entre 0 a 19 anos, virgens de terapia, admitidos no Centro Infantil Boldrini, Campinas - SP. Foram avaliados quanto à adequação da ingestão alimentar, estado nutricional (EN), dosagem de glutationa (GSH) eritrocitária, hemograma, produção de citocinas no plasma e em cultura de células, imunoglobulina A salivar e evolução da mucosite. Foram comparados os resultados bioquímicos dos pacientes com um grupo controle de indivíduos saudáveis da mesma faixa etária. Utilizou-se o software SPSS para as análises estatísticas (p<0,05). O protocolo de pesquisa foi aprovado pela Comissão Nacional de Ética em Pesquisa (CONEP) com registro 14097. Tomados em conjunto os resultados indicaram que o WPC apresentou efeito positivo sobre o EN dos pacientes, porém não influenciou na composição corporal. A distribuição % dos macronutrientes estava adequada em todos os tempos para os grupos WPC e maltodextrina (placebo), mas o % de adequação dos micronutrientes não atendia às recomendações em sua maioria. As análises bioquímicas não evidenciaram superioridade do WPC em relação ao placebo. Não houve diferença significativa entre os grupos sobre a avaliação clínica da mucosite oral. Na comparação dos pacientes com LMA e indivíduos saudáveis (controle) pode-se constatar níveis estatisticamente superiores do controle nos parâmetros bioquímicos de albumina, pré-albumina, eritrócitos, hematócrito, hemoglobina, plaquetas, produção de fator de necrose tumoral alfa (TNF-?), interleucina 6 (IL-6), interleucina 10 (IL-10) e interferon gama (IFN-?) quando estimuladas pela vacina BCG liofilizada e produção de IL-6 quando estimulada por fitohemaglutinina (PHA). No entanto, para a concentração de GSH eritrocitária e produção espontânea das citocinas TNF-??e IFN-??verificou-se que os pacientes com LMA apresentaram níveis estatisticamente superiores em relação ao controle saudável. No capítulo 3 foi elaborado um protocolo de indução de mucosite em ratos Wistar testando-se os quimioterápicos 5-Fluoruracila (5-FU) e sulfato de vincristina (SV), em administrações de diferentes concentrações e número de doses. Através desse experimento concluiu-se que o SV não foi um bom agente indutor de mucosite, ao contrário do 5-FU. A partir desses resultados foram realizados experimentos com 5-FU visando ajuste das condições dos experimentos inclusive do número, periodicidade e concentração das doses do quimioterápico. O melhor modelo de indução de mucosite gastrointestinal foi obtido através da administração de 3 doses de 5-FU com intervalo de 3 dias entre cada dose, nas concentrações entre 50 e 70 mg/Kg/dose. Os índices bioquímicos não foram influenciados pelo efeito da peletização da dieta e, de modo geral não houve diferença significativa entre os grupos tratados com WPC e com caseína. Independente da natureza da proteína (WPC ou caseína) observou-se maior proteção contra o 5-FU quando os animais receberam as dietas previamente à administração do quimioterápico. Ao contrário do esperado, o estímulo imunológico com hemáceas de carneiro não promoveu aumento nos níveis de GSH nos eritrócitos. As proteínas do soro do leite protegeram a mucosa, em termos de promover menor intensidade de mucosite, nos períodos mais críticos (72h após a 2ª e a 3ª dose de 5-FU) nas regiões de maior prevalência, duodeno e jejuno, quando se comparou com os resultados obtidos para os grupos tratados com caseína. Para os experimentos in vivo, do capítulo 4, utilizaram-se células de leucemia linfóide aguda (LLA) pediátrica humana, isoladas de pacientes do Centro Infantil Boldrini, as quais foram inoculadas em camundongos NOD/SCID. Nesses experimentos, utilizou-se dieta AIN-93G com WPC ou caseína e dieta comercial e o quimioterápico SV. Avaliou- se: peso corporal, consumo de dieta, razão entre peso dos órgãos (rim, baço e fígado) e peso corporal, evolução da leucemia, tempo de sobrevida, hemograma e níveis de glutationa em eritrócitos do sangue periférico. Nos experimentos in vitro foram usadas 6 linhagens celulares: K562 (Leucemia Mieloide Crônica), Nalm-6 (Leucemia Linfóide Aguda do tipo B), Jurkat (Leucemia Linfóide Aguda do tipo T), CEM (Leucemia Linfóide Aguda do tipo T), RAMOS (Linfoma Burkitt) e HL-60 (Leucemia Mieloide Aguda). Foram testadas a citotoxicidade (IC50) do WPC, a viabilidade celular e os níveis de glutationa total nas células leucêmicas estudadas em 4 tempos (0, 24, 48 e 72h) e 4 condições diferenciadas (células; células + WPC; células + WPC + ARAC-C; células + ARA-C). Todos os resultados, tanto do capítulo 3 como do 4, foram analisados através do software ¿Statística: Basic Statistics and Tables¿. Não houve diferença entre o WPC e a caseína em relação aos parâmetros avaliados nos experimentos in vivo, exceto na análise de Doença Residual Mínima (DRM), na qual a caseína se mostrou mais efetiva que o WPC. Constatou-se superioridade do WPC sobre os resultados de peso corporal, razão dos rins e baço pelo peso e evolução da leucemia em relação aos animais tratados com dieta comercial. Não foi possível verificar um efeito sinergístico entre o WPC e o quimioterápico sulfato de vincristina. No que se refere aos experimentos in vitro, o WPC apresentou IC50 para Nalm-6 de 6,72 mg/mL e para a linhagem CEM de 11,84 mg/mL. Verificou-se que as linhagens K562, Nalm-6 e HL-60 apresentaram perfis semelhantes em relação à viabilidade celular. No entanto, em relação à produção de glutationa total, cada linhagem comportou-se de maneira diferenciada, sendo que a maior produção em todas as condições e tempos estudados, foi para a linhagem HL-60 seguida pela K562 / Abstract: The objective of this research was to evaluate the effect of a bovine milk whey protein concentrate (WPC), enriched with transforming growth factor beta (TGF-ß) and lactoferrin in pediatric patients with Acute Myeloide Leukemia (AML) (Chapter 2); in a chemotherapic-induced gastrointestinal mucositis model in Wistar rats (Chapter 3); evaluate in human leukemic cells in culture or transplanted into immune-deficient mouse NOD/SCID (Chapter 4). WPC enriched with TGF-ß and lactoferrin was donated by Hilmar Cheese Company (Cal, USA). Chapter 2 describes a randomized, double-blind, placebo controlled, prospective clinical trial of nutritional intervention with participation of 21 therapy-naïve patients with AML aged 0-19 years from Centro Infantil Boldrini, Campinas, SP. Food intake, nutritional status, red blood cell glutathione (GSH) concentration, haemogram, cytokine concentration in plasma and cell cultures, salivary immunoglobulin A (IgA) and evolution of mucositis were studied. Health individuals at the same age range were used as a control group. The statistical analysis was done using SPSS software (p < 0.05). The research protocol was approved by the National Committee of Ethics in Research (CONEP), resgistered by the number 14097. WPC showed a positive effect in the nutritional status of the patients, but it did not influence their body composition. Percentual distribution of macronutrients was adequate in all times of analyses for both WPC and maltodextrin (placebo) groups, but the percentage of adequacy of the majority of micronutrients did not reach recommendation. Other laboratorial analyses and evolution of mucositis did not show any difference between WPC and placebo groups. Comparison of AML patients with a group of healthy control showed higher concentrations of albumin, prealbumin, haematocrit, hemoglobin, platelets, Bacillus Calmette-Guérin (BCG) vaccine-stimulated tumor necrosis factor alpha (TNF-a), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFN-?) and phytohemaglutinin (PHA)- stimulated IL-6 in controls. On the other hand, patients with AML had higher red blood cell glutathione concentration and spontaneous TNF-a and IFN-??production in comparison to controls. Chapter 3 describes the experiments envolved in the development of a chemotherapic-induced model of mucosites, in Wistar rats, in which 5-Fluoruracila (5-FU) and Vincristine Sulfate (VS) were used, in various concentrations and number of doses. Through this experiment it was concluded that the VS was not a good promoter of mucositis in rats, unlike 5-FU. From these results, experiments were performed with 5-FU considering conditions of the experiments including the number, frequency and concentration of the doses of chemotherapic. The best model was obtained by the administration of 3 doses of 5-FU, each with a 3-days interval and concentrations in the range of 50 to 70 mg/Kg/dose. Haematological parameters and erythrocyte glutathione (GSH) were not influenced by pelletization of the diet and no difference was found between WPC and casein groups. Independent of the protein type (casein or WPC) the effect of 5-Fluoruracila was less deleterious when the diets were offered prior to the chemotherapic treatment. Contrary to expected, sheep red blood cells did not stimulate higher production of erythrocyte GSH by the WPC. WPC decreased mucosites intensity better than casein protecting the mucosa during the most critical periods (72h after 2nd and 3rd doses of 5-FU) in the intestinal regions of major prevalence of mucositis (duodenum and jejunum). More studies are required to support the WPC benefit in the protection and recovery of the mucosa GIT in experimental animal models, and possibly in humans. In chapter 4, cells originated from patients with Acute Lymphoid Leukemia (ALL) from Centro Infantil Boldrini were inoculated in NOD/SCID mice. The animals were fed with commercial diet or AIN-93G diet with WPC or casein as the only protein source and used the chemotherapic vincristine sulfate. Body weight, diet ingestion, ratio between organs (kidney, spleen and liver) and body weight, leukemia evolution, survival time, haemogram and glutathione levels were evaluated. The cell lineages K562 (Chronic Myeloid Leukemia), Nalm-6 (B cell, ALL), Jurkat (T cell, ALL), CEM (T cell, ALL), RAMOS (Burkitt¿s Lymphoma) and HL-60 (AML) were used for the in vitro assays, in which WPC cytotoxicity (IC50), cell viability and glutathione levels were accessed in four different times (0, 24, 48 and 72h) and conditions (cells; cells + WPC; cells + WPC + ARA-C; cells + ARA-C). Data were submitted to statistic analysis by using the: Basic Statistics and Tables software. There was no differences between WPC and casein in relation to the parameters evaluated in vivo, with the exception of the analysis of Minimal Residual Disease (MRD), in which casein seemed to be more effective than WPC. Results on body weight, ratio between organs and body weight and evalution of leukemia were better for the WPC group when compared with animals fed commercial diet. No synergistic effect between the WPC and vincristine sulfate could be observed. In the in vitro experiments, WPC IC50 was 6,72 mg/mL for Nalm-6 and 11,84 mg/mL for CEM cells. K562, Nalm-6 and HL-60 cell lineages showed similar viability profiles. However, each lineage produced different total glutathione concentration in the culture medium, with a higher production achieved by HL-60 cells, followed by K562 cells / Doutorado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Doutor em Alimentos e Nutrição

Orientadores: Karin Spat Albino Barcellos Silveira, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T03:35:28Z (GMT). No. of bitstreams: 1 Bernusso_VanessaAline_M.pdf: 2366309 bytes, checksum: d89f227d922fcfaba6696c0e1af0fdae (MD5) Previous issue date: 2013 / Resumo: VASP (Vasodilator-stimulated phosphoprotein) e Zyxin são proteínas reguladoras de actina que controlam a adesão célula-célula. Zyxin dirige a montagem da actina através da interação e recrutamento da VASP a sítios específicos da adesão. A fosforilação da VASP ou da Zyxin altera suas atividades nas junções aderentes. PKA fosforila VASP em serina 157, regulando, assim, importantes funções celulares de VASP. VASP interage com ABL e é substrato da oncoproteína BCR-ABL. A presença da proteína BCR-ABL promove a oncogênese em pacientes com leucemia mieloide crônica (LMC) devido à ativação constitutiva da atividade tirosina quinase. Apesar de já descrita alteração da expressão de VASP e Zyxin em diferentes tumores epiteliais, o papel de VASP e Zyxin na LMC, na via de sinalização BCR-ABL e a participação destas proteínas na hematopoiese são desconhecidos. Desta maneira, demonstramos aqui ausência de p-VASP ser157 em células de medula óssea de pacientes com LMC, em contraste com a presença de p-VASP ser157 em doadores saudáveis. Pacientes com LMC em remissão, responsivos a inibidores de tirosina quinase, apresentam p-VASP ser157, enquanto os pacientes resistentes não expressam p-VASP ser157. Utilizando células K562 inibidas para VASP ou Zyxin, observamos que VASP e Zyxin modulam as proteínas anti-apoptóticas BCL-2 e BCL-XL da via de sinalização do BCR-ABL. Em adição, células K562 silenciadas para a VASP apresentam diminuição na atividade de FAK y925 e demonstramos que VASP interage com FAK. A expressão de VASP e Zyxin e de suas formas ativas aumenta durante a diferenciação megacariocítica e a inibição de VASP implica em diminuição na expressão do marcador CD61. Identificamos no presente estudo a participação de VASP e Zyxin na via do BCR-ABL, regulando a expressão de proteínas efetoras anti-apoptóticas e, também, na diferenciação megacariocítica. Desta maneira, a expressão alterada da atividade de VASP nos pacientes com LMC pode contribuir para a patogênese da doença, seja afetando a diferenciação celular ou a adesão das células leucêmicas / Abstract: VASP (vasodilator-stimulated phosphoprotein) and Zyxin are actin regulatory proteins that control cell-cell adhesion. Zyxin directs actin assembly by interacting and recruiting VASP to specific sites of adhesion. The phosphorylation of VASP and Zyxin modifies their activity in cell-cell junctions. PKA phosphorylates VASP at serine 157 regulating VASP cellular functions. VASP interacts with ABL and VASP is a substrate of BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. It has been described an altered expression of VASP and Zyxin in different types of tumor; however the function of VASP and Zyxin in CML, in BCR-ABL pathway and in hematopoiesis remains unknown. We describe here the absence of p-VASP ser157 in CML bone marrow cells, in contrast to p-VASP ser157 expression in healthy donors. Patients responsive to tyrosine kinase inhibitors present p-VASP ser157, while resistant patients do not have p-VASP ser157. In K562 cells we observed that VASP and Zyxin modulate anti-apoptotic proteins BCL-2 and BCL-XL. VASP depletion in K562 cells decreases FAK y925 activity and VASP interacts with FAK. Expression of VASP, p-VASP, Zyxin and p-Zyxin increases during megakaryocyte differentiation and VASP inhibition affects this differentiation through reduced CD61 expression in VASP depleted cells. We identify here the participation of VASP and Zyxin in BCR-ABL pathway affecting anti-apoptotic proteins and, also, in megakaryocyte differentiation. Then, the altered expression of VASP activity in CML patients may contribute to CML pathogenesis, affecting cellular differentiation or leukemic cell adhesion / Mestrado / Fisiopatologia Médica / Mestra em Ciências

Orientador: Jorg Kobarg / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T03:42:39Z (GMT). No. of bitstreams: 1 Migueleti_DeividLucasdosSantos_M.pdf: 8057948 bytes, checksum: ef239a4533686a90e511e97afd0f97ba (MD5) Previous issue date: 2012 / Resumo: A proteína UNC-76 foi identificada como necessária para a fasciculação e elongação de axônios do verme Caenorhabditis elegans durante o desenvolvimento do sistema nervoso. A homóloga de mamíferos FEZ1 apresenta altos níveis de expressão em tecidos neuronais e camundongos knockout para o gene FEZ1 apresentam desvios de comportamento que remetem a desordens neurológicas. O papel de FEZ1 no desenvolvimento do sistema nervoso parece residir na sua associação com elementos do citoesqueleto e vias de sinalização (e.g., PKC?, E4B, DISC1) que conduzem o crescimento axonal e a polarização celular. Trabalhos do grupo mostram que FEZ1 é uma proteína multifuncional (hub), capaz de interagir com mais de 50 parceiros através de seus domínios coiled-coil. Além disso, a superexpressão de FEZ1 em células HEK293 provoca o aparecimento de núcleos multilobulados, um fenótipo comum em alguns tipos de leucemia. Nesse trabalho foi investigado o papel de FEZ1 nos mecanismos causadores dos núcleos multilobulados e as consequências funcionais de sua superexpressão na viabilidade celular, tentando extrapolar esse modelo para leucemias. Análises in silico de diversas leucemias mostraram que FEZ1 está superexpressa em LMAs e que isso pode se relacionar à ocorrência da fusão 11q23/MLL. A expressão de FEZ1 na linhagem leucêmica THP-1 foi detectada por Western blotting, mas, a expressão em PBMCs de pacientes ainda permanece sem provas empíricas. Para avaliar as consequências funcionais da superexpressão, uma linhagem com expressão estável e indutível foi obtida e utilizada em ensaios de proliferação e resistência a quimioterápicos. Porém, não foram observadas diferenças entre as linhagens expressando a fusão FLAG-FEZ1 e as que expressavam o FLAG tag apenas. Em um ensaio de IP-MS utilizando tais linhagens, foram identificadas proteínas cuja interação com FEZ1 pode ser modulada pela atividade de PKCs. Finalmente, a cotransfecção de FEZ1 inteira com coiled-coils C-terminais diminui a formação de núcleos multilobulados em quase 40%. A transfecção com o mutante FEZ1 nocys contendo 5 cisteínas mutadas não teve o mesmo efeito, mas, novos experimentos são necessários para determinar o potencial de sinergismo que esses dois componentes podem ter sobre a ocorrência desse fenômeno / Abstract: The protein UNC-76 was identified as necessary for fasciculation and elongation of axons of the worm Caenorhabditis elegans during development of the nervous system. The mammalian homologue FEZ1 is mostly expressed in neuronal tissues and FEZ1 knockout mice present behavior abnormalities that resemble neurological disorders. The role of FEZ1 in the development of the nervous system seems to lie in its association with cytoskeletal elements and signaling pathways (e.g., PKC?, E4B, DISC1) regulating axon outgrowth and cell polarization. The studies of our group have shown that FEZ1 is a hub, able to interact with more than 50 partners through its coiled-coil domains. Furthermore, overexpression of FEZ1 in HEK293 cells causes the appearance of flower-like nuclei, a common phenotype to certain types of leukemia. In this work the role of FEZ1 in the mechanisms of flower-like nuclei formation and functional consequences of its overexpression on cell viability were investigated, attempting to extrapolate this model for leukemias. In silico analysis of several leukemias showed that FEZ1 is overexpressed in AML patients and that this may relate to the occurrence of 11q23/MLL genetic fusion. FEZ1 expression in leukemic THP-1 cells was detected by Western blotting, but the expression in PBMCs of leukemic patients still lacks empirical evidence. To assess the functional consequences of overexpression, cell lineage with stable and inducible expression of FEZ1 was obtained and used in proliferative assays. However, it was not observed any differences between lineages expressing FLAG-FEZ1 fusion protein or FLAG tag alone. IP-MS assay using these lineages identified proteins whose interaction with FEZ1 could be modulated by the activity of PKCs. Finally, cotransfection of C-terminal coiled-coils and FEZ1 full-length decreases flower-like nuclei formation to nearly 40%. Transfection with FEZ1nocys mutant containing five substituted cysteines did not play the same, but further experiments are needed to determine the potential synergism these two components may have on this phenomenon / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

La Leucémie Myéloïde Chronique (LMC) est une maladie clonale caractérisée par la présence du chromosome Philadelphie codant pour Bcr-Abl, une tyrosine kinase constitutivement active responsable de la leucémogenèse. Bien que très efficaces, les inhibiteurs de tyrosine kinase (ITKs) restent cependant inactifs sur les cellules souches leucémiques. Ce travail de thèse montre que la signalisation calcique, connue pour réguler de nombreux processus dans les cellules saines et cancéreuses, est importante dans la signalisation cellulaire au décours de la LMC. Le rôle des entrées calciques dépendantes des stocks (SOCEs) médiées par STIM1 (STromal Interaction Molecule 1) et les canaux Orai1 et TRPC1 ainsi que des entrées calciques induites par la thrombine a été étudié dans la leucémogenèse. Nous avons observé une diminution de ces entrées dans les cellules exprimant Bcr-Abl pouvant être expliquée par le changement de stœchiométrie Orai1/STIM1. Ceci entraîne la diminution de l'activation de NFAT (Nuclear Factor of Activated T-cells) ainsi que des conséquences sur la prolifération et la migration cellulaire mais pas sur l'apoptose. De plus, les SOCEs sont restaurées dans les cellules cancéreuses après traitement à l'Imatinib, le principal ITK. Nous proposons alors que l'expression de Bcr-Abl joue un rôle sur l'homéostasie calcique en entraînant une dérégulation générale des fonctions cellulaires dans les cellules leucémiques notamment via la voie PKC (Protein Kinase C). Ainsi, ces résultats montrent une dérégulation des entrées calciques dans les cellules exprimant Bcr-Abl, suggérant que la signalisation calcique puisse être une cible thérapeutique en parallèle avec les ITKs. / Chronic Myeloid Leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia chromosome encoding for Bcr-Abl, a constitutively active tyrosine kinase responsible for leukemogenesis. Although Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the therapy of Ph+ leukemia, the complete eradication of CML is limited by the emergence of resistance in hematopoietic stem cells. This thesis proposes that calcium (Ca2+) signaling pathways, known to govern a large number of functions in normal and cancer cells, may be important in CML cell signaling. Therefore, we studied the role of Store Operated-Calcium entry (SOCE) (i.e. STromal Interaction Molecule 1 (STIM1), Orai1 and TRPC1 channels) and thrombin induced Ca2+ entry in leukemogenesis. We found a decrease in both calcium entries in Bcr-Abl-expressing cells compared to normal cells. The reduced SOCE seems related to a change in stoichiometry of Orai1/STIM1. This leads to a reduction of the Nuclear Factor of Activated T-cells (NFAT) translocation and functional consequences on cell proliferation and migration but not on apoptosis. Moreover, we showed that SOCE is restored in malignant cells after treatment with Imatinib, the main TKI. We proposed that Bcr-Abl expression could impact on Ca2+ homeostasis enhancing a general disorganization of cell functions in leukemia cells notably via Protein Kinase C (PKC) pathway. Altogether this work shows a deregulation of Ca2+ entry in Bcr-Abl-expressing cells, suggesting that the Ca2+ signaling pathway could be a therapeutic target in parallel with TKIs.

La technologie iPSC (induced pluripotent stem cells) permet l’obtention d’une source cellulaire illimitée pour la modélisation et la thérapie de maladies génétiques, la médecine régénérative, l’étude pharmacologique et récemment la modélisation et l’étude du cancer. La leucémie myéloïde chronique (LMC) est la pathologie modèle du concept de « cellule souche cancéreuse » : des cellules souches LMC (CS-LMC) s’avèrent résistantes aux inhibiteurs de tyrosine kinases (ITK), traitement conventionnel de ce syndrome myéloprolifératif. Elles sont responsables de la persistance d’une maladie résiduelle et de rechutes lors de l’interruption du traitement. Malheureusement, leur isolement est difficile. Nous proposons dans ce travail de modéliser les CS-LMC par les iPSC LMC. Nous montrons pour la première fois dans les iPSC LMC que BCR-ABL1 réprime l’expression des facteurs clés de la pluripotence (OCT4,NANOG, le cluster miR302-367) via les kinases ERK1/2. Les ITK, en bloquant l’activité deBCR-ABL1, entraîne une hausse des niveaux d’expression de ces marqueurs. L’effet « prosouche» des ITK, découvert dans les iPSC LMC, est également observé lors du traitement de CS-LMC primaires. L’agent thérapeutique pourrait ainsi, en maintenant ou renforçant le compartiment LMC immature, participer paradoxalement à la persistance de la maladie résiduelle. Étonnamment, les ITK augmentent également l’expression des marqueurs« souches » dans les iPSC et CS hématopoïétiques normales. Ce résultat suggère un effet« pro-souche » généralisé à plusieurs types cellulaires. / IPSC (induced pluripotent stem cells) offer renewable source of biologically relevant human cells for genetic diseases modelling and therapy, regenerative medicine, pharmacological study and, recently, cancer research. The proof of « cancer stem cell concept » was established in chronic myeloid leukemia (CML) : CML stem cells (CML-SC) are resistant to treatment (tyrosine kinase inhibitors (TKI)). They are involved in residual disease persistence and relapse when treatment is discontinued in the majority of patients. We modelled CML-SC with CML iPSC. We showed, for the first time, that BCR-ABL1 acts as a repressor of key stemness markers (OCT4, NANOG, miR302-367 cluster) via ERK1/2 in human CML iPSC. By blocking BCR–ABL1 activity, TKI increase pluripotency gene expression. Interestingly, a similar pro-stemness effect was observed during CML-SC treatment. By inducing a more primitive stemness phenotype, TKI could promote residual disease and relapse. Interestingly, an increase of stemness gene expression was also observed during TKI treatment of healthy cells (iPSC and hematopoietic stem cells). These data suggest a global TKI pro-stemness effect in other stem cell types.

Les leucémies aiguës myéloïdes (LAM) avec dysplasie, identifiées par la classification OMS 2008 sous le nom de LAM-MRC (« AML with myelodysplasia-related changes »), sont actuellement définies par la présence de critères cliniques, cytologiques et cytogénétiques. Elles forment un groupe hétérogène tant sur le plan biologique que pronostique. Nous avons fait l'hypothèse que la caractérisation moléculaire des LAM-MRC pourrait permettre d'identifier des marqueurs spécifiques associés à ces pathologies et d'en distinguer différents sous-groupes. Nous avons mis en évidence que les LAM-MRC de risque cytogénétique intermédiaire présentent un profil mutationnel spécifique caractérisé par un taux élevé de mutation d'ASXL1 et une faible proportion de mutations de DNMT3A, NPM1 et FLT3. Les LAM-MRC de risque cytogénétique défavorable, essentiellement complexes et/ou monosomales, sont quant à elle associées aux mutations de TP53. Alors que les critères actuels des LAM-MRC ne permettent pas d'en stratifier le pronostic, nous avons montré que les mutations d'ASXL1 ou de TP53 sont des facteurs pronostics péjoratifs majeurs. Ainsi, une reclassification basée sur la présence de ces altérations moléculaires exclusives entre elles permettrait d'affiner le diagnostic et la stratification pronostique de ces maladies. Enfin, dans une stratégie de médecine personnalisée combinant le séquençage à haut débit à des tests de sensibilité thérapeutique in vitro, l'identification de tels marqueurs moléculaires permettraient de prédire la réponse aux traitements, de guider les choix thérapeutiques et d'orienter le développement de nouvelles drogues. / Acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) as reported in the WHO 2008 classification are defined by the presence of clinical, morphological and cytogenetic criteria. AML-MRCs are heterogeneous diseases with prognostic heterogeneity. We hypothesized that molecular characterization of AML-MRC could identify specific molecular markers and disease subgroups. We showed that AML-MRCs with intermediate cytogenetic risk harbor a specific mutational profile characterized by a high frequency of ASXL1 mutations and a low incidence of DNMT3A, NPM1 and FLT3 mutations. Unfavorable cytogenetic risk AML-MRCs, especially due to complex and/or monosomal karyotypes, are associated with TP53 mutations. While WHO criteria do not stratify the prognosis of AML-MRC patients, we showed that the mutations of ASXL1 or TP53 are major poor prognostic factors. The criteria defining AML-MRC do not identify distinct clinical and biological subgroups and do not predict outcome of patients with AML-MRC. In contrast, ASXL1 and TP53-mutated AML identify two distinct biological subgroups of AML-MRC with very poor outcome. This molecular characterization could be useful to redefine AML-MRC in a future classification aiming at merging biological characterization and specific prognostic value. Finally, we showed that a personalized treatment approach combining next generation sequencing and in vitro drug screening could be useful to predict therapeutic response and to guide both treatment choices and new targeted drug developments.

Orientador: Katia Borgia Barbosa Pagnano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T19:42:43Z (GMT). No. of bitstreams: 1 Ribeiro_BeatrizFelicio_M.pdf: 2588401 bytes, checksum: 356f862f97d6467c69db0ac9b13f28d5 (MD5) Previous issue date: 2015 / Resumo: A Leucemia Mielóide Crônica (LMC) é uma neoplasia mieloproliferativa crônica caracterizada pela presença do cromossomo Filadélfia (Ph) e produção da fusão proteica BCR-ABL que possui atividade tirosina quinase. O tratamento da LMC é realizado com inibidores de tirosina quinase (TKIs) e apesar das altas taxas de respostas obtidas com imatinibe, alguns pacientes são resistentes ou intolerantes ao tratamento, sendo necessário a troca para um TKI de segunda geração (nilotinibe ou dasatinibe). O monitoramento das respostas obtidas ao longo do tratamento permite identificar os pacientes que estão em um "estado seguro" (resposta ótima) e os que podem falhar ao tratamento. Os mecanismos de resistência ao tratamento são multifatoriais e a identificação desses mecanismos pode contribuir para o desenvolvimento de novas estratégias para o tratamento dos casos resistentes. Em um trabalho anterior do nosso grupo foram identificados diversos genes e RNAs longos não codificantes diferencialmente expressos em pacientes responsivos e não responsivos ao dasatinibe, entre eles os genes ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2. O objetivo desse trabalho foi avaliar a resposta molecular em pacientes com LMC em uso de inibidores de tirosina quinase de segunda geração (dasatinibe ou nilotinibe) após falha ou intolerância a um ou dois TKIs e avaliar a expressão dos genes ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2 em pacientes responsivos e resistentes ao dasatinibe. A metodologia utilizada para avaliação dos níveis de trancritos BCR-ABL e da expressão gênica foi o PCR quantitativo em tempo real. A pesquisa de mutações no domínio quinase do BCR-ABL foi realizada nos casos resistentes, através da técnica de sequenciamento direto. Setenta e um pacientes tratados com dasatinibe ou nilotinibe após falha ou intolerância ao imatinibe foram avaliados quanto à resposta molecular. Sessenta e sete porcento, 43% e 33% dos pacientes em fase crônica (FC), acelerada (FA) e crise blástica (CB) obtiveram resposta molecular maior (RMM), respectivamente, ao longo do tratamento. Os pacientes com respostas moleculares precoces (transcritos BCR-ABL <10% aos 3 meses e <1% aos 6 meses) apresentaram maiores SLP e SLE do que os casos que não alcançaram esses níveis de transcritos BCR-ABL aos 3 e/ou aos 6 meses. A avaliação molecular aos 3 meses e aos 6 meses permitiu a melhor identificação dos pacientes com pior prognóstico. Foram também avaliados 25 pacientes tratados com dasatinibe/nilotinibe após falha ou intolerância a dois TKIs. RMM foi obtida em somente 24% dos casos (em FC, um paciente em FA). As taxas de SG e SLP em 5 anos para pacientes em FC foram de 94% e 94%, respectivamente. Poucos pacientes alcançam respostas e essas respostas não são duráveis. Embora seja uma opção para pacientes não elegíveis ao TMO, é necessário o desenvolvimento de um tratamento mais eficaz para esses pacientes. Para avaliação da expressão dos genes ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2 foram utilizadas amostras de 9 pacientes ao diagnóstico (sem tratamento prévio), 39 pacientes tratados com dasatinibe (25 responsivos com RCC e 14 resistentes) e 13 doadores saudáves. Não houve diferença de expressão do gene ncSLC44A2 entre os grupos avaliados. Os genes ALOX15B e ncMYLIP estavam hipoexpressos em pacientes com LMC ao diagnóstico em relação aos pacientes com LMC tratados com dasatinibe e ao grupo controle. O gene ncFOXO3A apresentou expressão diminuída em pacientes com LMC ao diagnóstico em relação aos pacientes tratados com dasatinibe. O genes ABCF2 e PAWR estavam hipoexpressos em pacientes ao diagnóstico e nos pacientes tratados com dasatinibe em relação ao grupo controle. Além disso, o gene PAWR estava pouco expresso em pacientes resistentes ao dasatinibe em relação aos pacientes responsivos. Portanto, os genes ABCF2, ALOX15B, PAWR, ncMYLIP e ncFOXO3A foram encontrados com expressão alterada nos grupos estudados e podem estar associados a mecanismos importantes relacionados ao desenvolvimento e resistência da LMC, o que deve ser elucidado em estudos prospectivos / Abstract: Chronic myeloid leukemia (CML) is a chronic myeloproliferative neoplasm characterized by the presence of Philadelphia chromosome (Ph) and production of BCR-ABL fusion protein that has tyrosine kinase activity. Currently, the treatment of CML is accomplished with tyrosine kinase inhibitors (TKIs). Despite the high rates of responses obtained with imatinib, some patients are resistant or intolerant to treatment and need to switch to second generation TKIs (dasatinib or nilotinib). Monitoring responses during treatment allows the identification of patients who are in a "safe haven" (optimal response) and patients who may fail to treatment. Mechanisms of resistance to treatment are multifactorial and identification of these mechanisms may contribute to the development of new strategies for treatment of resistant cases. In a previous report of our group were identified several genes and long non-coding RNAs differentially expressed in CML patients responders and non-responders to dasatinib, including ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP and ncSLC44A2 genes. The aim of this study was to evaluate molecular responses in CML patients treated with second generation TKIs (dasatinib or nilotinib) after failure or intolerance to one or two TKIs and to evaluate the expression of ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP and ncSLC44A2 genes in patients responsive and resistant to dasatinib. The methodology used to evaluate BCR-ABL transcript leves and the gene expression was quantitative real time PCR. BCR-ABL kinase domain mutations analysis was performed in resistant cases by direct sequencing. Seventy-one patients treated with dasatinib/nilotinib after failure or intolerance with imatinib were evaluated according to molecular responses. Sixty-seven percent, 43% and 37% of chronic phase (CP), accelerated phase (AP) and blast crisis (BC) CML patients achieved major molecular response (MMR), respectively, during treatment. Patients with early molecular responses (BCR-ABL1<10% at 3 months and <1% at 6 months) had superior PFS and EFS than patients that not achieved these landmarks. The evaluation at 3 and 6 months allows better identication of patients with worse prognosis. We analyzed molecular responses in 25 patients treated with dasatinib/nilotinib after failure or intolerance with two TKIs. MMR was achieved in 24% of cases (all in CP, one in AP). Five-year OS and PFS rates for CP-CML patients were 94% and 94%, respectively. Few patients achieved responses and these responses are not durable. Although, dasatinib/nilotinib had been options for patients not elegible for bone morrow transplantation, its necessary the development of a treatment more effective to these patients. To evaluate the expression of ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2 genes was used samples of 9 patients newly diagnosed (without treatment), 39 patients treated with dasatinib (25 responsives with CCyR and 14 resistant) and 13 healthy donors. There¿s no difference of ncSLC44A2 gene expression between the groups analized. ALOX15B and ncMYLIP genes were down-regulated in CML patients newly diagnosed in comparison with CML patients treated with dasatinib and control group. ncFOXO3A gene presented decreased expression in CML patients at diagnosis in comparison with patients treated with dasatinib. ABCF2 and PAWR genes were down-regulated in CMl patients newly diagnosed and in CML patients treated with dasatinib in comparison with control group. Moreover, PAWR gene was down-regulated in CML patient¿s resistants to dasatinib in comparison with responsives. ABCF2, ALOX15B, PAWR, ncMYLIP and ncFOXO3A were found with altered expression between the groups evaluated and may be associated with mechanisms related to the development and resistance of CML, which should be elucidated in prospective studies / Mestrado / Clinica Medica / Mestra em Ciências

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa resultante da expansão clonal da célula hematopoética precursora. Sua fisiopatologia está associada ao cromossomo (cr) Philadelphia (Ph) originado da t(9;22) e ao oncogene bcr-abl1 que codifica a proteína Bcr-Abl1 com constitutiva atividade de tirosinoquinase (TK). A expressão de Bcr-Abl determina a leucemogênese por meio da alteração da adesão das células progenitoras leucêmicas ao estroma medular e resistência à apoptose. Os inibidores de TK, o mesilato de imatinibe, dasatinibe e nilotinibe são utilizados no tratamento da LMC, entretanto, casos de resistência têm sido relacionados à presença de mutações em Bcr-Abl1, duplicação do cr Ph e superexpressão do gene bcr-abl1. A resistência ou refratariedade de alguns pacientes ao tratamento com inibidores de TK impulsiona a realização de estudos para melhor conhecimento da fisiopatologia da LMC e descrição de novos alvos terapêuticos. Nesse contexto, o presente estudo investigou a participação de microRNAs na modulação da expressão de genes que regulam a apoptose. O objetivo geral deste trabalho foi investigar o efeito de bcr-abl1 e da atividade tirosinoquinase de Bcr-abl na expressão desses miRNAs em linhagens celulares e pacientes com LMC. O RNA das linhagens celulares, de pacientes e controles foram obtidos por meio da extração com Trizol® e o cDNA sintetizado com o kit High Capacity cDNA reverse transcription. A expressão dos microRNAs e dos genes alvoss foi quantificada por PCR em tempo real utilizando o kit SYBR Green PCR Master Mix® e TaqMan Universal PCR Master Mix®. A inibição de Bcr-Abl1 na linhagem HL-60.Bcr-Abl1 tratada com o mesilato de imatinibe aumentou a expressão de miR-let-7d, miR-15a, miR-130a e miR-145 e diminuiu os níveis de miR-21. O tratamento com dasatinibe aumentou a expressão de miR-let-7e, miR-15a, miR-16, miR-21, miR-30e, miR-130a e miR-142-3p. O nilotinibe aumentou a expressão de miR-let-7e, miR-15a, miR-16, miR-130a e miR-145 e, diminuiu os níveis de miRlet- 7d e miR-21. Os resultados obtidos da análise entre os de pacientes com LMC em diferentes fases da doença mostraram elevados níveis de miR-15a, miR-130b e miR-145 em pacientes na fase crônica versus controles e baixos níveis de miR-16, miR-26a e miR-146a. Pacientes em fases avançadas versus controles apresentaram baixa expressão de miR-let-7d, miR-16, miR-142-3p, miR-145 e miR-146a. Baixos níveis de miR-let-7d, miR-15a, miR-16, miR-29c, miR-142-3p, miR-145 e miR-146a foram observados nas fases avançadas da LMC em relação a fase crônica. Os genes anti-apoptóticos a1, bcl-2, c-flip, ciap-1 e ciap-2 estavam mais elevados na fase crônica do que nos controles. A expressão do gene c-flip estava diminuída e dos genes a1, ciap-1 e mcl-1 aumentada nas fases avançadas em relação aos controles e a fase crônica. Pacientes com LMC resistentes ao MI apresentaram menores níveis de miR-26a, miR-29c, miR-130b, miR-146a e dos genes anti-apoptóticos ciap-1 e mcl-1. Os dados obtidos sugerem que a TK Bcr-Abl modula a expressão de microRNAs que possuem como alvos genes que regulam a apoptose celular / Chronic myeloid leukemia (CML) is a myeloproliferative disease resulting from clonal expasion of hematopoietic precursor cells. Its physiopathology is associated to Philadelphia (Ph) chromosome (cr) originated from the t(9;22) and bcr-abl1 oncogene that encodes the Bcr-Abl protein with constitutive tyrosine kinase activity (TK). The Bcr-Abl1 expression determines leukemogenesis by altering the leukemic progenitor cells´ adhesion by bone marrow stroma and apoptosis resistance. TK inhibitors imatinib mesylate, dasatinib and nilotinib are used to treat CML, however, cases of resistance have been linked to mutation in Bcr-Abl1, duplication of the cr Ph and overexpression of the bcr-abl1. The resistance or refractoriness of some patients to treatment with TK inhibitors drives the studies to better understand the CML physiopathology and description of new therapeutic targets. In this context, this study investigated the participation of microRNAs in modulating expression of the genes that regulate apoptosis. The aim of this study was to investigate the effect of Bcr- Abl1 and its kinase activity in the expression of miRNAs in cell lines and CML patients. The RNA from cell lines, patients and controls were obtained by extraction with Trizol® and cDNA was synthesized with the kit High Capacity cDNA reverse transcription. The expression of miRNAs and target genes was quantified by real time PCR using SYBR Green PCR Master Mix® kit and TaqMan Universal PCR Master Mix®. The Bcr-Abl1 inhibition in the cell line HL-60.Bcr-Abl1 treated with imatinib mesylate increased the expression of miR-let- 7d, miR-15a, miR-130a and miR-145 and decreased miR-21 levels. Treatment with dasatinib increased the expression of miR-let-7e, miR-15a, miR-16, miR-21, miR-30e, miR-130a and miR- 142-3p. Nilotinib increased the expression of miR-let-7e, miR-15a, miR-16, miR-130a and miR- 145 and, decreased miR-let-7d and miR-21 levels. The results of the analysis among patients with CML in different stages of disease showed high levels of miR-15a, miR-130b and miR-145 in chronic phase versus controls and low levels of miR-16, miR-26a and miR-146a. Patients in advanced phases versus controls showed low expression of miR-let-7d, miR-16, miR-142-3p, miR-145 and miR-146a. Low levels of miR-let-7d, miR-15a, miR-16, miR-29c, miR-142-3p, miR-145 and miR-146a were observed in CML advanced phases when compared with chronic phase. The antiapoptotic genes a1, bcl-2, c-flip, ciap-1 and ciap-2 were higher in chronic phase than in controls. The c-flip expression was decreased and a1, ciap-1 and mcl-1 expression was increased in advanced phases when compared to controls and chronic phase. CML patients resistant to imatinib mesylate presented low levels of miR-26a, miR-29c, miR-130b, miR-146a and ciap-1 and mcl-1 antiapoptotic genes. The data obtained suggest that Bcr-Abl1 TK modulates the miRNA expression which has target genes involved in the apoptosis´ regulation.

Introduction: Acute myeloid leukemia (AML) is a highly heterogeneous disease which renders risk stratification at diagnosis of high importance to personalize therapy. Allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance for sustained remission in most AML patients, but usually comes at the risk of a significant treatment-related mortality. The red cell distribution width (RDW) is an universally accessible parameter that identifies individuals with a higher mortality in many diseases, including some hematological entities. However, the impact of diagnostic RDW levels in AML – especially in the context of a HSCT consolidation - has not been evaluated so far. Purpose: To evaluate the prognostic impact of RDW levels at AML diagnosis. Methods: A total of 294 newly diagnosed AML patients (median age 60.6, range 14.3-76.5 years), with available diagnostic RDW levels were retrospectively included in this analysis. All patients received a consolidation therapy with an allogeneic HSCT in curative intention between August 2007 and December 2020 at the University Medical Center Leipzig. The RDW was measured in all patients at AML diagnosis before the start of cytoreductive therapies. Results: RDW levels at diagnosis were highly variable (median 16.6%, range 12%-30.6%) and above the upper level of normal (>15%) in 73% of the analyzed AML patients. Patients with RDW levels above 15% did not have worse outcomes compared to patients with low diagnostic RDW levels. However, when the cohort was dichotomized according to a receiver operating characteristic (ROC)-based optimal cut-point (20.7%), patients with high RDW levels had a significantly higher non-relapse mortality (NRM), shorter overall survival and a trend for shorter event-free survival, while the risk of relapse or disease progression was similar in both groups. In multivariate analyses, the RDW remained an independent prognostic factor for higher NRM after adjustment for the body mass index at diagnosis. Patients with a higher RDW were more likely to harbor a secondary AML, as well as to harbor secondary AML-associated gene mutations (i.e. JAK2, ASXL1, or spliceosome mutations, especially SRSF2). Conclusion: High RDW levels at diagnosis represent an independent risk marker for a higher mortality following allogeneic HSCT. When confirmed in prospective clinical trials, the RDW might help to personalize AML consolidation therapy including conditioning regimens before allogeneic HSCT.:1. Bibliographische Beschreibung 2. Abkürzungsverzeichnis 3. Einführung / Introduction 3.1. Acute Myeloid Leukemia 3.1.1. Definition 3.1.2. Epidemiology and etiology 3.1.3. Clinical presentation 3.1.4. Diagnosis of AML 3.1.4.1. Morphology 3.1.4.2. Immunophenotyping 3.1.4.3. Cytogenetic and molecular analyses 3.1.5. AML classification according to WHO classification 3.1.6. Prognostic factors in AML 3.1.6.1. Patient-related risk factors 3.1.6.2. Genetic risk factors 3.1.6.3. Measurable residual disease 3.1.7. Treatment of AML 3.1.7.1. Induction therapy in curative intention 3.1.7.2. Consolidation therapies 3.1.7.3. Palliative treatment approaches 3.1.7.4. New substances 3.2. Allogeneic HSCT 3.2.1. Principles of allogeneic HSCT 3.2.2. Conditioning regimens 3.3. Red cell distribution width 4. Aufgabenstellung / Objectives 5. Materialien und Methoden / Materials and Methods 5.1. Patients and treatments 5.1.1. Treatment protocols 5.1.2. Allogeneic HSCT and immunosuppression 5.1.3. Assessment of GvHD 5.2. Disease characterization 5.2.1. Evaluation at AML diagnosis 5.2.1.1. Morphology 5.2.1.2. Flow cytometry 5.2.1.3. Genetic analyses 5.2.1.4. Evaluation of RDW levels 5.2.2. Evaluation at HSCT 5.2.2.1. Definition of remission status at HSCT 5.2.2.2. Evaluation of measurable residual disease at HSCT 5.3. Statistical Analyses 5.3.1. Associations 5.3.2. Clinical endpoints 5.3.3. Definition of an optimal cut-point for RDW levels 5.3.4. Multivariate analyses 6. Ergebnisse / Results 6.1. Overall outcomes of the patient cohort 6.2. RDW levels at AML diagnosis regarded as continous parameter 6.3. The role of RDW levels at diagnosis as a predictor for outcomes after allogeneic HSCT 6.4. Associations of RDW levels at diagnosis 7. Diskussion / Discussion 8. Zusammenfassung / Summary 9. Literaturverzeichnis / References 10. Erklärung über die eigenständige Abfassung der Arbeit 11. Curriculum Vitae 12. Komplette Publikationsliste (Peer-reviewed) 13. Danksagung