Les Leucémies Aigues Lymphoblastiques T (LAL-T) sont des hémopathies malignes causées par la prolifération de cellules immatures T bloquées à un stade donné de leur différenciation. Leur oncogenèse est multigénique. Une anomalie de type A (à l’origine du blocage de différenciation) ainsi que des anomalies de type B (gains de fonctions de type prolifération, métabolisme, résistance à l’apoptose…) sont souvent retrouvées chez le même patient. Ces leucémies reproduisent individuellement les différentes étapes de la maturation thymique humaine. En fonction de l’immunogénétique (phénotype et réarrangement des loci des TCR), 3 groupes de LAL-T sont ainsi identifiables : les LAL-T immatures (correspondant aux formes non T-restreintes), les LAL-T corticales (bloquées autour de la b-sélection) et les LAL-T matures TCR/CD3+. Mon travail de thèse a consisté à déterminer à quel point l’activation et la signalisation du TCR pouvaient être impliquées dans la biologie de cette hémopathie. Nous avons utilisé le modèle transgénique Marilyn TCR-HY et montré in vitro (lignée TLX+) et in vivo (modèle de leucémogénèse TEL-JAK2) que l’activation du TCR par la reconnaissance de son peptide spécifique (DBY, peptide présent uniquement dans les souris mâles) empêche le développement et le maintien de la leucémie. L'induction de la signalisation du TCR par des anticorps monoclonaux dirigés contre la chaîne de signalisation CD3e (anti-CD3e humain, OKT3 ; anti-CD3e murin, 145-2C11) entraîne également une mort massive des cellules leucémiques en induisant un programme d'expression génique et de phosphoproteomique ressemblant à la sélection négative thymique. In vitro dans des LAL-T primaires, la stimulation du complexe CD3/TCR par un anticorps anti-CD3 entraîne la mort cellulaire des LAL-T CD3/TCR+ et non des TCR/CD3- quelque soit leur mécanisme oncogénétique sous-jacent. Finalement, le traitement anti-CD3 in vivo empêche la leucémogenèse chez les souris transplantées avec des LAL-T murines et humaines. Ces données fournissent un fort rationnel en faveur d’une thérapie ciblée, basée sur le traitement anti-CD3 des LAL-T matures CD3/TCR+. Ce travail démontre aussi que des étapes-clés du développement (comme la sélection négative) peuvent être des cibles thérapeutiques et sont actionnables malgré l’accumulation d’altérations géniques et épigénétiques dans les cellules cancéreuses. Par ailleurs, j’ai étudié la fréquence et l’impact pronostique des anomalies de la signalisation du TCR/pré-TCR dans une grande série protocolaire de LAL-T de l’adulte (GRAALL-2003 et -2005). Les voies de prolifération RAS/MAPK et PI3K/PTEN/AKT participent à la signalisation du TCR/pré-TCR et ont été rapportées comme dérégulées dans des séries de LAL-T pédiatriques. Dans notre série, j’ai identifié des délétions/mutations perte de fonction de PTEN (12 %) ou des mutations activatrices de KRAS/N-RAS (11%) montrant que les anomalies du pré-TCR/TCR sont fréquentes dans les LAL-T puisqu’elles sont retrouvées dans 23% des cas. Les anomalies de RAS/PTEN sont associées à un pronostic défavorable. Leur impact pronostique en fonction du statut mutationnel NOTCH1/FBXW7 (N/F) a également été étudié et montre que les anomalies de RAS/PTEN abrogent le bon pronostic des mutations N/F. Ce travail permet de proposer une classification oncogénétique basée sur les anomalies de N/F et RAS/PTEN. Cette classification définit les patients de bas risque comme ceux ayant N/F muté mais sans anomalie de RAS et PTEN (51 %) et les hauts risques qui regroupent tous les autres patients (49 %). Cette classification oncogénétique est dorénavant utilisée dans le nouveau protocole GRAALL-2014 des LAL-T de l’adulte. / T-cell acute lymphoblastic leukemias (T-ALL) are rare lymphoid neoplasms characterized by the proliferation of T lymphoblasts arrested at specific stages of maturation. Leukemic transformation of maturating thymocytes is caused by a multistep pathogenesis involving numerous genetic abnormalities that drive normal T cells into uncontrolled cell growth and clonal expansion. Depending on immunogenetic, T-ALLs are classified in 3 groups: immature, cortical (blocked around b-selection) and mature (CD3/TCR+) T-ALL. My work was to determine if activation and TCR signalling are involved in the biology of this disease. We demonstrate in T-ALL that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent TCR signalling has anti-leukemic properties and enforces a molecular and phosphoproteomic program resembling thymic negative selection, a major developmental event in normal T cell development. Using mouse models of T-ALL, we show that induction of TCR signalling by high affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3e chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of TCR-expressing T-ALL patients and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. Besides, I studied frequency and prognostic impact of anomalies concerning pre-TCR/TCR signalling in a large cohort of adult T-ALL included in GRAALL trials. RAS/MAPK and PI3K/PTEN/AKT pathways are involved in pre-TCR/TCR signalling and are reported as deregulated in pediatric T-ALL. I identified deletion/mutation loss-of-function of PTEN (12%) and activating mutations of KRAS/N-RAS (11%) in 23% of patients. These anomalies predict poor outcome and abrogate the good prognosis of NOTCH1/FBXW7 mutations. We proposed a purely genetic stratification of patients based on N/F/RAS/PTEN status, identifying low-risk patients (51%) with N/F mutations without RAS/PTEN anomalies and high-risk patients (49%) composed by the remaining cohort. This stratification will be used for the next protocol of adult-T-ALL.

Este estudo teve como objetivo avaliar o estado nutricional relativo ao selênio (Se) de pacientes na fase de pós-tratamento da leucemia linfóide aguda (LLA) e sua relação com o estresse oxidativo. Foram selecionados 24 pacientes no pós-tratamento da LLA (9,2 ± 1,9 anos) atendidos no Instituto de Oncologia Pediátrica da Universidade Federal de São Paulo e 60 indivíduos saudáveis (9,5 ± 1,3 anos) da Escola de Aplicação da Universidade de São Paulo. Foram coletados 10 mL de sangue venoso para análise de Se plasmático e eritrocitário, glutationa peroxidase (GPx), superóxido dismutase (SOD), α- tocoferol, malondialdeído (MDA) e 8-oxo-desoxiguanosina (8-oxo-dGuo). A urina de 24 horas foi coletada para análise da excreção de Se, e três recordatórios de consumo alimentar de 24 horas para avaliação do Se ingerido. Os resultados obtidos quanto aos parâmetros bioquímicos de avaliação de Se não apresentaram diferença significativa entre os grupos de pacientes e controles, e foram respectivamente: Se plasmático, 44,4 ± 9,0 µg/L e 48,7 ± 12,0 µg/L (p = 0,122); Se eritrocitário, 49,9 ± 15,9 µg/L e 45,0 ± 15,9 µg/L (p = 0,202); Se urinário, 19,6 ± 14,8 µg Se/g de creatinina e 18,6 ± 9,6 µg Se/g de creatinina (p = 0,820). O consumo médio de Se foi de 27,4 ± 8,7 µg/dia e 28,0 ± 1,5 µg/dia (p = 0,756), respectivamente. Os grupos estudados foram considerados deficientes em Se, considerando os pontos de corte adotados. A atividade da GPx foi significativamente menor nos pacientes do que nos controles (33,3 ± 11,1 U/g Hb e 76,9 ± 25,9 U/g Hb) (p = 0,000), e a atividade da SOD não diferiu entre pacientes e controles (1796,9 ± 257,8 U/g Hb e 1915,9 ± 473,9 U/g Hb) (p = 0,145), assim como as concentrações de MDA (1,7 ± 0,3 µmol/L e 1,8 ± 0,4 µmol/L) (p = 0,053). A concentração de α-tocoferol foi estatisticamente maior nos pacientes que nos controles (17,7 ± 4,7 µmol/L e 10,6 ± 3,2 µmol/L) (p =0,000), bem como a concentração de 8-oxo-dGuo (43,6 ± 28,0 8-oxo/106 dG e 21,3 ± 22,9 8-oxo/106 dG) (p = 0,014). Os resultados apresentados apontam que os participantes deste estudo estão deficientes em Se e, em especial os pacientes no pós-tratamento da LLA estão sujeitos a um aumento do estado de estresse oxidativo, pois apesar das concentrações de MDA serem semelhantes entre os pacientes e os controles, a atividade da GPx dos pacientes foi reduzida e a concentração de 8-oxo-dGuo e α-tocoferol estavam aumentadas em relação aos controles. / This study aimed to evaluate the nutritional status of selenium (Se) in patients in post-treatment of acute lymphoblastic leukemia (ALL) and its relationship with oxidative stress. We selected 24 patients in post-treatment of ALL (9.2 ± 1.9 years) at the Pediatric Oncology Institute of Federal University of São Paulo and 60 healthy individuals (9.5 ± 1.3 years) of the School of Application at the University of São Paulo. We collected 10 mL of venous blood for analysis of Se in plasma and erythrocytes, glutathione peroxidase (GPx), superoxide dismutase (SOD), α-tocopherol, malondialdehyde (MDA) and 8-oxo-deoxyguanosine (8-oxo-dGuo). The 24-hour urine was collected for analysis of Se excretion and Se intake was evaluated by using three non-consecutive days of 24- hour recall. The results regarding biochemical evaluation of Se did not differ significantly between patients in post-treatment of ALL and controls, and the results were respectively: Se in plasma, 44.4 ± 9.0 µg/L and 48.7 ± 12.0 µg/L (p = 0.122); Se in erythrocytes, 49.9 ± 15.9 µg/L and 45.0 ± 15.9 µg/L (p = 0.202); Se in urine, 19.6 ± 14.8 µg Se/g creatinine and 18.6 ± 9.6 µg Se/g creatinine (p = 0.820). The average intake of selenium was 27.4 ± 8.7 mg/day and 28.0 ± 1.5 mg/day (p = 0.756), respectively. Both groups were considered deficient in selenium, according to the cut-off points adopted. The GPx activity was significantly lower in patients than in controls (33.3 ± 11.1 U/g Hb and 76.9 ± 25.9 U/g Hb) (p = 0.000), and no difference in SOD activity was observed between groups (1796.9 ± 257.8 U/g Hb and 1915.9 ± 473.9 U/g Hb) (p = 0.145). MDA concentrations were not different between patients and controls (1.7 ± 0.3 µmol/L and 1.8 ± 0.4 µmol/L) (p = 0.053) and α-tocopherol concentration was statistically higher in patients (17.7 ± 4.7 µmol/L and 10.6 ± 3.2 µmol/L) (p = 0.000), as well the concentration of 8-oxo-dGuo (43.6 ± 28.0 8-oxo/106 dG and 21.3 ± 22.9 8-oxo/106 dG) (p = 0.014). These results indicate that the participants in this study are deficient in Se mainly those who are in post-treatment of ALL are exposed to an increased state of oxidative stress, because although the concentrations of MDA were similar between patients and controls, the GPx activity of the patients was reduced and the concentration of 8-oxo-dGuo and α-tocopherol were increased compared to controls.

A crisantaspase é uma enzima do tipo asparaginase (ASNase) produzida pela bactéria Erwinia chrysanthemi e utilizada como biofármaco no tratamento da leucemia linfoblástica aguda (LLA) em casos de hipersensibilidade à ASNase de E. coli. As principais desvantagens deste biofármaco são a curta meia-vida (10 horas) e imunogenicidade. Nesse sentido, sua forma peguilada (PEG-crisantaspase) não só reduziria o efeito imunogênico, como também melhoraria a meia-vida plasmática. Atualmente, somente a ASNase de E. coli está disponível comercialmente na forma peguilada e essa, por ter sido uma das primeiras proteínas a serem peguiladas, é resultado de um processo de peguilação aleatória em resíduos de lisina. Portanto, apresenta alto grau de polidispersão em relação à quantidade de cadeias de PEG ligadas à enzima. Nesse trabalho desenvolvemos um processo de obtenção de crisantaspase peguilada de maneira sítio-específica, no grupamento N-terminal (PEG-crisantaspase). A crisantaspase foi obtida de forma recombinante na cepa E. coli BL21, cultivada em agitador metabólico e biorreator, em meio Luria Bertani. A produtividade volumétrica no biorreator aumentou 37% em comparação com o agitador metabólico (460 e 335 U·L-1·h-1 respectivamente). A crisantaspase foi recuperada por choque osmótico e purificada por cromatografia de troca catiônica (coluna HiTrap SP FF, 5 mL, eluição em pH 7,5), apresentando atividade específica de 694 U·mg-1, fator de purificação de 31 e rendimento de 69%. A crisantaspase purificada foi peguilada com mPEG-NHS 10 kDa, em tampão fosfato 100 mM, 22 °C, razão molar enzima:PEG 1:50 durante 30 min e sob diferentes valores de pH (6,5-9,0). O melhor rendimento de peguilação N-terminal (50%) foi em pH 7,5 com menor formação de estruturas poli-peguiladas (7%). A PEG-crisantaspase foi isolada por cromatografia de exclusão molecular, retendo 50% da atividade específica (357 U·mg-1) com valor de kM três vezes maior do que o da crisantaspase (150 e 48,5 µM respectivamente). Entretanto, apresentou maior estabilidade em altas temperaturas. Em duas semanas, a crisantaspase perdeu 93% de sua atividade específica enquanto que a PEG-crisantaspase foi estável por 20 dias. Portanto, a enzima PEG-crisantaspase desenvolvida representa uma alternativa promissora para o tratamento da LLA. / Crisantaspase is an asparaginase enzyme (ASNase) produced by Erwinia chrysanthemi bacterium and used as biopharmaceutical in the treatment of acute lymphoblastic leukemia (ALL) in case of hypersensivity to E. coli ASNase. The main disadvantages of this biopharmaceutical are the short half-life (10 hours) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce the immunogenic effect but also improve plasma half-life. Currently, only E. coli ASNase is commercially available in its pegylated form. Since ASNase was one of the first proteins to be pegylated, it corresponds to a random PEGylation process on lysine residues and consequently preparations are highly polydisperse. In this work we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21 strain, grown in shaker and bioreactor, in Luria Bertani medium. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U·L-1·h-1 respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography (HiTrap SP FF column, 5 mL, elution at pH 7.5), presenting specific activity of 694 U·mg-1,31 purification fold and an yield of 69%. Purified crisantaspase was PEGylated with 10 kDa mPEG-NHS in 100mM phosphate buffer, 22°C, enzyme:PEG molar ratio of 1:50 for 30 min, and at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with less poly-PEGylated forms (7%). PEG-crisantaspase was purified by size-exclusion chromatography, retaining 50% of specific activity (357 U·mg-1) with a kM value 3 times higher than crisantaspase (150 and 48,5 µM respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over the time. In two weeks, crisantaspase lost 93% of its specific activity, while PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme developed represents a promising alternative for the treatment of ALL.

A crisantaspase é uma enzima do tipo asparaginase (ASNase) produzida pela bactéria Erwinia chrysanthemi e utilizada como biofármaco no tratamento da leucemia linfoblástica aguda (LLA) em casos de hipersensibilidade à ASNase de E. coli. As principais desvantagens deste biofármaco são a curta meia-vida (10 horas) e imunogenicidade. Nesse sentido, sua forma peguilada (PEG-crisantaspase) não só reduziria o efeito imunogênico, como também melhoraria a meia-vida plasmática. Atualmente, somente a ASNase de E. coli está disponível comercialmente na forma peguilada e essa, por ter sido uma das primeiras proteínas a serem peguiladas, é resultado de um processo de peguilação aleatória em resíduos de lisina. Portanto, apresenta alto grau de polidispersão em relação à quantidade de cadeias de PEG ligadas à enzima. Nesse trabalho desenvolvemos um processo de obtenção de crisantaspase peguilada de maneira sítio-específica, no grupamento N-terminal (PEG-crisantaspase). A crisantaspase foi obtida de forma recombinante na cepa E. coli BL21, cultivada em agitador metabólico e biorreator, em meio Luria Bertani. A produtividade volumétrica no biorreator aumentou 37% em comparação com o agitador metabólico (460 e 335 U·L-1·h-1 respectivamente). A crisantaspase foi recuperada por choque osmótico e purificada por cromatografia de troca catiônica (coluna HiTrap SP FF, 5 mL, eluição em pH 7,5), apresentando atividade específica de 694 U·mg-1, fator de purificação de 31 e rendimento de 69%. A crisantaspase purificada foi peguilada com mPEG-NHS 10 kDa, em tampão fosfato 100 mM, 22 °C, razão molar enzima:PEG 1:50 durante 30 min e sob diferentes valores de pH (6,5-9,0). O melhor rendimento de peguilação N-terminal (50%) foi em pH 7,5 com menor formação de estruturas poli-peguiladas (7%). A PEG-crisantaspase foi isolada por cromatografia de exclusão molecular, retendo 50% da atividade específica (357 U·mg-1) com valor de kM três vezes maior do que o da crisantaspase (150 e 48,5 µM respectivamente). Entretanto, apresentou maior estabilidade em altas temperaturas. Em duas semanas, a crisantaspase perdeu 93% de sua atividade específica enquanto que a PEG-crisantaspase foi estável por 20 dias. Portanto, a enzima PEG-crisantaspase desenvolvida representa uma alternativa promissora para o tratamento da LLA. / Crisantaspase is an asparaginase enzyme (ASNase) produced by Erwinia chrysanthemi bacterium and used as biopharmaceutical in the treatment of acute lymphoblastic leukemia (ALL) in case of hypersensivity to E. coli ASNase. The main disadvantages of this biopharmaceutical are the short half-life (10 hours) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce the immunogenic effect but also improve plasma half-life. Currently, only E. coli ASNase is commercially available in its pegylated form. Since ASNase was one of the first proteins to be pegylated, it corresponds to a random PEGylation process on lysine residues and consequently preparations are highly polydisperse. In this work we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21 strain, grown in shaker and bioreactor, in Luria Bertani medium. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U·L-1·h-1 respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography (HiTrap SP FF column, 5 mL, elution at pH 7.5), presenting specific activity of 694 U·mg-1,31 purification fold and an yield of 69%. Purified crisantaspase was PEGylated with 10 kDa mPEG-NHS in 100mM phosphate buffer, 22°C, enzyme:PEG molar ratio of 1:50 for 30 min, and at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with less poly-PEGylated forms (7%). PEG-crisantaspase was purified by size-exclusion chromatography, retaining 50% of specific activity (357 U·mg-1) with a kM value 3 times higher than crisantaspase (150 and 48,5 µM respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over the time. In two weeks, crisantaspase lost 93% of its specific activity, while PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme developed represents a promising alternative for the treatment of ALL.

O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações. / The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications.

O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações. / The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications.

Made available in DSpace on 2016-08-19T18:16:04Z (GMT). No. of bitstreams: 1 dissertacao Thaiana.pdf: 2704967 bytes, checksum: e27a6cd767b0d6ac2256bec85730e19a (MD5) Previous issue date: 2012-08-13 / Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children and adolescents younger than fifteen years, with incidence peak between two and five years old. Several parameters have been evaluated for their importance in the prognostic evaluation of pediatric patients with ALL, of these, the evaluation of the anomalous myeloid expression prognosis of these pathologies. The aim of this work was to analyze the influence of myeloid anomalous expression in prognosis of children and adolescents with ALL of the state of Maranhão. It was evaluated 65 patients under 18 years diagnosed with ALL at the Instituto de Oncologia Maranhense Aldenora Belo (IMOAB) in São Luís, Maranhão. Laboratory data were obtained at diagnosis, made on the basis of morphological/ cytochemical and immunophenotype criteria. Other data were obtained from medical records. The sample was divided into groups with and without aberrant expression of myeloid antigens CD13 and CD33 for analysis with prognostic variables. The results indicated that the average age in the sample was 6,5 years, with the majority (69,2%) it was male children. The B-ALL was the most frequent type (83,1%). The mean percentage of blasts in our series was 75,8% in bone marrow and 46,6% in peripheral blood. Over ninety percent (90,8%) patients had L1 type morphology. The profile of blood test indicated the mean values of 32.373 leukocytes/mm3; 8,26 g / dl hemoglobin and 52.498 platelets/mm3. The anomalous myeloid expression occurred in 49,2% of the sample. The GBTLI-99 classification showed 56,9% of subjects with low risk of recurrence. At the end of the induction treatment period, 72,3% of children have responded positively in remission. The platelet count was significantly lower in without myeloid anomalous expression group (33.627 platelets/mm3, p = 0.01) compared to group that expressed this marker. About eighty-nine percent (88.9%) children with ALL-B and without myeloid aberrant expression had less than 50.000 platelets/mm3 (p = 0.01). Children with expression of CD33 were significantly older than those who did not express this marker (9,4 years, p = 0.01). It is concluded that the platelet count may be an important parameter to be analyzed in the prognosis of children with myeloid aberrant expression. The CD33 may be important in population with ALL and aberrant expression, since the CD13 was present in almost all cases. / A leucemia linfóide aguda (LLA) representa a neoplasia hematológica mais frequente em crianças e adolescentes menores de quinze anos, com pico de incidência entre dois e cinco anos de idade. Diversos parâmetros têm sido avaliados quanto à sua importância na avaliação prognóstica da população pediátrica com LLA, dentre estes, a avaliação da expressão anômala mielóide no prognóstico destas patologias. O objetivo deste trabalho foi analisar a influência da expressão anômala mielóide no prognóstico de crianças e adolescentes com LLA do estado do Maranhão. Foram avaliados 65 pacientes menores de 18 anos com diagnóstico de LLA assistidos no Instituto Maranhense de Oncologia Aldenora Belo (IMOAB), em São Luís, Maranhão. Os dados laboratoriais foram obtidos ao diagnóstico, realizado com base nos critérios morfológicos/citoquímicos e imunofenotípicos. Os demais dados foram obtidos a partir de prontuários. A amostra foi dividida em grupos com e sem expressão anômala mieloide dos antígenos CD13 e CD33 para análise com as variáveis prognósticas. Os resultados obtidos indicaram que a média de idade na amostra foi de 6,5 anos, sendo que a maioria (69,2%) se tratava de crianças do sexo masculino. A LLA-B foi o tipo mais frequente (83,1%). A porcentagem média de blastos em nossa casuística foi de 75,8% na medula óssea e de 46,6% em sangue periférico. Mais de noventa por cento (90,8%) dos pacientes apresentava morfologia do tipo L1. O perfil do hemograma indicou os valores médios de 32.373 leucócitos/mm3; 8,26 g/dl de hemoglobina e 52.498 plaquetas/mm3. A expressão anômala mielóide ocorreu em 49,2% da casuística. A classificação GBTLI-99 indicou 56,9% de sujeitos com baixo risco de recidiva. No final do período de indução do tratamento, 72,3% das crianças responderam positivamente com remissão da doença. A contagem de plaquetas foi estatisticamente inferior no grupo sem expressão anômala mielóide (33.627 plaquetas/mm3, p = 0,01) em relação ao grupo que expressou este marcador. Cerca de oitenta e nove por cento (88,9%) das crianças com LLA-B e sem expressão anômala mielóide apresentavam menos de 50.000 plaquetas/mm3 (p = 0,01). Crianças com expressão do CD33 foram significativamente mais velhas do que aquelas que não expressavam este marcador (9,4 anos, p = 0,01). Conclui-se que a contagem de plaquetas pode ser um importante parâmetro a ser analisado no prognóstico de crianças sem expressão anômala mielóide. O CD33 pode ser importante na caracterização da população com LLA e expressão anômala, já que o CD13 esteve presente em quase todos os casos.

Les leucémies aigües lymphobastiques (LAL) représentent un tiers des leucémies et constituent le cancer pédiatrique le plus fréquent chez l’enfant. Les LAL de type T (LAL-T)sont caractérisées par l’expansion anormale de progéniteurs de lymphocytes T. Aujourd’hui,la réponse curative aux traitements est proche de 80% chez l’enfant et 50% chez l’adulte. La rechute reste donc fréquente et souvent de mauvais pronostic. Pour ces raisons,l’identification de nouvelles voies de signalisation en vue de développer de nouvelles stratégies thérapeutiques est cruciale afin d’améliorer le traitement des LAL-T.Les résultats précédents du laboratoire ont révélé l’activation soutenue de la voie calcineurine (Cn)/NFAT dans des échantillons humains de lymphomes et de LAL, ainsi que dans des modèles murins de ces pathologies. Le laboratoire a ensuite montré que Cn est intrinsèquement requise pour la capacité des cellules leucémiques de LAL-T à propager la maladie (activité LIC « Leukemia Initiating Cells ») dans un modèle murin de LAL-T induit parun allèle activé de NOTCH1 (ICN1). Puisque l’inhibition pharmacologique de Cn induit de nombreux effets secondaires, la recherche de cibles thérapeutiques en aval de Cn constitue un axe de recherche important. J’ai participé à une étude du laboratoire montrant que l’expression à la surface cellulaire de CXCR4 est régulée par Cn et requise pour la migration des cellules de LAL-T, mais non suffisante pour rétablir le potentiel de ré-initiation suggérant que d’autres effecteurs doivent être impliqués dans cette activité.Les facteurs de transcription NFAT (NFAT1, NFAT2 et NFAT4) sont des effecteurs importants de Cn en réponse à la signalisation calcique lors du développement des thymocytes, mais également dans les lymphocytes T. L’essentiel de ce travail de thèse a utilisé des LAL-T induites par ICN1 dans lesquelles l’inactivation génique des trois facteurs NFAT par recombinaison homologue. Nous avons ainsi montré que (i) les facteurs NFAT sont requis en aval de Cn pour le potentiel LIC des LAL-T-ICN1 in vivo, (ii) leur inactivation altère la survie, la prolifération et la migration des cellules de LAL-T in vitro, (iii) NFAT1,NFAT2 et NFAT4 ont une fonction largement redondante dans les LAL-T. Nous avons également par une approche transcriptomique identifié deux gènes dont l’expression estsous contrôle des facteurs NFAT et impliqués dans la régulation de la survie et de la prolifération des LAL-T in vitro : CDKN1A et MAFB.Tout comme la voie Cn/NFAT, les CaMKs sont des protéines kinases activées en aval de la signalisation calcique dans les lymphocytes T. Nous avons montré par une approche pharmacologique que l’inhibition des CaMKs dans les LAL-T-ICN1 in vitro altère la survie etla prolifération des cellules leucémiques. L’inhibition spécifique par une approche d’ARN interférence de deux isoenzymes CaMKIIγ et CaMKIIδ suggèrent que ces protéines jouent dans le maintien des cellules leucémiques in vitro. / T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T cell progenitors. Despite initial response to chemotherapy, relapses remain frequent in children and adults. Previous results identify sustained activation of Calcineurin (Cn)/NFAT signaling pathway in human T-ALL and murine T-ALL models. Importantly, they also demonstrated Cn is essential for T-ALL Leukemia Initiating Cells (LIC) activity in a murine model of T-ALL induced by an activated allele of NOTCH1 (ICN1). Since pharmacologic inhibition of Cn induces side effects, we aim to identify downstream effectors involved in T-ALL. NFAT (Nuclear Factor of Activated T cells) factors play crucial roles downstream Cn during development and activation of T cells. To address their role in T-ALL, we generated mouse ICN1-induced T-ALL in which NFAT genes can be inactivated either single or in combination following Cre-mediated gene deletion. We demonstrated that (i) NFAT factors are required downstream Cn for LIC activity in T-ALL in vivo (ii) ex vivo NFAT factors deletion alters survival, proliferation and migration of T-ALL (iii) NFAT1, 2 and 4 have a largely redundant function in T-ALL. Moreover, the NFAT-dependant transcriptome allowed to identify important targets (CDKN1A, MAFB) involved in T-ALL survival and proliferation in vitro. Calmodulin-dependant kinases (CaMK) are kinases activated by calcium signaling in T cells. We showed that pharmacologic inhibition of CaMKs in ICN1-induced T-ALL alters survival and proliferation of T-ALL in vitro. Beside, specific inhibition by RNA interference of CaMKIIg and CaMKIId suggests a putative role of these kinases in T-ALL maintenance.

Mon travail de thèse concerne l’étude des leucémies aiguës lymphoblastiques T (LAL-T). Il se décline en deux projets. Le premier, Le Saracatinib affecte les LAL-T humaines en ciblant la kinase LCK dans les cellules riches en radeaux lipidiques, nous a permis d’identifier une nouvelle voie métabolique importante pour la prolifération des cellules leucémiques. Nous avons montré que la kinase LCK est intégrée dans les radeaux lipidiques et impliquée dans la croissance des cellules leucémiques. L’inhibiteur de LCK Saracatinib affecte les cellules de LAL-T in vitro et in vivo en ciblant particulièrement les cellules les plus agressives ayant beaucoup de radeaux lipidiques à leur surface. Ces résultats permettent d’envisager une nouvelle stratégie thérapeutique pour traiter les LAL-T et ont fait l’objet d’une publication parue dans Leukemia en janvier 2018. Le second projet, Les leucémies aiguës lymphoblastiques T produisent l’interleukine 7 de manière autocrine, démontre pour la première fois que pour la plupart des LAL-T, les cellules sont capables de sécréter elles-mêmes la cytokine IL-7. Nous avons complété cette étude par une analyse des mécanismes épigénétiques impliqués dans la régulation de cette sécrétion autocrine. Nos résultats montrent qu’elle est activée par la fixation des facteurs de transcription IRF-1 (Interferon Regulatory Factor 1) et IRF-2 au niveau du promoteur du gène IL 7, lorsque celui-ci est peu méthylé. Grâce à l’inactivation du gène IL-7 dans un de nos modèles de LAL-T, nous avons pu démontrer que la sécrétion autocrine favorise le développement de la leucémie chez la souris xénogreffée en impactant la prise de greffe et le nombre de cellules initiatrices de leucémie. Ainsi, la régulation épigénétique de la sécrétion autocrine d’IL-7 pourrait être impliquée dans les premières étapes de la leucémogenèse des LAL-T. / My PhD work concerns T-cells acute lymphoblastic leukemia (T-ALL) and includes two projects. The first one, Saracatinib impairs maintenance of human T-ALL by targeting the LCK tyrosine kinase in cells displaying high level of lipid rafts, allow us to identify a new signaling pathway important for the proliferation of T-ALL cells. We showed that LCK is localized into lipid rafts and is involved in the growth of T-ALL cells. The LCK inhibitor Saracatinib affects T-ALL cells in vitro and in vivo by targeting the most aggressive cells displaying high level of lipid rafts. These results highlight a new therapeutic strategy to treat T-ALL and were published in Leukemia in January 2018. The second project, T cell acute lymphoblastic leukemia produces autocrine interleukin 7, demonstrated for the first time that T-ALL cells are able to produce IL-7 cytokine. We performed an analysis of epigenetic mechanisms involved in the regulation of this autocrine secretion. Our results showed that when the IL-7 gene promoter is low methylated, Interferon Regulatory Factor 1 (IRF-1) and (IRF-2) transcription factors bind IRF-E sequence and upregulate IL-7 gene transcription. Thanks to IL 7 gene inactivation in one of our T ALL models, we demonstrated that autocrine secretion promotes leukemia development on xenografted mice through increasing engraftment cells capacity and leukemia initiating cells number. Thus, epigenetic regulation of IL-7 autocrine secretion may be involved in the leukemogenesis of T-ALL.

<p>The aims of the studies described in this thesis were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL).</p><p>Leukemic cells from 370 children with newly diagnosed ALL were tested by the Fluorometric Microculture Cytotoxicity Assay to measure their resistance to each of ten standard cytotoxic drugs. In the high-risk group, increased in vitro resistance to each of the drugs dexamethasone, etoposide and doxorubicin was associated with a worse clinical outcome. Combining the results for these drugs yielded a drug resistance score, showing a relative risk of relapse in the most resistant group that was 9.8 times higher than in the most sensitive group. In the standard-risk and intermediate-risk groups, final evaluation must await longer follow-up.</p><p>The new cytotoxic agent CHS 828 was equally active in vitro in samples from children with acute myeloblastic leukemia (AML) and ALL, with 50% cell kill at concentrations achievable in vivo. In AML samples CHS 828 also displayed high frequencies of synergistic interactions with four standard drugs. The well-known differences in clinical outcome between Down´s syndrome (DS) and non-DS children with acute leukemia may partly be explained by our finding of differences in drug resistance at the cellular level.</p><p>Pharmacokinetic studies were performed at the start of induction treatment of ALL. Doxorubicin was assayed by reversed-phase liquid chromatography with fluorometric detection, and vincristine by high performance liquid chromatography with electrochemical detection. Plasma doxorubicin concentrations were measured in 107 children after 23 h of a 24-h infusion. The median steady-state concentration in children 4-6 years old, a group known to have a favorable outcome of treatment, was about 50% higher than in those 1-2 and >6 years old Vincristine pharmacokinetics was evaluated in 98 children. There was no correlation between age and total body clearance or any other pharmacokinetic parameters.</p><p>In vitro testing of cellular drug resistance might be useful in predicting the outcome in high-risk ALL. The further exploration of CHS 828 in childhood leukemia seems warranted. There is no pharmacokinetic rationale for the common practice of administering relatively lower doses of vincristine to adolescents than to younger children.</p>