Orientador: Cármino Antonio de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T09:59:01Z (GMT). No. of bitstreams: 1 Almeida_MariaHelenade_D.pdf: 2031022 bytes, checksum: 4ca81cfeb49f5ad6382582a5201e8b80 (MD5) Previous issue date: 2013 / Resumo: A leucemia mieloide crônica e uma doença clonal, caracterizada por uma translocação recíproca entre os cromossomos 9 e 22. A introdução do tratamento com inibidores de tirosino quinase, administrado por via oral, revolucionou e melhorou drasticamente o tratamento destes pacientes, porem a eficácia deste tratamento esta diretamente relacionada ao uso desta medicação a longo prazo. Pacientes e métodos: 137 pacientes com leucemia mieloide crônica, em uso de inibidores de tirosino quinase, tiveram a sua aderência avaliada em três diferentes momentos: seis meses retrospectivo, seis e doze meses prospectivos. Três diferentes métodos indiretos foram utilizados para a avaliação da aderência: o teste de Morisky, o diário de medicação e o índice de posse da medicação. Resultados: não houve diferença significante quando consideramos os três diferentes momentos em que avaliação foi realizada e o método considerado mais efetivo para a avaliação da aderência foi considerado o índice de posse da medicação (p=0.0001). Considerando as informações acima, o método utilizado para a avaliação da aderência foi o índice de posse da medicação avaliado apos, aproximadamente, 12 meses de analise. A mediana de aderência observada neste estudo foi de 96,5%. Os fatores que influenciaram a aderência foram: nível socioeconômico, qualidade de vida, tempo de uso dos inibidores de tirosino quinase e participação em protocolos de pesquisa clinica. Sendo que pacientes com níveis socioeconômicos mais elevados, com melhor qualidade de vida e que estavam inseridos em protocolos clínicos estavam relacionados com melhores índices de aderência (p=0.01, p=0.007 e p=0.02 respectivamente). Enquanto que pacientes com tempo mais prolongado de uso dos inibidores de tirosino quinase estavam relacionados a piores índices de aderência (p=0.03). Para os pacientes em uso de imatinibe por um período de 24 a 48 meses, em primeira linha de tratamento a aderência foi significativamente melhor em pacientes que apresentavam resposta molecular maior quando comparado ao grupo que não havia atingido este beneficio terapêutico (p=0.04). Apenas 24% da população avaliada mantenve-se completamente aderente no período de avaliação. Conclusões: a manutenção de uma aderência adequado em pacientes com leucemia mieloide crônica em uso de inibidores de tirosino quinase esta relacionado com a obtenção de melhores índices de resposta molecular e consequentemente, melhores resultados terapêuticos. A monitorização continua da aderência nestes pacientes e fundamental para que se atinjam os objetivos x xi terapêuticos. Informações sobre os fatores que podem influenciar a aderência, uma equipe multidisciplinar especializada e treinada para assistir este paciente pode fazer a diferença na obtenção de melhores resultados terapêuticos / Abstract: Chronic myeloid leukemia is a clonal disorder caused by the reciprocal translocation between cromossomes 9 and 22.The introduction of oral tyrosine kinase inhibitors has dramatically revolucioned and improved outcomes in these patients. However, treatment success is directly related to good long-term adherence and questions relating to adherence to prescribed therapy have become an important issue. Patient and Methods: adherence to tyrosine kinase inhibitors therapy was evaluated in 137 chronic myeloid leukemia patients in three different times: six months retrospective, six and twelve month prospectively. Three different methods were used to evaluate adherence: the Morisky Questionnaire, the medication diary and the medication possession ratio. Results: there was no difference between the three different time of evaluation and the medication possession ratio was the most effective method of assessing adherence (p=0.0001); the median adherence was 96.5%. Duration of tyrosine kinase inhibitors treatment was the variable that most impacted adherence (p=0.03); the medication possession ratio was inversely correlated to the duration of therapy. Additionally, participation in clinical trials, better quality of life as reported by patients and higher socioeconomic status were all related to better adherence (p=0.02, p=0.007 and p=0.01, respectively). For patients treated with imatinib for 24 to 48 months (n=22), individuals with major molecular response had a significantly better medication possession ratio than those who failed to achieve MMR (p=0.04). In this group the mean MPR was 87% for the population without apparent molecular response and 96% for those achieving major molecular response, however only 24% of the patients were completely adherent to tirosine kinase inhibitors treatment. Conclusions: higher adherence in chronic myeloid leukemia patients using imatinib is related with best molecular response and therapeutic outcomes. Monitoring the adherence treatment is critical to improving de therapeutics results. A specialized, trained multidisciplinary team that is capable of gaining the trust of patients may persuade them to comply with treatment and detect those who require better monitoring to optimize treatment / Doutorado / Clinica Medica / Doutora em Clínica Médica

BCR-ABL1 transforms hematopoietic stem cells into leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase. Expression of BCR-ABL1 stimulates production of elevated levels of reactive oxygen species (ROS), which induce oxidative DNA damage. CML cells accumulate excessive amounts of ROS-induced DNA damage which can be converted to potentially lethal DNA double strand breaks (DSBs). BCR-ABL1 stimulates enhanced Rad51-mediated DSB repair by the homologous recombination repair (HRR) pathway. In these studies we show BCR-ABL1-transformed cells depend on Rad52-mediated HRR to promote repair of ROS-induced DSBs and that this activity is dependent on Rad52 binding to single-stranded DNA (ssDNA). Our results show in the absence of Rad52, BCR-ABL1-positive hematopoietic cells accumulated elevated numbers of DSBs as detected by enhanced γ--H2AX foci formation compared to cells with wild-type Rad52 which resulted in a decrease in proliferation and expansion of the Rad52-null LSC population. Expression of wild-type Rad52 in Rad52-null cells decreased the accumulation of DSBs and restored expansion of the LSC population. Inhibition of ROS with the antioxidants Vitamin E or N-acetyl cysteine exerted similar effects on the LSC population of Rad52-null cells as restoration of wild-type Rad52. Our studies also show Rad52's ssDNA-binding activity is required for the proliferation of CML cells as evidenced by the accumulation of DSBs and impairment of clonogenic potential in cells in which the Rad52-F79A ssDNA-binding deficient mutant was expressed. Inhibition of Rad52 DNA binding activity by a peptide aptamer targeting Rad52-F79 resulted in a synthetic lethal phenotype in BCR-ABL1-positive cells due to impairment of the Rad52-dependent HRR pathway, as demonstrated by immunofluorescence and HRR repair assays. Altogether we identify Rad52 as a novel target in the treatment of CML, and other BRCA1- and/or BRCA2-deficient cancers, by showing induction of synthetic lethality in proliferating BCR-ABL1-positive cells in which Rad52 ssDNA-binding activity is inhibited. / Biology

Chronic Myelogenous Leukemia (CML) is a hematological disease originated with a chromosomal translocation t(9;22)(q34;q11) in a pluripotent hematopoietic stem cell. CML typically evolves in 3 different clinical phases: chronic and accelerated phases, and blast crisis. Disease progression is associated with the acquisition of secondary mutations that can be of very diverse origins, including inactivation of tumor suppressor genes, as well as inhibition of differentiation, DNA repair and telomere maintenance. While current therapies are very often successful, the remaining issues of resistance and the fact that therapy will not cure CML make it important that new therapy capable of effectively curing it be developed. The early growth response-1 (Egr-1) gene is a zinc-finger transcription factor localized to the human chromosome 5. Egr-1 belongs to a family of early response genes whose expression is rapidly stimulated by growth factors, hormones and neurotransmitters. In addition, Egr-1 is a myeloid differentiation primary response (MYD) gene, and is a positive regulator of terminal myeloid differentiation that potentiates macrophage differentiation. It also has been shown that Egr-1 plays a role in the development, growth control and survival of several cell types, such as T cells, B cells, and neuronal cells in addition to myeloid cells. There is a large amount of evidence consistent with Egr-1 behaving as a tumor suppressor in hematopoietic cells, both in vivo & in vitro, in both humans & mice, making it a prime candidate for a role in CML. In this study we asked if Egr-1 would behave as a tumor suppressor in CML. To answer that we investigated the function of Egr-1 in BCR-ABL driven leukemia using a mouse m bone marrow transplantation (BMT) model. We observe that loss of Egr-1 accelerates the onset of BCR-ABL driven CML. Furthermore, through Facs analysis we showed that most animals developed myeloid leukemia, determined by the observation that the majority of GFP+ cells in the BM were positive for Gr-1 and negative for B220. Interestingly a small cohort of mice developed B-cell acute lymphoid leukemia (B-ALL); this included both WT BCR-ABL and Egr-1 KO BCR-ABL BM-transplanted groups. In addition, we demonstrated that the loss of Egr-1 caused a more aggressive leukemia, which resulted not only in more rapid onset of disease but also greater enlargement of spleen and liver, as well as a tendency to more aggressive lung infiltration of leukemic cells. We also showed that decreased apoptosis, increased proliferation rates and resulting increased viability are consistent with, and probably contribute to, the increased leukemic potential of Egr-1 KO BCR-ABL BM. In addition, we demonstrated that Egr-1 expression was downregulated in BCR-ABL expressing BM cells in vitro, and in spleens of transplanted leukemic mice. Moreover, a very interesting observation, consistent with the rapid onset and aggression of disease, was that the bone marrow of leukemic mice caused by Egr-1 KO BCR-ABL BM transplantation, were enriched with lineage negative BCR-ABL-expressing cells, significantly more so than what was observed in WT BCR-ABL-transplanted mice. That this is also an enrichment of leukemia initiating cells was demonstrated using bone marrow from primary transplantation in a secondary bone marrow transplantation assay. Furthermore, using serial replating assays of colony forming units (CFUs), it was demonstrated that Egr-1 KO BCR-ABL-expressing BM had higher self-renewal ability than WT BCR-ABL-expressing BM, exhibiting an enrichment of primitive stem cells and fewer differentiated cells relative to WT counterparts. Finally, we also analyzed expression of Egr-1 in samples of CML human patients; the results are intriguing but due to small sample size inconclusive. Further inquiry on Egr-1 in CML, including expanding the study of human CML, signaling analysis, interaction of Egr gene family members in leukemia, and gain of function experiments should identify novel players that can impact on the aggressiveness of the disease, predict outcome for currently established therapies, as well identify targets for treatment regimens or adjunct therapy. In addition, these studies can provide a paradigm for understanding how Egr-1 functions as a tumor suppressor for other cancers and types of leukemia, and also delineate pathways that can be activated/inhibited by drugs, including reactivating Egr-1 expression. / Molecular Biology and Genetics

Le gène hybride BCR-ABL1, responsable de la leucémie myéloïde chronique (LMC), code une protéine à activité tyrosine kinase constitutive. Lors d’une étude transcriptomique menée au laboratoire sur des patients résistants secondaires à l’imatinib, les deux gènes codant les protéines des rétrotransposons LINE-1 ont été trouvés sous exprimés d’environ 20 fois lorsque les patients rechutent. Le rôle des transposons n’a jamais été clairement défini, ils assurent certainement une fonction importante puisqu’ils sont conservés au cours de l’évolution et présents chez tous les organismes. Le but de ce travail a été d’étudier l’implication de LINE-1 dans la LMC. La sous-expression de LINE-1 est-elle une conséquence de la présence de BCR-ABL1 ou une cause de son apparition ? Différents groupes ont montré que les rétrotransposons LINE-1 possédent la capacité de réparation des cassures double-brin de l’ADN. Nous avons fait l’hypothèse qu’une diminution de l’expression des gènes codés par les rétrotransposons LINE-1 entraînerait l’instabilité génétique observée dans la LMC. Une étude réalisée chez des patients atteints de LMC et des sujets contrôles a montré une correlation inverse entre l’expression de LINE-1 et celle de l’oncogène BCR-ABL1. Parallèlement, une étude sur des lignées cellulaires leucémiques humaines BCR-ABL positives et négatives a été réalisée. Nous avons recherché le lien qui existe entre l’expression de LINE 1, de BCR-ABL1 et la réparation des cassures double-brin de l’ADN. Nous avons montré d’une part qu’une inhibition de l’expression de BCR-ABL1 induit une augmentation de l’expression des transposons LINE-1 D’autre part, une diminution de l’expression de LINE-1 entraîne une apparition du transcrit BCR-ABL1 dans les cellules BCR-ABL negatives. / BCR-ABL1 fusion gene, responsible of the chronic myeloid leukemia (CML) encodes a constitutively activated tyrosine kinase protein. Expression of both LINE-1 retrotransposon ORFs were found decreased at the time of imatinib resistance in a comparative transcriptional study focused on secondary resistant patients. The role of retrotransposons is unclear. They are conserved through evolution. This project focuses on the involvement of LINE-1 in CML. Is LINE-1 under expression a result of BCR-ABL1 expression or is it at the origin of BCR ABL1? Different groups have shown that LINE-1 retrotransposons were able to repair DNA double strands breaks. We suggest that LINE-1 under expression could be responsible of genetic instability observed in CML. We show in a study on CML patients and healthy subjects that LINE-1 expression is inverse correlated to BCR-ABL1 expression. Moreover, study on BCR-ABL+ and BCR-ABL- human leukemic cell lines was carried on. First, we show that decrease of BCR-ABL1 expression induces increase of LINE-1 expression. Then that decrease of LINE-1 expression generates BCR-ABL1 transcript in BCR-ABL negatives cell lines.

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.

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.

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa, resultante da expansão clonal da célula-tronco hematopoética pluripotente. A fisiopatologia da LMC está associada a uma translocação entre os braços longos dos cromossomos 9 e 22, o que promove o aparecimento do neogene bcr-abl, cujo gene codifica uma proteína denominada Bcr-Abl. A oncoproteína Bcr-Abl possui atividade tirosina-quinase constitutiva que é a responsável pelo fenótipo maligno da célula, incluindo resistência à apoptose. O tratamento da LMC pode ser realizado com hidroxiuréia, IFN- associado à citarabina, inibidores de TK (mesilato de imatinibe e dasatinibe) e transplante de medula óssea. O tratamento de escolha para pacientes com LMC na fase crônica é o inibidor de tirosina-quinase mesilato de imatinibe e para os refratários utiliza-se o dasatinibe. Apesar do conhecimento acerca do mecanismo de ação dos inibidores de TK, pouco se sabe sobre seu efeito na maquinaria apoptótica. Sendo assim, no presente trabalho foi detectada a expressão dos genes e proteínas anti- (A1, Bcl-2, Bcl-Xl, Bcl-W, C-Flip, Ciap-1, Ciap-2 e Mcl-1) e pró-apoptóticos (Bad, Bak, Bax, Bcl-Xs, Bid, Bik, Bimel, Bmf, Bok, Fas, Fasl, Noxa e Puma) em células mononucleares de 32 indivíduos saudáveis e 26 pacientes com LMC antes e após 12 meses da terapia com mesilato de imatinibe e dasatinibe. Dentre os 26 pacientes avaliados, 13 eram do sexo feminino e 13 do sexo masculino, três eram negros, um amarelo e 22 brancos, com idade média de 48 anos (faixa etária de 25 a 77 anos). O grupo controle foi composto por 32 indivíduos, 16 do sexo feminino e 16 do sexo masculino, 26 eram brancos, quatro negros e dois amarelos, com idade média de 45 anos (idade de 23 a 77 anos). O isolamento das células mononucleares foi realizado pelo método de Ficoll-Hypaque, a determinação da expressão gênica por PCR em tempo real e a protéica por western-blot. Os resultados foram expressos em unidade relativa de expressão (U.R.E.), comparados entre os diferentes grupos (controle e pacientes pré- e pós-tratamento), associados à resposta aos medicamentos e correlacionados ao índice de prognóstico de SOKAL. Na comparação dos dados de expressão gênica entre pacientes e controles, verificou-se que os pacientes apresentaram maior expressão dos genes bcl-xL, c-flip, mcl-1 e fas e níveis reduzidos de bik. O mesilato de imatinibe modulou significativamente a transcrição dos genes bcl-xL, bok, mcl-1 e noxa, enquanto que o dasatinibe agiu sobre a expressão dos genes a1, bmf, c-flip, ciap-1, ciap-2 e mcl-1. Os pacientes refratários ao mesilato de imatinibe apresentaram níveis elevados de expressão de a1 e c-flip e reduzida expressão de bcl-2, ciap-2, bak, bax, bid e fasl em relação aos pacientes em remissão. A expressão protéica refletiu os dados da quantificação do RNAm dos genes. Os dados da presente investigação indicam que as células mononucleares dos pacientes com LMC apresentam desregulação do processo de apoptose celular. Essa alteração pode ser parcialmente associada ao fenótipo de resistência das células leucêmicas Bcr-Abl+ à apoptose e ausência de resposta aos inibidores de TK. Os dados revelam ainda que os inibidores de tirosina-quinase interferem na transcrição e tradução das moléculas envolvidas na regulação do processo de apoptose. / Chronic myeloid leukemia (CML) is a myeloproliferative disease resultant of a clonal expansion of pluripotent hematopoietic stem cells. The CML physiopathology is associated with a translocation between chromosomes 9 and 22 long arms, promoting the formation of a bcr-abl neogene, which codifies the Bcr-Abl protein. The Bcr-Abl oncoprotein presents tyrosine-kinase activity that is responsible for the malign phenotype which includes apoptosis resistance. CML treatment may be performed with hydroxyurea, IFN- plus cytarabine, tyrosine-kinase inhibitors (imatinib mesylate and dasatinib) and bone marrow transplantation. The standard treatment for CML patients in chronic phase is the tyrosine-kinase inhibitor imatinib mesylate (IM) and for IM-refractory patients dasatinibe is employed. Despite of the knowledge related to the mechanism of action of tyrosine-kinase inhibitors, little is known about its effects on the apoptosis machinery. In this work we characterized both the mRNA and protein patterns of expression of the anti- (A1, Bcl-2, Bcl-Xl, Bcl-W, C-Flip, Ciap-1, Ciap-2 e Mcl-1) and pro-apoptotic (Bad, Bak, Bax, Bcl-Xs, Bid, Bik, Bimel, Bmf, Bok, Fas, Fasl, Noxa e Puma) regulators in peripheral blood mononuclear cells (PBMC) from 32 healthy individual and 26 CML patients before and after 12 months of imatinib mesylate and dasatinibe therapy. Thirteen patients were female and thirteen were male, three were black, one was japanese and 22 were white, the mean age was 48 years (varying from 25 to 77 years). The control group was composed of 32 individuals, 16 were female and 16 were male, 26 were white, four black and two japanese, the mean age was 45 years (varying from 23 to 77 years). PBMC isolation was performed by Ficoll-Hypaque, gene expression was assessed by real time PCR and protein expression was carried out by western-blot methodology. Results were given by the relative expression, after comparison between the different groups (control and patients before and after treatment), associated with drug response and related to Sokal index. The comparison of gene expression profiles between patients and controls showed that CML patients present increased levels of bcl-xL, c-flip, mcl-1 and fas expression and reduced levels of bik gene expression. The imatinib mesylate significantly modulated the transcription of bcl-xL, bok, mcl-1 and noxa, whereas dasatinib affected the expression of a1, bmf, c-flip, ciap-1, ciap-2 and mcl-1. MI-refractory patients present higher levels of a1 and c-flip and lower levels of bcl-2, ciap-2, bak, bax, bid and fasl when compared to patients who achieved complete cytogenetic response. Overall, the patterns of protein expression agree with the profiles of mRNA expression. Taken together, the results described in this investigation indicate that PBMC from CML patients present deregulation in cell death pathways. These alterations could partly account for the apoptosis resistance phenotype observed in Bcr-Abl+ leukemic cells and for lack of response to tyrosine kinase inhibitors. Furthermore, our data also reveal that tyrosine kinase inhibitors interfere in the transcription and translation of molecules that regulate the apoptosis process.

Na leucemia mielóide crônica (LMC) a proteína Bcr-Abl possui atividade tirosina-quinase constitutivamente ativada, induzindo a mieloproliferação e a resistência das células à apoptose. A maioria dos pacientes na fase crônica da LMC tratados com inibidores de tirosina-quinase (TKIs), como o mesilato de imatinibe, apresenta remissão citogenética completa da doença, mas uma parcela desses pacientes tem se mostrado resistente à terapia. A fisiopatologia da LMC e os mecanismos celulares e moleculares envolvidos na resistência à terapia com TKIs são diversos e precisam ser melhor estudados. Nesse contexto, o objetivo do presente estudo foi quantificar os níveis de expressão do gene DIDO, incluindo suas diferentes isoformas (DIDO 1, 2 e 3) e promotores (DIDO PP e PD) em controles e em pacientes com LMC nas diferentes fases da doença, tratados ou não com TKIs, bem como em linhagens celulares BCR-ABL1+ sensíveis (S) e resistentes (R) ao mesilato de imatinibe (MI). A literatura relata que DIDO 1 participa do processo de apoptose e que alterações na expressão de DIDO 2 e DIDO 3 podem estar associadas com o desenvolvimento de neoplasias mielóides. Dessa forma, foram estudados 60 pacientes com LMC e 57 controles, assim como as linhagens celulares HL-60, HL-60.Bcr-Abl+, LAMA 84 S, LAMA 84 R, KCL 22 S e KCL 22 R. Foram separadas as células mononucleares de sangue periférico dos pacientes e controles, com posterior extração de RNA e síntese de cDNA, que foi então empregado nas reações de PCR em tempo real (qPCR) para quantificação das expressões gênicas de DIDO 1, 2, 3, PP, PD e ORF. As linhagens celulares foram tratadas por 4h com TKIs e então a expressão das diferentes isoformas de DIDO foi também quantificada por qPCR. A avaliação da expressão proteica de Bcr-Abl, c-Abl e proteínas fosforiladas nas linhagens foi realizada por Western-blotting. A expressão de DIDO 1 e 2 foi maior nos pacientes nas fases avançadas da LMC e nos pacientes na fase crônica da doença tratados com TKIs (mesilato de imatinibe ou dasatinibe) do que nos controles. Os pacientes na fase crônica da LMC tratados com MI expressaram mais DIDO 1 e 3 do que os pacientes na fase crônica sem tratamento. Houve uma correlação positiva entre expressão de BCR-ABL1 e de DIDO 2 e entre índice de Sokal dos pacientes e expressão de DIDO 2. Na linhagem HL60.Bcr-Abl+, a expressão de proteínas fosforiladas reduziu após tratamento de 4h com TKIs, mas não houve alteração na expressão gênica de DIDO. Nas linhagens celulares S e R, houve aumento da expressão de DIDO 1 após o tratamento de 4h com MI. Conclui-se, portanto, que as diferentes isoformas de DIDO parecem exercer funções distintas na leucemia mielóide crônica; que o tratamento de pacientes e linhagens BCR-ABL1 positivas com inibidores de tirosina-quinase aumenta expressão de DIDO 1; e que a expressão de DIDO 2 correlaciona-se positivamente à expressão de BCR-ABL1 e ao índice de Sokal dos pacientes. / In chronic myeloid leukemia (CML) the Bcr-Abl protein has constitutively activated tyrosine kinase activity, that induces to myeloproliferation and apoptosis resistance of the cells. Most patients in chronic phase of CML treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate have a complete cytogenetic remission, but a portion of these patients have been shown to be resistant to therapy. The pathophysiology of CML and the cellular and molecular mechanisms involved in resistance to TKIs therapy are diverse and require further study. In this sense, the aim of this study was to quantify the expression levels of the DIDO gene, including its isoforms (DIDO 1, 2 and 3) and promoters (DIDO PP and PD) in controls and in patients with CML in different phases of the disease treated or not treated with TKIs, as well as in cell lines BCR-ABL1+ sensitive (S) and resistant (R) to imatinib mesylate (IM). The literature reports that DIDO 1 is involved in apoptosis process and that alterations of DIDO 2 and DIDO 3 expression may be associated with the development of myeloid neoplasms. Thus, 60 CML patients, 57 control individuals and the cell lines HL-60, HL-60.Bcr-Abl+, LAMA 84 S, LAMA 84 R, KCL 22 S and KCL 22 R were studied. Peripheral blood mononuclear cells of patients and controls were isolated and RNA extraction and cDNA synthesis were performed. The cDNA samples were used in Real-Time PCR reactions (qPCR) to quantify the DIDO 1, 2, 3, PP, PD and ORF gene expression. The cell lines were treated during 4h with TKIs and then the expression of DIDO different isoforms was also quantified by qPCR. The assessment of protein expression of Bcr-Abl, c-Abl and phosphorylated proteins in this cell lines was performed by Western blotting. The DIDO 1 and 2 expression was higher in advanced phases patients and in chronic phase patients CML treated with TKIs (imatinib mesylate and dasatinib) than in controls. Chronic phase CML Patients treated with IM expressed more DIDO 1 and 3 than chronic phase untreated patients. There was a positive correlation between BCR-ABL1 expression and DIDO 2 expression and between Sokal score prognostic and DIDO 2 expression. In HL60.Bcr-Abl+ cells the expression of phosphorylated proteins was lower after treatment during 4h with TKIs, but there was no change in DIDO gene expression. There were an increase of DIDO 1 expression in all S and R cell lines after treatment during 4h with IM. Therefore we conclude that the DIDO different isoforms may have different functions in chronic myeloid leukemia; the treatment of patients and BCR-ABL1+ cell lines with TKIs increases DIDO 1 expression; and that the DIDO 2 expression is positively correlated to the BCR-ABL1 expression and Sokal score prognostic of CML patients.

BMI1 est une protéine appartenant à la famille des polycombs impliquée dans la régulation épigénétique de la transcription. Il a été montré que cette protéine est essentielle à la régulation de la prolifération, de la sénescence et du métabolisme ainsi qu’à l’auto-Renouvellement des cellules souches hématopoïétiques et cancéreuses. Ce répresseur transcriptionnel au fort potentiel oncogénique est retrouvé surexprimé dans de nombreux types de cancer ; dans le cas de la Leucémie Myéloïde Chronique (LMC) le niveau d’expression de BMI1 augmente avec l’aggravation de la pathologie. Cependant, les voies de signalisation impliquées dans sa surexpression et le rôle qu’il joue au sein de cette maladie demeurent méconnus. En réprimant l’expression de BMI1 par ARN interférence nous avons pu mettre en évidence que ce polycomb était essentiel à la prolifération cellulaire ainsi qu’au potentiel clonogénique des cellules de LMC. Nous avons également démontré pour la première fois que BMI1 soutenait la croissance tumorale à travers la répression d’un processus autophagique délétère pour la cellule cancéreuse. Une approche transcriptomique nous a permis d’identifier la cible transcriptionnelle impliquée dans ce processus, la Cycline G2. Nous avons, pour finir, trouvé une molécule, via une approche bioinformatique, capable de réinduire l’expression de la Cycline G2 dans les cellules de LMC, l’alexidine dihydrochloride. Cette molécule induit une forte autophagie dans les cellules cancéreuses ainsi que de l’apoptose. Elle s’est également montrée capable de resensibiliser à l’imatinib (un inhibiteur de BCR-ABL) une lignée pourtant résistante. / The polycomb protein Bmi1 is a major epigenetic regulator. It has been shown that this protein is essential for the regulation of cell proliferation, senescence and metabolism but also self-Renewal of hematopoïetic and cancer stem cells. This transcriptional repressor, with a strong oncogenic potential, is overexpressed in many types of cancer. In case of Chronic Myeloid Leukemia (CML) the expression level of BMI1 is associated with worsening prognosis. However, the signaling pathways involved in its overexpression and its role in this disease remains unclear. By using RNAi to repress BMI1 expression we highlighted that this polycomb was essential for proliferation and clonogenicity of CML cells. We also demonstrated, for the first time, that BMI1 supported tumor growth through repression of deleterious cancer cell autophagy. A transcriptomic approach allowed us to identify a transcriptional target involved in this process: the Cyclin G2. Through a bioinformatic approach, we finally found a molecule capable of expression re-Induction of Cyclin G2 in CML cells : alexidine dihydrochloride. This molecule induced a high level of autophagy as well as apopotosis in cancer cells. It had also been able to re-Sensitize to imatinib a resistant cell line. In conclusion, our results revealed a new role for the polycomb BMI1 in supporting the CML pathology. Moreover, our work allowed the identification of two new approaches for therapeutically targeting this oncogene functions.

<p>The aims of the studies described in the thesis were to investigate different treatment strategies in chronic myeloid leukemia (CML) patients. Furthermore, activity of imatinib was investigated by <i>in vitro</i> cytotoxicity assay, and the gene expression pattern in interferon treated patients.</p><p>In a randomized prospective national study, we examined the influence of busulphan (n=89) versus hydroxyurea (n=90) treatment on time to blast crisis, and survival. There was no significant difference in survival between hydroxyurea and busulphan treated patients; median survival was 3.5 and 3.2 years, respectively. The 26 patients who underwent allogeneic stem cell transplantation had a significantly longer median survival (4.7 years) than those who were not transplanted.</p><p>We investigated the feasibility of mobilizing Philadelphia chromosome negative blood stem cells with intensive chemotherapy and lenograstim in CML patients. Twenty-three patients (62%) were successfully mobilized. Twenty-one of these patients underwent autologous stem cell transplantation later on, with a 5-year overall survival at 68%.</p><p>Fluorometric Microculture Cytotoxicity Assay was used to analyze 32 tumor cell samples from CML patients, (26 chronic phase and 6 blast crisis). Imatinib showed a higher <i>in vitro</i> activity and more positive drug interactions in cells from blast crisis than from chronic phase. Interferon, daunorubicin and arsenic trioxide had the greatest benefit from a combination with imatinib.</p><p>Microarray-based gene expression analyses were performed on diagnostic CML samples prior to interferon treatment. We identified six genes that were differentially expressed in responders and non-responders to interferon. It might prove possible to use gene expression analysis to predict future response to interferon.</p>