Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 18, 2009). Includes bibliographical references.

Although cure rates have significantly improved for children with T-cell acute lymphoblastic leukemia (T-ALL), 20-30% undergo induction failure or relapse with most succumbing to disease. Leukemia-initiating cells (L-ICs) are hypothesized to be resistant to conventional chemotherapy and radiation and are thereby responsible for disease recurrence. Using an in vivo limiting dilution assay, we previously showed that the murine T-ALL L-IC is quite rare, with only 0.003-0.05% of cells capable of initiating disease, and demonstrated that the L-IC is a subset of the leukemic DN3 thymic progenitor population. Work described in this thesis validates the L-IC assay using two transplantation methods to rule out effects of homing and/or microenvironment on T-ALL L-IC survival and maintenance. Using this assay, we demonstrate that sustained Notch1 signaling is required for T-ALL initiation in vivo and show that treatment with a Notch1 inhibitor reduces or in some cases eliminates the L-IC population. We further analyze the effects of inhibiting c-Myc, a Notch1-regulated gene, on L-IC frequency and uncover an essential role for c-Myc in L-IC survival and expansion. Suppressing c-Myc by using specific shRNAs or a c-Myc inhibitor reduces the L-IC population and interferes with leukemia initiation. Together, these findings reveal a critical role of the Notch1-c-Myc pathway in T-ALL initiation and suggest that therapeutics targeted at this pathway could be used to treat and/or prevent disease relapse in patients.

Orientador: José Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T05:50:44Z (GMT). No. of bitstreams: 1 Jotta_PatriciaYoshioka_D.pdf: 8922035 bytes, checksum: 3734371a320410ba431d6e6ce6579e55 (MD5) Previous issue date: 2012 / Resumo: A leucemia linfóide aguda (LLA) é o câncer mais frequente na infância, e destas, 15% são do tipo T (LLA-T). A hiperativação da via PI3K/Akt tem sido amplamente descrita em tumores e em linhagens celulares de LLA-T. PTEN é o principal regulador negativo dessa via e frequentemente encontra-se inativado em cânceres humanos. Com frequência, pacientes com LLA-T apresentam mutações ativadoras de NOTCH1. NOTCH1 pode regular transcricionalmente PTEN, contudo ainda não está claro como as mutações ativadoras de NOTCH1 influenciariam a expressão de PTEN nas LLA-T. Nós encontramos uma ocorrência de 11 (17,7%) mutações no éxon 7 do PTEN em 62 casos de LLA-T estudados consecutivamente. Contudo, nenhuma mutação foi encontrada na análise de 71 casos de LLA-B derivada. A maioria das mutações de PTEN apresentavam inserções/deleções de mais de 3 nucleotídeos. Não encontramos associação entre mutações em PTEN e o gênero, a idade e a contagem de glóbulos brancos ao diagnóstico. Pacientes com alterações no PTEN apresentaram uma tendência a pior sobrevida global (OS, p=0.07). Dentre os pacientes de LLA-T classificados como alto risco (n=56), aqueles possuindo anormalidades no PTEN mostraram-se associados significativamente a menor OS (p=0.019) e sobrevida livre de leucemia (LFS 47% vs 76%; p=0.045). As curvas de LFS foram significativamente diferentes (p=0.003), mesmo considerando apenas pacientes que atingiram a remissão no dia 28 do tratamento para a análise. Nosso estudo também mostrou que pacientes com mutações em NOTCH1 apresentavam aumento na transcrição de MYC e menor expressão de PTEN mRNA comparados a pacientes com NOTCH1 selvagem. Nós recentemente demonstramos que células de LLA-T apresentavam fosforilação de PTEN mediada por CK2, resultando na estabilização e consequentemente inativação da proteína PTEN. Corroborando ao estudo anterior, os casos de LLA-T analisados, independente do status de mutação em NOTCH1, expressam níveis significativamente mais altos de proteína PTEN do que controles normais. Para avaliar o impacto da regulação transcricional de NOTCH e a inativação postranscricional por CK2 de PTEN, nós tratamos as células de LLA-T com inibidores de gamma-secretase (DAPT e de CK2 (DRB/TBB). Nosso estudo enfatiza a relevância biológica e clínica da regulação do PTEN em LLA-T. E sugerimos o uso combinado de inibidores de gamma-secretase e CK2 devem possuir potencial terapêutico nas LLA-T / Abstract: T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of pediatric ALL. Patients with T-ALL are at increased risk of relapse compared with children treated for B-cell precursor ALL. Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. PTEN is the main negative regulator of the PI3K/Akt survival pathway. T-ALL patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. We report that PTEN exon 7 mutations occurred in 11 (17.7%) out of 62 consecutive pediatric T-cell acute lymphoblastic leukemia (T-ALL) but in none of 71 precursor B-ALL patients. Most PTEN mutations were insertions/deletions of more than 3 nucleotides. No associations were found between PTEN mutation and age, gender, WBC at diagnosis, early response to therapy and remission rate. Patients with PTEN mutation (n=11) had a tendency toward worse overall survival (OS, p=0.07). Remarkably, PTEN mutations were significantly associated with lower OS (p=0.019) and leukemia-free survival (LFS 47% vs 76%, p=0.045) within patients classified in the high risk group (n=56). LFS curves were significantly different (p=0.003) even if only patients who reached remission on day 28 were considered for analysis. We compared patients with or without NOTCH1mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of NOTCH transcriptional and CK2 post-translational inactivation of PTEN, we treated TALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL. And emphasize the biological and clinical relevance of PTEN regulation in pediatric T-ALL / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular