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

Notch receptors and ligands are single-pass transmembrane proteins which play important roles in cell-cell communication. Notch in ‘harmony’ with other signaling pathways regulate the entire diversity of metazoan life (Artavanis-Tsakonas & Muskavitch, 2010). These signaling pathways also play key roles in regulatingseveral developmental processes. Given the importance of Notch signaling in various developmental decisions, it is not surprising that aberrant gain or loss-of-function of Notch pathway leads to several human diseases including cancer (Ranganathan et al, 2011). Notch signaling has also been implicated in various human cancers, most notably in T-cell acute lymphoblastic leukemia (T-ALL) (Weng et al, 2004). In view of the importance of Notch signaling in cancers, therapeutic molecules targeting this pathway are making their way into clinical trials (Rizzo et al, 2008). This underscores the importance of understanding the mechanism of Notch receptor activation in normal and patho-physiological conditions. In this thesis, antibodies against different domains of human Notch1 receptor have been used as tools to understand the mechanism of receptor activation. This work has provided insights into the role of Notch1 extracellular domain in ligand-dependent receptor activation. Further, the mechanism of ligand-independent receptor activation in T-ALL associated mutant Notch1 has also been investigated. This understanding of ligand-dependent and independent receptor activation facilitated development of mechanistic inhibitors of Notch signaling for therapeutic targeting of the cancer stem cells (CSCs) across the pectrum of cancers. The thesis is divived into two parts. Part-I focuses on understanding the role of Notch1 extracellular domain in receptor-ligand interactions using antibodies as a tool. In part-II, implications of these antibodies in therapeutic targeting of CSCs has been investigated. Part-I Unfolding the Mechanism of Notch1 Receptor Activation The extracellular domain of Notch1 receptor consists of 36 EGF-like repeats that contribute to ligand binding (Kopan & Ilagan, 2009). Despite extensive studies on the downstream consequences of Notch signaling, the initial events of ligandreceptor interactions have not been clearly elucidated. In the absence of structural insights into the receptor-ligand interactions, it was important to decipher the roles of various receptor domains in ligand-binding and consequent signaling. In this study, antibodies have been employed as tools for in-depth analyses of Notch receptorligand, interactions. Studies in Drosophila Notch receptor suggest that EGF-like repeats 11-12 are necessary and sufficient for ligand binding (Rebay et al, 1991). However, the role of these repeats in human Notch1 receptor-ligand interaction(s) was not clearly elucidated. Antibodies were generated against Notch1 EGF-like repeats 11-15. Further, these antibodies were characterized for their specificity for Notch1 receptor in various ligand-binding and signaling assays. The results suggest that the monoclonal antibodies (MAbs) against EGF-like repeats 11-12 were more potent inhibitors of ligand-binding compared to the antibodies against EGF-like repeats 13-15. As a part of these investigations, the Notch ligands Jagged1 and Jagged2, Delta-like1 and Delta-like4 were purified and characterized in various assays. Ability of these ligands to interact with Notch1 EGF-like repeat 11-15 was determined using Surface Plasmon Resonance. The Jagged family of ligands demonstrated higher affinity for this recept or fragment when compared to the Delta family of ligands. The relatively low affinities (μM) of all the ligands suggested possibile involvement of other EGF-like repeats in ligand-binding. This was further investigated using antibodies against other EGF-like repeats of Notch1. In Drosophila Notch EGF-like repeats 24-29 have been implicated in the ligand-dependent gain-of-function phenotype, suggesting a plausible involvement of this region in receptor activation (Pei & Baker, 2008). Therefore, role of human Notch1 EGF-like repeats 21-30 in ligand-binding and signaling was investigated. These EGF-like repeats demonstrated specific interaction with the ligand-binding domain (EGF-like repeats 11-15). This suggested that in the absence of the ligand, these inter-domain interactions keep the receptor in an auto-inhibited conformation. Further, ligand binding to EGF-like repeats 11-15 dissociated pre-formed interdomain interactions. These results suggested that, the binding of ligand to EGF-like repeat 11-12 overcomes the negative constraint imposed by the intra-domain interactions which might lead to receptor activation. Next, to understand the role of EGF-like repeats 21-30 in ligand binding, polyclonal antibodies were generated against the same and extensively characterized in various solid-phase and cell-based assays. These antibodies demonstrated partial inhibition of ligand-binding. Further, using immunoaffinity purified antibodies it was demonstrated that antibodies against EGF-like repeats 25-26 were most potent inhibitors of ligand-binding compared to antibodies against EGF-like repeats 21-24 and 27-30. These results provided novel insights into Notch1 receptor activation. The model proposed on the basis of these results suggested that ligand-binding to EGF-like repeats 11-12 competes with the inter-domain interaction, in turn dissociating EGF-like repeats 21-30 from the ligandbinding domain. It emerged that this altered conformation of the receptor creates a secondary ligand-binding site at EFG-like repeats 25-26. Overall these results provided novel insight into the mechanism of Notch receptor-ligand interaction(s). Part-II Implication in Cancer Stem Cell Targeting Recent studies have suggested existence of the CSC population in various cancers (Clevers, 2011). Notch signaling plays an important role in maintenance of these CSCs (Pannuti et al, 2010). Thus, targeting Notch signaling may provide a potential therapeutic tool for CSC targeting. Several studies have indicated that Notch1 receptor and ligands are overexpressed in breast cancer cells compared to the normal breast epithelium (Mittal et al, 2009; Reedijk et al, 2005; Reedijk et al, 2008). Moreover, it has been suggested that Notch1 signaling plays a key role in breast carcinogenesis (Stylianou et al, 2006). Monoclonal antibodies (MAbs) were used as mechanistic inhibitors of aberrant Notch1 signaling for therapeutic targeting of CSCs. One such antibody, MAb 602.101, against Notch1 ligand-binding domain (EGF-like repeat 11-12) inhibited proliferation and depleted breast CSCs. This MAb also modulated genes associated with stemness and epithelial to mesenchymal transition (EMT). Furthermore, MAb 602.101 irreversibly inhibited the sphere-forming potential of breast cancer cells by modulating long-term self renewing capacity of breast CSCs. Inhibition of Notch1 signaling by the MAb also depleted the chemoresistant CD44Hi/CD24Low sub-population in breast cancer cells. Interestingly, antibody treatment led to elevated expression of genes associated with myoepithelial lineage, which suggested that inhibition of Notch1 signaling might induce a differentiation program leading to reduction in the CSC population. This study demonstrated the importance of Notch1 signaling in CSCs and effectiveness of antibodies as a tool for specific targeting of individual Notch receptors in cancer therapeutics. While aberrant expression of receptors and ligands leads to breast cancer (Reedijk et al, 2005), gain-of-function mutations are associated with 40-50% of TALL\ patients (Weng et al, 2004). These mutations lead to ligand-independent receptor activation (Malecki et al, 2006). Despite several attempts of successful antibodymediated therapeutic targeting of Notch1 (Aste-Amézaga et al, 2010; Wu et al, 2010), specific antibodies recognizing T-ALL associated mutant Notch1 remains elusive. Using homology modeling, the mutation induced conformational change in T-ALL associated mutant Notch1 was predicted. These results suggested that mutation led to conformational changes in the Notch1 negative regulatory region (NRR) This conformation change might result in the constitutive activation of Notch1 signaling leading to pathogenesis. Next, MAbs were generated against the wild-type Notch1 NRR and characterized in flow-cytometry based assays for identification of conformation specific antibodies. These antibodies were classified as either wild-type specific, mutant specific or unbiased to receptor conformations. One such mutant specific MAb 604.107 demonstrated higher binding to mutant Notch1 in flowcytometer and SPR based experiments. This MAb also demonstrated specific inhibition of T-ALL associated mutant Notch1 signaling without affecting the wildtype signaling. Moreover, antibody treatment also inhibited proliferation and depleted leukemia initiating sub-population in patient derived T-ALL cells. Taken together, this study provides a novel tool for specific targeting of mutant Notch1 receptors in TALL. CSCs are inherently chemo-resistant and lead to tumor relapse (Chen et al, 2012). Recent studies have demonstrated a strong correlation between Notch1 signaling in lung CSCs and chemotherapy resistance (Hassan et al, 2013). In this study, Notch1 heterogeneity in solid tumors viz. breast and colon cancers was investigated. Using the antibodies generated previously in this study, Notch1High and Notch1Low sub-populations from MDA-MB-231 (breast cancer) and HCT-116 (colon cancer) cell lines were flow-sorted. It was demonstrated that the Notch1High subpopulation represented the sphere-forming CSCs in breast and colon cancer. The Notch1High sub-population also demonstrated chemo-resistant properties and expressed higher level of EMT and stemness markers. These results suggested explicit involvement of Notch1 signaling in EMT and maintenance of CSCs subpopulation in these cancers. The anti-Notch1 MAb also inhibited proliferation of the chemo-resistant Notch1High sub-population. Further, treatment with MAb inhibited expression of ABCC1 transporters in these drug-resistant cells leading to augmentation of chemotherapeutic response. Using mouse xenograft assays, it was demonstrated that Notch1 signaling plays an important role in the maintenacne of tumor-initiating sub-population in breast and colon cancer cells. Prior exposure of breast and colon cancer cells to MAb inhibited the tumor forming potential of these cells in xenotransplantation assays. Treatment with MAb alone or in combination with chemotherapy led to regression of pre-formed tumors in breast and colon xenograft models. These results demonstrated existence of Notch1 heterogeneity in breast and colon cancer cells and emphasised the importance of targeting Notch1 signaling to overcome drug-resistance in these cancers. The results described above have provided important insights into Notch1 receptor activation and this understanding was translated into therapeutic targeting of CSCs. This “proof-of-principle” demonstration has significant mechanistic and applied implications in Notch and cancer biology.