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Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

O timo é o órgão linfóide primário responsável pela maturação dos linfócitos T onde se observam estruturas e células especializadas. A sinalização intra-tímica durante a maturação de linfócitos T não é bem esclarecida. Entre outras, a via de sinalização Notch, composta por receptores (Notch 1 a 4) e ligantes (DLL1, 3 e 4, Jagged 1 e 2), pode regular este processo. Neste trabalho temos como objetivo avaliar a expressão gênica e proteica de receptores e ligantes Notch nas diferentes fases de maturação de linfócitos T, linfócitos T reguladores naturais (nTreg) e células dendriticas tímicas (tDC). Para tanto, timos de 10 crianças submetidas à cirurgia cardíaca corretiva foram manipulados e as populações CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4-CD8+, nTreg e tDC foram purificadas por citometria de fluxo. O RNA total foi isolado e os genes NOTCH1, 2 e 3, DLL1 e 4, JAG1 e 2, FOXP3 foram amplificados por RT-PCR. Alguns fragmentos de tecido foram avaliados por imunohistoquímica, quanto a expressão dos receptres Notch 1, 2, 3 e 4 e dos ligantes, DLL1 e 4, Jagged 1 e 2 em timócitos totais, células FOXP3+ e células S100+ em cada região tímica. Todos os genes de receptores e ligantes Notch foram expressos nas populações estudadas. Na população CD4-CD8- o gene NOTCH1 é mais expresso em comparação as outras populações de timócitos imaturos. Na população CD4+CD8+ o gene NOTCH2 é menos expresso, e o gene JAG2 mais expresso quando comparados à população CD4+CD8-. Os demais genes de receptores ligantes Notch são expressos de maneira similar entre as populações de timócitos. A avaliação relativa dos genes Notch nas populações de timócitos mostrou uma maior expressão do gene DLL1 na população CD4+CD8- e JAG1 na população CD4-CD8+ em comparação à CD4-CD8-. A expressão dos receptores e ligantes Notch no tecido mostrou ser homogênea entre as regiões. As nTreg expressam o gene do ligante JAG1 em maior nível entre os avaliados. A expressão gênica relativa das nTreg mostrou uma maior expressão de NOTCH1, NOTCH2, DLL4 e JAG1 e menor expressão de NOTCH3, DLL1 e JAG2 em relação as CD4-CD8-. Quando a relação utilizou a população CD4+CD8- observamos que as nTreg expressam mais os genes NOCTH1, NOCTH2, NOTCH3, DLL4 e JAG1, e em níveis similares DLL1 e JAG2. A avaliação histológica mostrou uma maior expressão de DLL4 em nTreg em comparação às demais proteínas avaliadas, e uma distribuição homogênea entre as regiões com exceção de Notch3 e Jagged2. As tDC mostraram uma maior expressão do gene JAG1 em comparação aos demais, e uma maior expressão da proteína DLL4. A expressão do receptor Notch2 em tDC difere entre as regiões tímicas. Em conjunto, nossos resultados mostraram que os receptores e ligantes Notch são expressos de forma gênica e proteica em todos os estágios de maturação de linfócitos T, nTreg e tDC do timo humano, com algumas variações quanto ao nível de expressão e distribuição no timo. Dados que se assemelham às avaliações murinas, porém com pontos a serem discutidos. Nossa inédita avaliação em nTreg propõem uma nova abordagem ao envolvimento da via Notch em sua maturação e a avaliação das tDCs sugere sua participação direta via Notch na maturação dos timócitos humanos / The thymus is a primary lymphoid organ responsible of maturing T lymphocytes, where specialized structures and cells are observed. The intrathymic signaling during lymphocytes maturation is unclear. Among them, Notch signaling pathway, which comprises in receptors (Notch1-4) and ligands (DLL1, 2 and 3, Jaaged1 and 2), may regulate this process. In this work our aim is to evaluate the expression of Notch receptors and ligands in different phases of T lymphocytes maturation, natural T regulatory cells (nTreg), and thymic dendritic cells (tDC). For this purpose, thymuses from 10 children who underwent corrective cardiac surgery were manipulated and populations CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4- CD8+, nTreg and tDC were sorted by flow cytometry. Total RNA was purified and genes NOTCH1, 2 and 3, DLL1 and 4, JAG1 and 2, FOXP3 were amplified by RT-PCR. Some thymic fragments were evaluated by immunohistochemistry and screened for expression of Notch 1, 2, 3 and 4 receptors, DLL1 and 4, Jagged and e 2 ligands in total thymocytes, FOXP3+ cells and S100+ cells in each thymic region. All Notch receptors and ligands genes were expressed in studied populations. In CD4-CD8- subset NOTCH1 gene is more expressed in comparison to others immature thymocytes. In CD4+CD8+ subset NOTCH2 gene is less expressed, and JAG2 gene is more expressed when compared to CD4+CD8- population. The other receptors and ligands genes were expressed in a similar level among developing lymphocytes subsets. The relative Notch genes evaluation in developing lymphocytes populations showed a higher expression of DLL1 gene in CD4+CD8- population and JAG1 gene in CD4-CD8+ subset in comparison to CD4-CD8- thymocytes. The Notch receptors and ligands expression in thymic tissue showed to be homogeneous between thymic regions. The nTreg cells express JAG1 ligand gene in highest level among evaluated genes. The relative gene expression in nTreg presented higher expression of NOCTH1, NOTCH2, DLL4 and JAG1 genes, and low levels of NOTCH3, DLL1 and JAG2 related to CD4-CD8- subset. When relative gene expression were performed using CD4+CD8- subset, we observed that nTreg cells expressed more NOCTH1, NOCTH2, NOTCH3, DLL4 and JAG1, and in a similar level of expression DLL1 and JAG2 genes. The histological analysis showed that DLL4 was more expressed in nTreg cells in comparison to others proteins evaluated in this work, and a homogeneous distribution in thymic regions, in exception Notch3 and Jagged2. tDC cells presented higher expression of JAG1 gene among the others, and a higher expression of DLL4 protein. Notch2 expression in tDC was different between thymic regions. All together, our results showed that both genes and proteins of Notch receptors and ligands are expressed in distinct developmental stages of the maturation of T lymphocytes and nTreg cells and in the tDC cells in human thymus, with some variations in levels of expression and distribution in the thymus. These data are similar to the murine evaluations, but with some issues to be discussed. Our unpublished assessment in nTreg propose a new approach about the involvement of Notch pathway in its maturation and the evaluation of tDC suggests its direct participation of Notch signaling in the process of human thymocytes maturation

O timo é o órgão linfóide primário responsável pela maturação dos linfócitos T onde se observam estruturas e células especializadas. A sinalização intra-tímica durante a maturação de linfócitos T não é bem esclarecida. Entre outras, a via de sinalização Notch, composta por receptores (Notch 1 a 4) e ligantes (DLL1, 3 e 4, Jagged 1 e 2), pode regular este processo. Neste trabalho temos como objetivo avaliar a expressão gênica e proteica de receptores e ligantes Notch nas diferentes fases de maturação de linfócitos T, linfócitos T reguladores naturais (nTreg) e células dendriticas tímicas (tDC). Para tanto, timos de 10 crianças submetidas à cirurgia cardíaca corretiva foram manipulados e as populações CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4-CD8+, nTreg e tDC foram purificadas por citometria de fluxo. O RNA total foi isolado e os genes NOTCH1, 2 e 3, DLL1 e 4, JAG1 e 2, FOXP3 foram amplificados por RT-PCR. Alguns fragmentos de tecido foram avaliados por imunohistoquímica, quanto a expressão dos receptres Notch 1, 2, 3 e 4 e dos ligantes, DLL1 e 4, Jagged 1 e 2 em timócitos totais, células FOXP3+ e células S100+ em cada região tímica. Todos os genes de receptores e ligantes Notch foram expressos nas populações estudadas. Na população CD4-CD8- o gene NOTCH1 é mais expresso em comparação as outras populações de timócitos imaturos. Na população CD4+CD8+ o gene NOTCH2 é menos expresso, e o gene JAG2 mais expresso quando comparados à população CD4+CD8-. Os demais genes de receptores ligantes Notch são expressos de maneira similar entre as populações de timócitos. A avaliação relativa dos genes Notch nas populações de timócitos mostrou uma maior expressão do gene DLL1 na população CD4+CD8- e JAG1 na população CD4-CD8+ em comparação à CD4-CD8-. A expressão dos receptores e ligantes Notch no tecido mostrou ser homogênea entre as regiões. As nTreg expressam o gene do ligante JAG1 em maior nível entre os avaliados. A expressão gênica relativa das nTreg mostrou uma maior expressão de NOTCH1, NOTCH2, DLL4 e JAG1 e menor expressão de NOTCH3, DLL1 e JAG2 em relação as CD4-CD8-. Quando a relação utilizou a população CD4+CD8- observamos que as nTreg expressam mais os genes NOCTH1, NOCTH2, NOTCH3, DLL4 e JAG1, e em níveis similares DLL1 e JAG2. A avaliação histológica mostrou uma maior expressão de DLL4 em nTreg em comparação às demais proteínas avaliadas, e uma distribuição homogênea entre as regiões com exceção de Notch3 e Jagged2. As tDC mostraram uma maior expressão do gene JAG1 em comparação aos demais, e uma maior expressão da proteína DLL4. A expressão do receptor Notch2 em tDC difere entre as regiões tímicas. Em conjunto, nossos resultados mostraram que os receptores e ligantes Notch são expressos de forma gênica e proteica em todos os estágios de maturação de linfócitos T, nTreg e tDC do timo humano, com algumas variações quanto ao nível de expressão e distribuição no timo. Dados que se assemelham às avaliações murinas, porém com pontos a serem discutidos. Nossa inédita avaliação em nTreg propõem uma nova abordagem ao envolvimento da via Notch em sua maturação e a avaliação das tDCs sugere sua participação direta via Notch na maturação dos timócitos humanos / The thymus is a primary lymphoid organ responsible of maturing T lymphocytes, where specialized structures and cells are observed. The intrathymic signaling during lymphocytes maturation is unclear. Among them, Notch signaling pathway, which comprises in receptors (Notch1-4) and ligands (DLL1, 2 and 3, Jaaged1 and 2), may regulate this process. In this work our aim is to evaluate the expression of Notch receptors and ligands in different phases of T lymphocytes maturation, natural T regulatory cells (nTreg), and thymic dendritic cells (tDC). For this purpose, thymuses from 10 children who underwent corrective cardiac surgery were manipulated and populations CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4- CD8+, nTreg and tDC were sorted by flow cytometry. Total RNA was purified and genes NOTCH1, 2 and 3, DLL1 and 4, JAG1 and 2, FOXP3 were amplified by RT-PCR. Some thymic fragments were evaluated by immunohistochemistry and screened for expression of Notch 1, 2, 3 and 4 receptors, DLL1 and 4, Jagged and e 2 ligands in total thymocytes, FOXP3+ cells and S100+ cells in each thymic region. All Notch receptors and ligands genes were expressed in studied populations. In CD4-CD8- subset NOTCH1 gene is more expressed in comparison to others immature thymocytes. In CD4+CD8+ subset NOTCH2 gene is less expressed, and JAG2 gene is more expressed when compared to CD4+CD8- population. The other receptors and ligands genes were expressed in a similar level among developing lymphocytes subsets. The relative Notch genes evaluation in developing lymphocytes populations showed a higher expression of DLL1 gene in CD4+CD8- population and JAG1 gene in CD4-CD8+ subset in comparison to CD4-CD8- thymocytes. The Notch receptors and ligands expression in thymic tissue showed to be homogeneous between thymic regions. The nTreg cells express JAG1 ligand gene in highest level among evaluated genes. The relative gene expression in nTreg presented higher expression of NOCTH1, NOTCH2, DLL4 and JAG1 genes, and low levels of NOTCH3, DLL1 and JAG2 related to CD4-CD8- subset. When relative gene expression were performed using CD4+CD8- subset, we observed that nTreg cells expressed more NOCTH1, NOCTH2, NOTCH3, DLL4 and JAG1, and in a similar level of expression DLL1 and JAG2 genes. The histological analysis showed that DLL4 was more expressed in nTreg cells in comparison to others proteins evaluated in this work, and a homogeneous distribution in thymic regions, in exception Notch3 and Jagged2. tDC cells presented higher expression of JAG1 gene among the others, and a higher expression of DLL4 protein. Notch2 expression in tDC was different between thymic regions. All together, our results showed that both genes and proteins of Notch receptors and ligands are expressed in distinct developmental stages of the maturation of T lymphocytes and nTreg cells and in the tDC cells in human thymus, with some variations in levels of expression and distribution in the thymus. These data are similar to the murine evaluations, but with some issues to be discussed. Our unpublished assessment in nTreg propose a new approach about the involvement of Notch pathway in its maturation and the evaluation of tDC suggests its direct participation of Notch signaling in the process of human thymocytes maturation

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.