While some rare and severe forms of platelet function disorders are now well characterized, many common types of platelet function disorders are not yet characterized. My hypothesis was that uncharacterized platelet function disorders that impair platelet function in aggregation and/or dense granule ATP release assays are associated with increased bleeding risk. The main goal of the thesis was to study the phenotype and bleeding risks for uncharacterized platelet function disorders, through analysis of the results from clinical laboratory tests of platelet function and for a detailed analysis of their reported bleeding symptoms. First, I assessed if lumi-aggregometry provides useful diagnostic information on platelet function and can be used to help decide if an individual has a bleeding disorder. Two cohorts of individuals were studied that had dense granule ATP release assessed in response to multiple agonists as part of a work-up for a bleeding disorder. Cohort I was comprised of individuals tested between January 2007 and June 2013 and cohort II was comprised of subjects tested at least twice by this assay prior to September 2015. Among subjects tested more than once for dense granule release defects as part of the work up for a bleeding disorder (cohort I; n=133; cohort II; n=17), normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), but impaired release with multiple agonists was not often confirmed (cohort I: 34%; cohort II: 54%) and even if it was present, the finding was not predictive of a bleeding disorder. Consequentially, it was recommended that lumi-aggregometry should not be used to diagnose platelet function disorders. Next, I studied the bleeding risks associated with uncharacterized platelet function disorders, by evaluating subjects who had abnormal findings by validated assays, namely subjects who had defective aggregation responses to two or more agonists and/or dense granule deficiency. Bleeding history was evaluated using the International Society for Thrombosis and Haemostasis bleeding assessment tool (ISTH BAT) and the likelihood for bleeding symptoms/ problems, was estimated using odds ratios (OR) collected using the clinical history assessment tool - platelet (CHAT-P) for all affected subjects, a subgroup family with a mutation RUNX1, unaffected family v members and general population controls. Individuals with platelet function disorders (n=29) and the affected members of the family with the RUNX1 mutation (n=6) had elevated ISTH BAT scores (median: 9; range:0-18 and median: 8.5, range 4-15, respectively) and an increased risk of abnormal bruising (OR 15-65 and 11-67), nosebleeds (OR 23-40 and 19-121), menorrhagia (OR 6.5-29) and excessive bleeding after trauma or dental/surgical procedures (OR 9.5-44 and 15-77 ) and wound healing problems (OR 13 and 38) compared to general population control (n=60) and unaffected (n=12) family members. Overall, the platelet function disorders in the study present with a significantly increased risk of mild, rather than severe bleeding problems. These findings are important for individuals and healthcare providers to promote evidence-based care of common uncharacterized inherited platelet function disorders for individuals with RUNX1 mutations, dense granule deficiency and/or impaired aggregation responses. / Thesis / Master of Science (MSc) / Platelets are small blood cells that help stop bleeding. People who have platelets that do not work properly are more likely to bleed. Determining who has platelet problems can be challenging as there are limitations to diagnostic tests for these conditions. Additionally, the risks for bleeding in individuals with platelet problems are unknown. We looked at individuals with bleeding problems and found that a recommended test to assess platelet dense granule release, called lumi-aggregometry, wasn’t able to reliably identify persons with bleeding problems. Based on this, we recommend that lumi-aggregometry should not be used to diagnose platelet function disorders. We also found that individuals with uncharacterized platelet function disorders have increased risks for wound healing problems and experiencing bruising, nosebleeds, menorrhagia, and excessive bleeding after dental or surgical procedures. These risks are common among other mild bleeding disorders and will be important to differentiate bleeding risk from other platelet disorders.

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第25213号 / 人健博第119号 / 新制||人健||8(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 錦織 桃子, 教授 岡 昌吾, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Les membres de la famille Homeobox jouent un rôle critique dans le développement hématopoïétique normal. L'expression ectopique des gènes Homeobox provoque des désordres dans l'hématopoïèse et le développement de leucémies. L'oncogène TLX3 s'exprime de manière ectopique exclusivement dans les Leucémies Aiguës Lymphoblastiques T (LAL-T), avec un blocage des thymocytes à un stade de différentiation précoce cortical CD4+CD8+ DP. De nombreuses études ont investigué les mécanismes d'action des oncogènes TLX1/3, mais plusieurs questions restent en suspens. Durant ma thèse, j'ai étudié l'impact de l'expression ectopique de l'oncogène TLX3 sur le développement lymphocytaire T et les mécanismes de transformation leucémique associés. L'expression de TLX3 a provoqué le blocage des thymocytes à un stade DN2 avec une immortalisation des clones preleucémiques. Les souris transplantées avec les cellules TLX3 ont développé des tumeurs similaires aux LAL-T. Les analyses de ChIP-Seq et d'expression génique ont identifié un recrutement de TLX3 sur les enhancers spécifiques aux cellules T par le motif de fixation Ets/Runx1. Nos résultats suggèrent que la fixation de TLX3 sur les éléments cis-régulateurs peut contribuer à la transformation maligne des thymocytes en perturbant les réseaux transcriptionnels responsables de l'oncogenèse LAL-T. / It is now well established that members of the homeobox gene family play a critical role in normal hematopoietic cell development and that their unbalanced or ectopic expression can lead to characteristic perturbations in haemopoiesis and the onset of leukaemia. TLX3 expression in human haematologic malignancies is exclusive to T-ALL, where it is almost universally associated with transformation of early cortical CD4+CD8+ DP thymocytes. Multiple studies intensively investigated the mechanisms by which TLX1/3 oncogenes could promote complex tumor development, but many questions remain still unclear. During my thesis I investigated the impact of ectopic TLX3 expression on T cell development, and the initiating mechanisms of T-cell transformation leading to leukemia onset. Forced expression of TLX3 disrupted the thymic develoment at DN2-like stage giving rise to immortalized preleukemic clones. Following the transfer into immunodeficient mice TLX3 preleukemic cells initiated malignant cell transformation resulting into leukemia-like disease. Applying a combination of ChIP sequencing and gene expression profiling, we identified TLX3 recruitment onto T-cell specific enhancers via interaction with Ets1/Runx1 composite motif sites as preferential molecular events in the initial steps of TLX3-induced transformation. Thus our findings suggest that the genome-wide binding properties of TLX3 on cis-regulatory elements may contribute to its ability to promote thymocyte preleukaemic state via perturbation of transcriptional regulatory networks responsible for T-ALL oncogenesis.

Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro. / Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro. / Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has demonstrated that these therapy-resistant cells possess stem cell-like properties and are therefore commonly referred to as breast cancer stem cells (BCSCs). A major hallmark of BCSCs is the cell surface expression of CD44 and lack of expression of CD24, the so-called CD24-/CD44+ phenotype. Research indicates that this dangerous and rare subpopulation of BCSCs may be responsible for cancer onset, recurrence, and ultimately metastasis that leads to death. Two different model systems were utilized in this research. The first was the MCF7 cell line, a luminal A tumor subtype representative of a mildly invasive breast ductal carcinoma with an ER+/PR+/-/HER2- immunoprofile. The second was the MCF10A breast cancer progression model, which consists of three cell lines: MCF10A, MCF10AT1, and MCF10CA1a. In this system, spontaneously immortalized, non-malignant MCF10A cells were transfected with constitutively active H-Ras to form pre-malignant MCF10AT1 cells, which were then subcutaneously injected into mice and allowed to metastasize in order to form the oncogenic MCF10ACA1a cell line. This thesis presents evidence of a CD24low/-/CD44+ BCSC subpopulation within the MCF10A breast cancer progression model system. Findings indicate that RUNX1 and RUNX2 expression levels are involved in maintaining the BCSC phenotype. Across two different model systems, qRT-PCR analysis revealed that decreased levels of RUNX1 expression and increased levels of RUNX2 expression are essential for the maintenance of the BCSC subpopulation. It was also shown that low expression levels of RUNX1 and high expression levels of RUNX2 are present in CD24low/-/CD44+ BCSCs as compared to CD24+/CD44+ non-BCSCs. Furthermore, shRNA knockdown of RUNX1 was shown to enhance tumorigenicity, while shRNA knockdown of RUNX2 repressed tumorigenicity in BCSCs, as measured by the tumorsphere-formation assay. This research lays the groundwork for future investigations into the roles of RUNX1 and RUNX2 in regulating stemness in breast cancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Translocation t(12;21) with the presence of the fusion gene ETV6-RUNX1 (TEL-AML1) is the most common chromosomal aberration found in acute lymphoblastic leukemia in childhood. The occurrence of the ETV6-RUNX1 is associated with excellent prognosis and high sensitivity to the treatment with the enzyme L-asparaginase (ASNase). Resistance to the drug aggravates the outlook of the patient and increases the risk of treatment failure, therefore, the CLIP working group has been for a long time involved in the identification of the mechanism of action of ASNase and the origin of the resistance to it. This thesis follows previous findings of the group and is devoted to the analysis of the importance of ETV6-RUNX1 and signalization and metabolic changes accompanying shifts in the L-asparaginase resistance. In the first part of the thesis, the knockout clones with stable increased resistance to ASNase have been established thanks to the CRISPR/Cas9 system, which created frameshift in the fusion gene. The accomplishment in this regard and removal of the fusion protein was confirmed on the level of DNA, mRNA a protein expression. The presence of other significant chromosomal aberrations affection the sensitivity to ASNase was ruled out by the means of SNP analysis. In the second part of the project, the signalization...

Translocation t(12;21) with the presence of the fusion gene ETV6-RUNX1 (TEL-AML1) is the most common chromosomal aberration found in acute lymphoblastic leukemia in childhood. The occurrence of the ETV6-RUNX1 is associated with excellent prognosis and high sensitivity to the treatment with the enzyme L-asparaginase (ASNase). Resistance to the drug aggravates the outlook of the patient and increases the risk of treatment failure, therefore, the CLIP working group has been for a long time involved in the identification of the mechanism of action of ASNase and the origin of the resistance to it. This thesis follows previous findings of the group and is devoted to the analysis of the importance of ETV6-RUNX1 and signalization and metabolic changes accompanying shifts in the L-asparaginase resistance. In the first part of the thesis, the knockout clones with stable increased resistance to ASNase have been established thanks to the CRISPR/Cas9 system, which created frameshift in the fusion gene. The accomplishment in this regard and removal of the fusion protein was confirmed on the level of DNA, mRNA a protein expression. The presence of other significant chromosomal aberrations affection the sensitivity to ASNase was ruled out by the means of SNP analysis. In the second part of the project, the signalization...

Breast cancer is the most prevalent cancer in women in the UK with over 50,000 new cases diagnosed each year. Almost all breast cancer deaths are due to metastatic disease. The RUNX1-CBFbeta transcription factor complex has been implicated in the development of human breast cancer and recent evidence from our laboratory indicated that it might have a role in metastasis. The aim of this project was therefore to determine the role of the RUNX1 transcription factor in breast cancer metastasis. Initial experiments to knockdown RUNX1 by shRNA also decreased the expression of RUNX2. Therefore, due to the off-target effect of shRUNX1, CRISPR-Cas9n was used to establish a RUNX1-negative cell line by targeting the first exon of the RUNX1 gene. Migration and invasion capacity of the cells decreased in the absence of RUNX1 and it was comparable to the absence of RUNX2 and CBFbeta respectively, which are known to play roles in migration and invasion of MDA-MB-231 cells. The cells also formed spherical clusters in 3D culture which was associated with the changes in cell morphology from stellate to round shape in the absence of RUNX1. The expression of the metastasis-related genes MMP13, MMP9, OPN and SLUG also decreased in parallel with the loss of the mesenchymal-like phenotype whilst the expression of the epithelial markers cytokeratin, desmoplakin and E-cadherin increased concomitantly. Importantly, re-expression of RUNX1 in the RUNX1-negative cell lines using an inducible expression system rescued migration and invasion. Therefore, RUNX1 is required to maintain the mesenchymal-like phenotype of MDA-MB-231 cells and hence is important for the epithelial to mesenchyme transition (EMT), a key characteristic of metastatic cells. The transcription factor SLUG is a known regulator of EMT. Data obtained shows that RUNX1 down-regulates the expression of SLUG. ChIP analysis demonstrated that RUNX1 was bound to the SLUG promoter and RUNX1 was subsequently shown to activate the promoter activity. Finally, experiments to inhibit the activity of the RUNX transcription factors pharmacologically showed changes in cell differentiation and also affected cell viability, possibly by off-target effects. Taken together, data presented in this work demonstrates that RUNX1 is required for EMT in the metastatic breast cancer cells and it is therefore a potential therapeutic target to prevent breast cancer metastasis.