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A new approach to treatment of patients with TEL/AML1 acute lymphoblastic leukemia
Starková, Júlia
Leukemia accounts for about 1/3 of all cancers in children. Treatment of acute lymphoblastic leukemia (ALL) has made great advance in recent years, whereby 98% of all treated children are currently in complete remission and more than 80% of them are fully treated. Unfortunately remaining 15-20% of children still relapse. The specific group of ALL consists of patients with the TEL/AML1 fusion gene resulting from translocation t (12; 21) (p13; q22) and is present in approximately 25% of patients with B-precursor ALL. This translocation is considered to be a favorable prognostic feature, a rapid response to the initial treatment and increased sensitivity to the cytotoxic agent commonly used in therapy, L-Asparaginase (L-Asp). In the dissertation thesis we focused on the study of current therapy and potential targeted approach. In the first part we tried to clarify the mechanism of L-Asp effect in this genotypically defined ALL subgroup. The main effect of L-Asp lies in the depletion of asparagine and glutamine in the serum of patients. The anti-leukemic effect is associated with a lower level of asparagine synthetase enzyme (ASNS) specifically in leukemic cells. Previous work has shown the relationship between lower sensitivity of L-Asp and higher ASNS in leukemic cells. However, our results of monitoring the...
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Regulation of HOX genes expression in hematopoesis and leukemogenesis
Rejlová, Kateřina ; Starková, Júlia (advisor) ; Machová Poláková, Kateřina (referee) ; Fišerová, Jindřiška (referee)
HOX gene expression is tightly regulated during hematopoiesis and it is gradually decreased during the differentiation of hematopoietic cells. By contrast in case of leukemic blasts the expression of HOX genes is often disrupted and dysregulated. Especially in acute myeloid leukemia (AML) different expression of HOX genes was described between different subtypes classified according to cytogenetics and molecular genetics. In this study, the cohort of childhood AML patients were screened for HOX gene expression and based on these valuesdivided into five clusters using unsupervised hierarchical clustering characterized mainly by presence or absence of the typical molecular aberrations. HOX gene expression was also tested in the healthy counterpart of hematologic cells equivalent to the particular morphological stages of leukemic cells. Based on these results, HOX gene expression directly or indirectly participate in leukemogenesis and it not only copies the developmental/morphological stage in which the hematopoietic cell was stopped during differentiation. It this thesis/study it was concluded that the HOX gene expression is dependent on the presence of specific molecular aberration. In the second part of our study, we investigated the HOX gene transcription regulation in AML patients with PML-RARα...
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The role of ETV6-RUNX1 fusion protein in the sensitivity of leukemic cells to L-asparaginase
Staněk, Petr ; Starková, Júlia (advisor) ; Burjanivová, Tatiana (referee)
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...
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Study of dysregulation of DLX1 protein in myeloid leukemia cells in in vitro and in vivo models
Jelínková, Alena ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
The heterogeneous nature of acute myeloid leukemia (AML) worsens the results of patients treated with standard therapy. Understanding the processes of leukemogenesis can contribute to identification of more appropriate treatment. Family of DLX genes (Distal-less homeobox), belonging to the homeobox genes, are associated with haematological malignancies and solid tumors. In the analysis of expression data, the low level of the DLX1 gene was associated with a worse prognosis of patients with AML. In this work we studied phenotypic changes of cell lines with different expression of the DLX1 gene. We silenced the DLX1 gene in AML cell line (sh cells) and compared it to the parental line with higher expression of DLX1 (NSC cells). By cell cycle analysis and apoptosis assays in vitro and in vivo, we have observed the arrest of sh cells in the G0 phase and a lower number of apoptotic cells. Differences were found when measuring the absolute number of cells in time. In in vitro conditions there were less sh cells, in in vivo environment there was significantly higher number of sh cells engrafted in comparison to NSC cells. Further results have shown that sh cells have lower levels of pro-apoptotic proteins and exhibit a higher level of TGF-β targeting PAI-1 gene that activates replicative senescence. We...
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A new approach to treatment of patients with TEL/AML1 acute lymphoblastic leukemia
Starková, Júlia
Leukemia accounts for about 1/3 of all cancers in children. Treatment of acute lymphoblastic leukemia (ALL) has made great advance in recent years, whereby 98% of all treated children are currently in complete remission and more than 80% of them are fully treated. Unfortunately remaining 15-20% of children still relapse. The specific group of ALL consists of patients with the TEL/AML1 fusion gene resulting from translocation t (12; 21) (p13; q22) and is present in approximately 25% of patients with B-precursor ALL. This translocation is considered to be a favorable prognostic feature, a rapid response to the initial treatment and increased sensitivity to the cytotoxic agent commonly used in therapy, L-Asparaginase (L-Asp). In the dissertation thesis we focused on the study of current therapy and potential targeted approach. In the first part we tried to clarify the mechanism of L-Asp effect in this genotypically defined ALL subgroup. The main effect of L-Asp lies in the depletion of asparagine and glutamine in the serum of patients. The anti-leukemic effect is associated with a lower level of asparagine synthetase enzyme (ASNS) specifically in leukemic cells. Previous work has shown the relationship between lower sensitivity of L-Asp and higher ASNS in leukemic cells. However, our results of monitoring the...
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Interplay between cellular signaling and metabolism in cancer cells.
Záhumenská, Romana ; Vališ, Karel (advisor) ; Starková, Júlia (referee)
Hippo signaling pathway represents organ size control mechanism constrained between all metazoans. Individual components of the Hippo signaling pathway were identified as key tumor-suppressors which phosphorylate and inhibit activity of several oncogenic factors and signaling pathways (such as YAP/TAZ, PI3K and mTOR). MST1 kinase is a part of central protein complex of the Hippo signaling pathway and its activation is involved in anti-cancer activity of several drugs. We have demonstrated activation of the MST1 kinase by natural compounds in leukemic cells followed by inhibition of proliferation and induction of apoptosis. Shikonin represents natural naphthoquinonic compound isolated from Lithospermum erythrorhizon which acts as inhibitor of glycolysis and mitochondrial respiratory chain in human cells. Shikonin induces fast activation of the MST1 protein in leukemic cells however mechanism of this activation remains unknown. Therefore, we tried to characterize posttranslational modifications of the MST1 kinase during shikonin treatment of leukemic cells. Firstly, we isolated MST1 kinase from control and shikonin-treated cells using immunoprecipitation. Then we characterized posttranslational modifications of the MST1 protein employing mass spectrometry. Using this approach we found out...
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The mechanism of action of L-asparaginase in childhood acute lymfoblastic leukemia
Heřmanová, Ivana ; Starková, Júlia (advisor) ; Šálek, Cyril (referee) ; Trbušek, Martin (referee)
Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. In this study, we wanted to elucidate the effect of ASNS expression level on the sensitivity of ALL cells to ASNase treatment. Our aim was also to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients' response. We used four ALL cell lines (NALM-6, RS4;11, REH, and UOCB-6) and 30 diagnostic bone marrow samples of ALL patients to study the relationship between ASNS expression and sensitivity to ASNase using MTS proliferation assay. RNA interference was used to study the effect of a range of ASNS levels on the response to ASNase treatment. Using a cell line model with a gradually knocked-down ASNS gene, we defined a cutoff level below which ASNS gene expression does not correlate with sensitivity to ASNase. Importantly, ASNS gene expression in patients' ALL blasts is below this level. We confirmed that there was no...
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Metabolic alterations in cancer cells and their implications in the therapy of acute leukemias
Harárová, Alžbeta ; Starková, Júlia (advisor) ; Mrvová, Silvia (referee)
Cancer metabolism differs from that of the healthy cells in several aspects. Aerobic glycolysis (e.g. converting pyruvate to lactate under normoxic conditions) was the first described metabolic alteration of cancer cells. Metabolic alterations have since been described in the tricarboxylic acid cycle, oxidative phosphorylation, in the metabolism of amino acids (especially glutamine, asparagine and serine) and also in the metabolism of fatty acids and cholesterol. The common feature of these changes is the tendency to prefer anabolic pathways, thus enabling fast proliferation of cancer cells. The study of cancer metabolism is particularly important in the case of cancer cells that show resistance to treatment, as their aberrant metabolism is not only a potential diagnostic marker but also a potential therapeutic target. The majority of metabolic alterations have been described for the first time in solid tumors, whereas only recently has the metabolism of acute leukamias gained more attention. Asparaginase is an example of a chemotherapeutic agent that targets a metabolic alteration of leukemic cells. Distinct metabolic profile is also associated with the glucocorticoid resistance. Detailled study of the metabolic alterations of leukemic cells has elucitated the mechanisms of the asparaginase and...
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Study of leukemic cells' metabolism in association with response to the therapy
Šimčíková, Markéta ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
Acute lymphoblastic leukemia (ALL) is the most common malignant dise- ase in children. Despite great advancements in treatment of this disease, around 15-20 % of patients suffer a relapse. One of the possible reasons for relapse is developed resistance to cytostatic drugs. L-asparaginase is an im- portant chemotherapy component for childhood ALL and resistance to this drug often complicates treatment. To date, causes of developing resistance have not been sufficiently described. This thesis is a part of a greater research project focusing on mechanisms of L-asparaginase's activity and reasons for developing resistance to this chemotherapeutic agent. Differential metabolic requirements of cancerous cells have been described as early as 1924 by O. H. Warburg and they have been subject to scientific inquiry since. This study aimed to describe the relationship between basal metabolic determinants of leukemia cells and their sensitivity to L-asparaginase. For this reason, two metabolic pathways, glycolysis and oxidative phosphorylati- on, were studied in detail using a Seahorse Bioanalyzer. Further, expression of specific genes involved in glycolysis was detected. Content of mitochon- drial reticulum in cells, expression of the asparagine synthetase gene, and cell size were also studied. Experiments were...
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