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Lineage plasticity of leukemic cells
Slámová, Lucie ; Mejstříková, Ester (advisor) ; Brdička, Radim (referee) ; Machová Poláková, Kateřina (referee)
So far, the lymphoid to myeloid lineage switch during the treatment of B cell precursor acute lymphoblastic leukemia (BCP ALL) was identified only rarely in patients with the MLL gene rearrangement. We discovered a novel BCP ALL subset switching to monocytoid lineage during an early phase of the treatment - swALL ("switching" ALL) with no MLL gene rearrangement. The proportion of swALL cases among BCP ALLs was unexpectedly high (3-4%). All swALLs have expressed the CD2 antigen (LFA-2). The upregulation of C/EBPα gene and hypomethylation of the CEBPA promoter were significant in blasts already at diagnosis, proceeding the lineage switch in the majority of the cases. SwALL patients were characterized by unique subpopulation of the cells coexpressing B lymphoid and monocytoid markers. Changes in the gene expression of M-CSFR, GM- CSFR and other genes accompanied the lineage switch. The lineage switch could be recapitulated in vivo and in vitro. Even if the children patient with swALL respond slowly to initial therapy, the prognosis is comparable to "other" BCP ALLs. Risk-based ALL therapy appears to be the treatment of choice for swALL. Powered by TCPDF (www.tcpdf.org)
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TEL/AML1, BCR/ABL and TEL/ABL Fusion genes in childhood acute lymphoblastic leukemia
Žaliová, Markéta ; Trka, Jan (advisor) ; Machová Poláková, Kateřina (referee) ; Pospíšilová, Dagmar (referee)
Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. It represents a group of clinically and biologically heterogenous malignancies that can be subclasified into several subtypes according to the presence of recurrent genetic aberrations. The typical genetic aberrations in childhood ALL are chromosomal translocation, that often result in creation of fusion genes encoding either chimeric kinases or chimeric transcription factors. These recurrent genetic aberations are aquired lesions, they are supposed to be the initial hits (that may arise even prenataly) with a causal role in the process of leukemogenesis, which is, however, in the majority of them not yet fully understood. They further represent specific markes used for the detection of leukemic cells and some of them have also prognostic significance and belong among the factors used for risk group stratification in treatment protocols. Risk group stratification and subsequent risk-adapted therapy together with introduction of new therapeutic approaches (intensive chemotherapeutic regimens involving intrathecal application, hematopoetic stem cell transplantation (HSCT), supportive therapy) account for the significant improvement of the treatment outcomes of childhood ALL in the last decades. In addition to genotype, several...
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Study of the regulatory properties of oncogenic microRNAs under normal and pathologically altered conditions in order to detect new tumors.
Dusílková, Nina Borisovna ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Rohoň, Peter (referee)
Oncogenic microRNAs (miRNAs) are small RNA molecules that inhibit post-translational regulatory mechanisms at the epigenetic level. miRNAs are often deregulated in malignancies and due to their stability are detectable in non-cellular fractions of peripheral blood. In our laboratory, we have performed several studies that have investigated and utilized miRNAs as biomarkers for various hematological tumors (e.g., chronic lymphocytic leukemia, Hodgkin`s lymphoma) and solid tumors (e.g., breast cancer). The aim of these studies was to find the association of miRNAs with pathophysiological and clinical aspects of each disease. Here, we confirmed the importance of particular miRNA or its complex during disease monitoring. Combining clinical, molecular biological and statistical analyses, we were able to find miRNA sets that fulfilled not only a diagnostic role but also a prognostic role beyond expectations. The main focus of this thesis is on the investigation of microRNAs in the diagnosis of a hematological malignancy - primary cutaneous T-cell lymphoma (CTCL). Tumor specificity of some miRNAs has been demonstrated. Their aberrant expression in tissue samples of CTCL patients obtained from skin biopsies, correctly distinguished malignant disease from control samples of benign skin lesions. Here, we...
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Identification and Characterization of Genetic Aberrations in Acute Childhood Leukemia
Lukeš, Julius ; Kubričanová Žaliová, Markéta (advisor) ; Machová Poláková, Kateřina (referee) ; Živný, Jan (referee)
Childhood acute leukemias are genetically complex disorders, with recurrent or random aberrations found in most patients. Their proper functional characterization is crucial for understanding the role they play in the process of leukemogenesis. We aimed to identify and characterize the genetic background of two leukemic entities. The transient myeloproliferative disorder (TMD) is a preleukemic condition that occurs in 10% of newborns with Down syndrome. Trisomy 21 together with in-utero gained mutations in the GATA1 gene are essential in TMD and represent an ideal "multi-hit" model to study leukemogenesis. We investigated an alternative pathogenic mechanism enabling TMD development in a confirmed absence of trisomy 21. Novel deletions in the GATA1 and JAK1 genes were described as potential drivers of this TMD. The deletion D65_C228 in GATA1 results in the expression of an aberrant isoform, which is predicted to lose transactivation potential and, more importantly, to partially lose the ability of recognizing physiological DNA binding sites, possibly triggering TMD alone. Our thorough characterization of JAK1 F636del questions its role in TMD development. Analysis of JAK/STAT signaling suggested decrease of kinase activity upon F636 loss. Cells harboring the aberrant JAK1 did not obtain cytokine-...
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Role of the oncogenic microRNAs miR-17-92 and miR-155 in the regulation of hematopoietic differentiation and leukemogenesis
Pospíšil, Vít ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee) ; Machová Poláková, Kateřina (referee)
(English version): Hematopoietic differentiation is highly ordered multistep process, where generation of terminal blood cells is dependent upon coordinated regulation of gene expression by key regulators: transcription factors and mikroRNAs. PU.1 (Sfpi1) is a versatile hematopoetic transcription factor required for the proper generation of both myeloid and lymphoid lineages. MikroRNAs represent a novel class of ~22 nucleotide long non-coding posttranscriptional regulators that inhibit expression of genes by blocking protein translation or by mRNA degradation. In this PhD thesis I present research data documenting novel mechanisms of regulation and function of two oncogenic mikroRNAs, miR-17-92 cluster and miR-155 and myeloid transcriptional factors PU.1 upon macrophage differentiation of myeloid progenitors. The miR-17-92 cluster (Oncomir1) encodes seven related mikroRNAs that regulate cell proliferation, apoptosis and development and is overexpressed in number of malignancies including myeloid leukemia. Presented PhD thesis documents novel macrophage specific regulatory mechanisms involving the oncogenic cluster miR-17-92. Using transgenic PU.1-/- myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant, the transcription...
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The role of transcription factors PU.1 a GATA-1 during leukemia differentiation.
Burda, Pavel ; Stopka, Tomáš (advisor) ; Kořínek, Vladimír (referee) ; Machová Poláková, Kateřina (referee)
Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...
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Mechanisms of resistance to tyrosin kinase inhibitors in treatment of patients with chronic myeloid leukemia
Polívková, Václava ; Machová Poláková, Kateřina (advisor) ; Zuna, Jan (referee) ; Bělohlávková, Petra (referee)
Mechanisms of resistance to tyrosin kinase inhibitors in treatment of patients with chronic myeloid leukemia The introduction of tyrosine kinase inhibitor (TKI) therapy represented a breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Despite a high success rate of the TKI therapy, 20-30 % of patients develop resistance to the treatment. The aim of this work was to study the mechanisms, related to the mutation development in BCR-ABL1 kinase domain (KD) or in the other genes responsible for TKI resistance, using the in vitro CML models. Furthermore, the effect of interferon alpha (IFN-α) and TKI sequential therapy on the presence of clones with multidrug-resistant mutations and a role of activation the immune response in achieving the treatment response were followed. Lastly, we studied the possibility of using the measurement of gene expression of hOCT1 and ABCB1 transporters at the time of diagnosis in CML patients as a predictive marker of the imatinib treatment response. KCL-22 CML cell line cells exposed to imatinib were repeatedly able to develop BCR-ABL1 kinase domain mutations. We demonstrated the existence of de novo mutagenesis in clones derived from the KCL-22 cell line. In four clones, which can proliferate in 4µM imatinib, mutations in BCR-ABL1 KD or KRAS were...
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Identification and Characterization of Genetic Aberrations in Acute Childhood Leukemia
Lukeš, Julius ; Kubričanová Žaliová, Markéta (advisor) ; Machová Poláková, Kateřina (referee) ; Živný, Jan (referee)
Childhood acute leukemias are genetically complex disorders, with recurrent or random aberrations found in most patients. Their proper functional characterization is crucial for understanding the role they play in the process of leukemogenesis. We aimed to identify and characterize the genetic background of two leukemic entities. The transient myeloproliferative disorder (TMD) is a preleukemic condition that occurs in 10% of newborns with Down syndrome. Trisomy 21 together with in-utero gained mutations in the GATA1 gene are essential in TMD and represent an ideal "multi-hit" model to study leukemogenesis. We investigated an alternative pathogenic mechanism enabling TMD development in a confirmed absence of trisomy 21. Novel deletions in the GATA1 and JAK1 genes were described as potential drivers of this TMD. The deletion D65_C228 in GATA1 results in the expression of an aberrant isoform, which is predicted to lose transactivation potential and, more importantly, to partially lose the ability of recognizing physiological DNA binding sites, possibly triggering TMD alone. Our thorough characterization of JAK1 F636del questions its role in TMD development. Analysis of JAK/STAT signaling suggested decrease of kinase activity upon F636 loss. Cells harboring the aberrant JAK1 did not obtain cytokine-...
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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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Epigenetic factors CTCF a SMARCA5 control expression of hematopoietic transcription factor SPI1 in cells of acute myeloid leukemia and myelodysplastic syndrome.
Dluhošová, Martina ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Kozák, Tomáš (referee)
CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on the non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement, despite mutations in CTCF and Cohesin complex were identified in leukemia, remains elusive. CTCF regulates transcription dependently on DNA methylation status and can if bound block interactions of enhancers and promoters. Here, we show that in hematopietic cells CTCF binds to the imprinting control region of H19/Igf2 and found that chromatin remodeller Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding and regulatory effects. Furthermore, Smarca5 supports CTCF functionally and is needed for enhancer-blocking effect at imprinting control region. We identified new CTCF-recognized locus near hematopoietic regulator SPI1 (PU.1) in normally differentiating myeloid cells together with members of the Cohesin complex. Due to DNA methylation, CTCF binding to the SPI1 gene is reduced in AML blasts and this effect was reversible by DNA methylation inhibitor 5-azacitidine.
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