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Patient-derived xenograft models of human lymphomas for experimental immunotherapy
Jakša, Radek ; Klener, Pavel (advisor) ; Škarda, Jozef (referee) ; Kalinová, Markéta (referee)
Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDX) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDX keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We derived 15 PDX models from patients with various subtypes of aggressive lymphomas. We implemented complex genetic and immunohistochemical analysis of the established PDX models head-to-head with the patient's primary lymphoma cells, from which the PDXs were derived. We clearly confirmed that the established PDX cells shared majority of somatic mutations with the patient's primary cells, from which they were derived. Thus, from the genetic perspective the PDX models represent relevant tools for the study of lymphoma biology. Immunohistochemistry analysis of selected antigens revealed some differences between the PDXs and patients' primary cells. Importantly, the analysis demonstrated complete loss of non-malignant cellular components of the tumor microenvironment frequently observed in lymphoma infiltrated lymph nodes,...
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Lineage plasticity of leukemic blasts. Importance for detection of minimal residual disease and study of hematopoesis
Vakrmanová, Barbora ; Mejstříková, Ester (advisor) ; Šálek, Cyril (referee) ; Klener, Pavel (referee)
Acute leukemia is the most common malignancy in children. According to the origin of the leukemic blasts, two types of leukemia are distinguished - lymphoid (ALL) and myeloid (AML). The focus of this thesis is lineage plasticity of the leukemic blasts. In about 2-5% of leukemias, blasts share immunophenotypic features of both lymphoid and myeloid lineages. In international retrospective study we showed superior overall survival in patients treated according to lymphoid type of protocol compared to patients treated to myeloid type of protocol, especially in cases with CD19 positivity on the blasts. Another type of the plasticity and diagnostic uncertainty in leukemia is ALL with early switch to monocytic lineage. About 8% of B cell precursor ALL underwent monocytic switch in our consecutive cohort. This phenomenon is more common among DUX4r, PAX5-P80R and ZNF384r leukemias. Discrepancy between minimal residual disease (MRD) measured by flow cytometry and quantitative assessment of immunoreceptor rearrangements method occurs because of the loss of B-lymphoid markers. We investigated transdifferentiation process by mass cytometry. By the multilabel panel we were able to determine the sequence of changes in proteins and transcription factors by new tviblindi algorithm. Targeted treatment, such as...
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Molecular events associated with resistance to tyrosine kinase inhibitors in leukemia cells.
Hrdinová, Tereza ; Vyoral, Daniel (advisor) ; Klener, Pavel (referee) ; Holoubek, Aleš (referee)
Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disease characterized by the expression of BCR-ABL oncoprotein with constitutive tyrosine kinase activity. Although the development of tyrosine kinase inhibitors (TKI) such as imatinib dramatically improved the treatment of CML, a certain subset of patients develops resistance to TKI drugs. The most common cause of TKI resistance are point mutations in the BCR-ABL1 gene, followed by other mutation-independent mechanisms. Survival and proliferation of CML cells in the presence of TKI drugs are accompanied by adaptive changes in their metabolism. Drug resistance can be maintained by extrinsic signals, among which exosomes, small vesicles released by (drug-resistant) cells, have been shown to play an important role. The aim of this thesis was to characterize two CML cell lines sensitive and resistant to imatinib, as well as the exosomes derived from imatinib-resistant CML cells by proteomic approaches. Identification of metabolic vulnerabilities in drug-resistant cells enables their targeting by clinically available drugs, thus offering potential therapeutic targets for their selective elimination. Analysis of exosomes derived from imatinib-resistant cells can identify specific membrane surface proteins exploitable as clinically relevant...
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Leukemia cell signaling and signaling of non-malignant B-cells
Kanderová, Veronika ; Kalina, Tomáš (advisor) ; Klener, Pavel (referee) ; Otáhal, Pavel (referee)
Acute leukemia (AL) is the most common pediatric cancer. Approximately 90 - 100 children is diagnosed every year in the Czech Republic. Acute leukemia is a complex disease that is pathologically manifested at the DNA, mRNA, protein and cellular level. Leukemic cells aberrantly express molecules that are found in other cell types under physiological conditions and their functional involvement in leukemic cells is unknow. We found that aberrantly expressed CEACAM6 increases the expression and affinity of integrins, increases the phosphorylation of intracellular kinases Akt, p38MAPK and p44/42 MAPK and triggers apoptosis in B-cell precursor acute lymphoblastic leukemia cells. Adaptor molecule NTAL, aberrantly expressed in T-cell acute lymphoblastic leukemia, signals through intracellular kinase p44/42 MAPK and potentiates corticosteroid induced apoptosis. Current leukemia research is focused mainly on monitoring of mutations at the DNA level, however, the functional consequences of these changes on cellular machineries are not straightforward. Since proteome analysis can provide link between gene sequence and cellular physiology, proteomics will contribute to elucidate mechanism of disease, prognosis and response to treatment. Protein microarrays technology is of major interest for basic proteomic research as...
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Resenzitalizace leukemických a lymfomavých buněk k trailerem indukované apoptóze
Molinský, Jan ; Klener, Pavel (advisor) ; Hyršlová Vaculová, Alena (referee) ; Vyoral, Daniel (referee)
Apoptosis serves as a natural barrier to cancer development, and the resistance to apoptosis represents one of the key capabilities acquired during tumor development or progression. Impairment of the intrinsic apoptotic pathway exemplifies one of the established mechanisms of constitutive or acquired drug resistance. As most of the currently used cytotoxic drugs initiate tumor cell death by direct or indirect triggering of the intrinsic apoptotic pathway, impairment of the intrinsic pathway is associated with therapy failure. Targeting of the death receptors, however, enables induction of apoptosis even in the chemotherapy resistant cancer cells. TRAIL is a death ligand belonging to the TNFα superfamily that specifically kills tumor cells while sparing healthy tissues. Much enthusiasm has been generated for TRAIL as a highly promising targeted anti-cancer agent. However, many primary tumors have been shown to be TRAIL resistant. In attempt to overcome such an intrinsic TRAIL resistance a wide array of agents have been shown to sensitize tumor cells to TRAIL. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. In this study we analyzed the sensitivity of diverse hematologic malignancies to TRAIL-induced apoptosis and measured the...
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Physiological and pathophysiological role of GCPII in the body
Sedlák, František ; Konvalinka, Jan (advisor) ; Klener, Pavel (referee) ; Smetana, Karel (referee)
Glutamate carboxypeptidase II (GCPII) is a metalloprotease responsible for cleaving the neurotransmitter N-acetyl-aspartyl-glutamate in the central nervous system to N-acetyl aspartate and glutamate. At the same time, in the human small intestine, it facilitates folate absorption by cleaving γ-linked glutamate from folyl-poly-γ-glutamate. In humans, GCPII is also expressed in a number of other organs (e.g., kidney and prostate) and tumors, where its physiological function is unknown. In an attempt to characterize the physiological function of the enzyme, we first characterized the commercially available monoclonal antibodies against GCPII. Further, we developed a fully synthetic replacement based on a hydrophilic polymer with bound GCPII inhibitors. We evaluated the suitability of using a murine biomodel to study GCPII function in vivo. We found the difference in GCPII expression profile in mouse and human. We did not observe GCPII in either the mouse prostate or small intestine. To assess physiological and pathophysiological functions of the enzyme we analyzed a GCPII-deficient mouse model. Apart from the observation of enlarged seminal vesicles in older males, we did not detect any other obvious phenotype. Similarly, we confirmed that GCPII cannot cleave amyloid peptides (Aβ1-40 and Aβ1-42)....
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The Role of Antiangiogenic Therapy in Mantle Cell Lymphoma
Kovaříková, Petra ; Klener, Pavel (advisor) ; Kalinová, Markéta (referee) ; Boudová, Ludmila (referee)
Mantle cell lymphoma (MCL) is a subtype of B-non-Hodgkin's lymphoma, characterized by often relapses. Despite an Ibrutinib (a Bruton's kinase inhibitor) implementation into salvage therapy, these patients often relapse with biologically highly aggressive disease and very poor prognosis. An increased activation of alternative metabolic pathways was described as one of ibrutinib-resistance mechanisms. Some of these pathways have also significant proangiogenic activity (e.g. PI3K-AKT-mTOR). In presented study, we established and standardized a real-time ultrasound and photoacoustic imaging of neovascularization and tissue oxygenation of subcutaneous MCL tumors in mice. Ultrasound and photoacoustic imaging is a fast, non-invasive method for angiogenesis evaluation in subcutaneous tumors with huge preclinical potential. Using MCL mice models, we also demonstrated the importance of CD31/PECAM-1 expression for engraftment, growth and spread of MCL cells in vivo. The level of CD31 expression in primary MCL cell (obtained directly from MCL patients) positively correlates with extent of extranodal involvement. CD31 facilitates survival and regulates extranodal spread of mantle cell lymphoma. We found that increased VEGFA expression causes not only increased microvessel density due to higher sprouting...
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Role of PIM oncogenes in the biology and chemoresistance of aggressive lymphomas
Kořínková, Klára ; Klener, Pavel (advisor) ; Petrák, Jiří (referee)
Proviral integration site for Moloney murine leukemia virus (PIM) kinases are serine/threonine kinases and oncoproteins involved in tumorigenesis of many solid and hematopoietic malignancies including mantle cell lymphoma (MCL) and diffuse-large B-cell lymphoma (DLBCL). They were shown to promote growth and survival of cancer cells by phosphorylation of proteins involved in cell cycle regulation, transcription, translation and apoptosis. Their potential as therapeutic target is, however, complicated by existence of overlapping signaling pathways. Here we show that inhibition of PIM kinases with AZD1208, highly selective pan-PIM inhibitor, reduces growth of cell lines derived from MCL and DLBCL patients. Inhibition of PIM kinases results in decreased phosphorylation of proteins involved in apoptosis and cap-dependent translation. We further showed that concurrent inhibition of PIM-kinases with AZD1208 and signaling from B-cell receptor with ibrutinib (a Bruton's tyrosine kinase (BTK) inhibitor) or idelalisib (a phosphatidylinositol 3-kinase (PI3K) inhibitor) synergistically reduces growth of MCL cell lines. Combination of AZD1208 and ibrutinib was accompanied by decreased phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and decreased expression of...
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Experimental therapy of B-cell Non-Hodgkin's lymphonas.
Klánová, Magdalena ; Klener, Pavel (advisor) ; Machová Poláková, Kateřina (referee) ; Froňková, Eva (referee)
1 ABSTRACT B-cell non-Hodgkin lymphomas (B-NHL) represent the most common mature lymphoproliferative diseases. B-NHL arise at different stages of B-cell development and represent their malignant counterpart. Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are aggressive types of B-NHLs. Deregulation of cell cycle control, inhibition of apoptosis or abnormal DNA damage response play a key role in the pathogenesis of DLBCL and MCL. Aberrant activation of several signaling pathways that further promote survival, cell proliferation or affect the tumor microenvironment have been recently recognized. Increased understanding of the oncogenic mechanisms implicated in pathogenesis of B-NHL lead to development of novel agents that target the oncogenic drivers of distinct lymphoma subtypes. MCL is an aggressive subtype of B-NHL associated with poor prognosis. In vivo models of human MCL for experimental therapy are however scarce. We established and characterized several mouse models of human MCL by xenotransplantation of either primary cells or established cell lines into immunodeficient mice (publication no 1). We demonstrated that engrafted MCL cells displayed complex changes of gene expression profile, phenotype and sensitivity to cytotoxic agents compared to the original in vitro growing...
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