Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Perfler, B.
Impact of EZH2 on the activation status of RAS-signaling cascades in RAS-mutated myeloid leukemias: a study using intracellular phospho-flow analysis in a murine leukemia model.
[ Diplomarbeit/Master Thesis (FH) ] FH Joanneum Graz; 2023. pp.91.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Zebisch Armin
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- Abstract:
- RAS mutations are found in about 20% of human cancers with NRAS being highly prevalent in myeloid malignancies. The RAS proteins belong to the RAS-MAPK signaling transduction pathway, which is involved in several cellular processes like proliferation, cell growth, and differentiation. Thus, a continuously activated signal transduction of this pathway is a favorable condition for tumorigenesis. In line with previous studies, our group was able to successfully show that NRASG12D mutation frequently co-exists with Enhancer of zeste homolog 2 (EZH2) inactivation in myeloid malignancies. By generating an EZH2 knock-down (KD) in a NRAS-mutated myeloid cell line, we were able to show that EZH2 KD cells in Western Blot analysis resulted in upregulated pERK bands compared to the controls. However, Western Blot reached the detection limit since it is analyzing the total cell population and the investigation of individual hematopoietic stem and progenitor cell (HSPC) compartmentsis notfeasible. Hematopoiesisis a hierarchical process that originates from hematopoietic stem cells (HSCs) and goes through various cell-developing stages. To gain more insightin leukemogenesis, it might be beneficial to study the hematopoietic cell compartments rather than the whole population of cells. For this reason, the aim in this work was to establish intracellular phospho-Fluorescence-activated cell sorting (FACS) for phosphorylated ERK (pERK) and two additional RAS-downstream targets in different HSPC compartments using a murine model. Additionally, we wanted to investigate the role of Ezh2 inactivation in the hyperactivation of the MAPK/ERK pathway in NrasG12D-mutated mice with Ezh2+/+ wild type (Nras) and Ezh2-/- homozygous loss (Nras/Ezh2). In this master thesis, hypothesized that in RAS-mutated myeloid malignancies, the inactivation of EZH2 increases the activity of RAS-signaling transduction pathways, which will be determined by increased phosphorylation levels in the HSPC compartments. (...)