Functional characterization of KIS and its role in leukemogenesis

• Grant number: 16/04372-8
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: April 01, 2016
• End date: January 31, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Leticia Fröhlich Archangelo
• Grantee: Vanessa Cristina Arfelli
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 14/01458-3 - Defining the functional role of the splicing factor regulator (KIS) during leukemogenesis using a murine bone marrow transplantion model, AP.JP

Abstract

KIS is a serine / threonine kinase that couples both functions of controlling the cell cycle and activity of splicing factors. Through its UHM domain, KIS interacts with splicing factors SF1 and SF3b1 and through its kinase activity phosphorylates these factors. Thus it is proposed that KIS function is related to RNA splicing control. The analysis of the genome and exome of patients with hematological malignancies showed somatic mutations in several components of the splicing machinery, suggesting that this process is a new and important mechanism in leukemogenesis. Phosphorylation of proteins involved in the mechanism of splicing regulation is fundamental in this process. Phosphorylation of splicing factors enables the interaction of these factors with other proteins and RNA, as well as its intracellular localization and its activity in alternative splicing. High levels of KIS were observed in samples from patients with leukemia and myelodysplastic syndrome. Our hypothesis is that increased expression of KIS can lead to excessive phosphorylation of its substrates (in this case, SF1 and / or SF3b) and it could contribute to the leukaemogenic process. The aim of this study is to investigate the role of KIS in leukemogenesis through the murine model of bone marrow transplantation. This research is justified because it will bring valuable contribution to defining the role of KIS in leukemogenesis and its implication as a potential molecular target for development of new target-specific therapeutic strategies. The methodology is based on the construction of a retroviral vector (MIY-KIS; MIY = MSCV (Murine Stem Cells Virus) + IRES (Internal Ribosome Entry Site) + YFP (Yellow Fluorescent Protein)) for the overexpression of KIS, and It will be used for transfection of Phoenix packaging cells 293, in which occurs the production of viral particles. These viral particles will infect the murine packaging cells GP+E86. GP+E86 cells transduced efficiently will be separated by FACS (Fluorescence Activated Cell Sorting) and after these ones will undergo single sorting to produce monoclonal populations of cells stable producing retrovirus. The more stable from these cell populations will be cultured with hematopoietic progenitor cells obtained from bone marrow of mice, that transduction occurs. The transduced cells will be transplanted intravenously into mice bone marrow. The animals will be observed for up to 12 months in search of developing leukemia. Those who develop the disease will be sacrificed and cells from bone marrow and spleen will be analyzed. Colony forming cell assays will be carried out with the aim of analyzing the transforming ability of the overexpressed KIS has on progenitor cells. To analyze the same ability in hematopoietic stem cells more primitive, transduced (with MIY-KIS and empty MIY) and purified progenitor cells will be injected into lethally irradiated animals and the formation of colonies in the spleen will be evaluated after 12 days. Also, pattern phosphorylation of splicing factors SF1 and SF3b1 in hematopoietic stem cells that overexpress KIS will be analyzed through 2D electrophoresis, as the global splicing pattern of these cells will be valued by RNA-Seq. (AU)