Chronic myeloid leukemia (CML) cytogenetic marker is the Philadelphia chromosome, which is formed by the reciprocal translocation between the 9 and 22 chromosomes. The Philadelphia chromosome encodes a Bcr-Abl hybrid oncogene, which is formed by the fusion of the 5'-region of the Bcr gene with the major 3'-part of the Abl gene, the product of the gene is Bcr-Abl oncoprotein with constitutive tyrosine kinase activity. Imatinib was the first to be introduced into clinical therapy as the tyrosine kinase inhibitor of Bcr-Abl oncoprotein, but despite its efficacy and satisfactory tolerability compared to other cytostatics, almost one-third of patients develop the resistance to such therapy. An alternative approach to treat CML is the selective reduction of the oncoprotein level, which provides a thorough understanding of molecular rearrangements, signaling cascades malignant cells, and detailed screening of Bcr-Abl partner proteins as novel therapeutic targets.
Presented dissertation is dedicated to the search for the new partner proteins of the Bcr-Abl oncoprotein for establishing their role in the development and progression of chronic myeloid leukemia.
In the dissertation, for the first time, the interaction of Bcr-Abl oncoprotein with USP1 protein was established in cells of a patient with blast crisis of CML. Immunofluorescence analysis and confocal microscopy revealed that the formation of the Bcr-Abl/USP1 protein complex occurs in the cell nucleus. The binding specificity of Bcr-Abl/USP1 is implemented by the interaction of the USP1 deubiquitinase with the PH domain of the Bcr part of the oncoprotein. In the dissertation, it was first demonstrated that inhibition of the functional properties of USP1 deubiquitinase correlates with a decreased level of Bcr-Abl oncoprotein in CML cells. This decrease coincides in time with the decrease or complete disappearance of Bcr-Abl/USP1 colocalization in K562 cells. For the first time, it has been experimentally confirmed that inhibition of USP1/UAF1 protein complex formation by ML323 shifts USP1 localization from nuclear to cytoplasmic. A hypothetical model was proposed, suggesting that USP1 by its deubiquitination activity prevents proteasomal degradation of Bcr-Abl oncoprotein. Therefore, USP1 seems to be a promising therapeutic target in CML treatment.
The direct interaction of the Bcr-Abl oncoprotein with GLG1 protein in K562 cells was also demonstrated. Colocalization of Bcr-Abl/GLG1 proteins in the Golgi complex was detected by immunofluorescence analysis and confocal microscopy. According to the results of bioinformatics analysis, tyrosine sites are predicted for GLG1 protein, which can be phosphorylated due to the tyrosine kinase activity of the Bcr-Abl oncoprotein. It is experimentally confirmed that the isoform of the GLG1 protein, which interacts with the Bcr-Abl oncoprotein, is tyrosine phosphorylated. It is known that the deregulation of GLG1 protein by protein interactions in the Golgi complex leads to disturbance of adhesion, mobility and cell migration. It is suggested that Bcr-Abl tyrosine kinase may uncontrollably activate GLG1 protein functions, leading to impairment of downstream signaling pathways and creating prerequisites for CML progression.
It was shown that the nuclear colocalization of the Bcr-Abl oncoprotein and the ZFP217 protein is involved in the regulation of cell proliferation and differentiation, responses to growth suppressors, genome stability of antiproliferative signaling, and so on. The inhibition of tyrosine kinase activity of Bcr-Abl by imatinib has been found to correlate with the low or complete absence of ZFP217 protein expression in K562 cells and affect the nuclear localization of the oncoprotein itself.
Thus, in the dissertation, the interactions between the Bcr-Abl oncoprotein with USP1, GLG1 proteins, and colocalization with ZFP217 protein in Ph-positive cells of CML were first demonstrated and characterized. The correlation between USP1 deubiquitinase activity and Bcr-Abl protein level was established. The possible effect of the uncontrolled tyrosine kinase activity of Bcr-Abl on the functional properties of partner proteins (USP1, GLG1, ZFP217) was determined. The immunofluorescence assay method for working with K562 suspension cells has been modified and adapted. The results obtained in the dissertation can be used to further study the etiology of CML and to develop a new treatment strategy based on therapeutic target proteins capable of selectively interacting with the Bcr-Abl oncoprotein and promoting its cellular proteolysis.
