The investigation of effects on leukemic cell response of stat5 inhibitor pimozide applied on CML cell which is sensitive and resistant to third generation tyrosine kinase inhibitor ponatinib

Chronic myeloid leukemia (CML), a clonal myeloproliferative disorder of the hematopoietic stem cell, is characterized by BCR/ABL oncogenesis resulting from t(9;22) reciprocal translocation. This oncogene causes continuous tyrosine kinase activity and is responsible for CML leukomogenesis. Tyrosine kinase inhibitor (TKI) İmatinib has opened a new era in the treatment of CML. However, it has been observed that CML cases develop resistance to İmatinib over time. For this reason II. (Dasatinib, Nilotinib and Bozutinib) and III. generation TKIs (Ponatinib) have been developed. Earned İmatinib resistance; Dasatinib, Nilotinib, Bosutinib and Ponatinib have also been associated with decreased efficacy and increased resistance. STAT proteins on the JAK / STAT pathway are transcription factors and play important roles in the development of leukemia. Particularly active STAT5 is the basic protein that ensures survival and proliferation of leukemic cells. Demonstration of leukemic cell apoptosis induced by suppression of increased STAT5 gene expression in the leukemia and a key role in the pathogenesis of resistance to TKI therapy; STATs have become the primary target of new anti-cancer drugs developed against CML. In our study, the FDA-approved psychotropic drug "Pimozide" was found to be a STAT5 inhibitor at the same time. The inhibition of STAT5 protein phosphorylation investigated the proliferation of CML cells, apoptosis, cell cycle arrest, the expression at the mRNA level of genes with various biological functions, and the effect of STAT5 on cellular responses such as protein expression. For this purpose, the KML cell line model ancestral K562 and its 1 nM Ponatinib-resistant K562 / Pon1 resistant cells were analyzed at the molecular level. In addition, Ponatinib + Pimozide combination experiments were investigated within the scope of the thesis, and cellular response analyzes were also performed for these experimental groups. The cytotoxic effect of the pimozide active agent in the leukemia cell lines used in this study was assessed as time and dose dependent using WST-1 analysis. The apoptotic effect of pimozide, ponatinib doses and combination doses on cells was assessed according to the Annexin V method and the effect on cell cycle was evaluated with "BD Cycletest Plus DNA Reagent Kit".The gene expression changes produced by the dose applications on the cells were determined by RT-qPCR with mRNAs from all dose groups and control groups. Finally, the western blot method was used for the quantitative determination of proteins. For the 72 hour IC50 doxed K562 cells of the pimozide active substance, 5.06 μM and 8.72 μM for K562 / Pon1 cells was determined. Our gene expression studies and western blot studies have shown that Pimozidenin STAT5 inhibitor is a common result and inhibits both STAT5A and STAT5B genes. We also think that proteins such as STAT3, JAK2, SOCS1, which have various functions in JAK / STAT pathway, are an important agent that can affect this pathway. It has been determined that the pimozide active substance has apoptotic effect on the cells and causes G0-G1 arrest in the cell cycle. Pimozidenin was found to be more effective in K562 / Pon1 cells than K562 cells. At this point, we think that CML may be an effective agent that can be used in the crushing of drug resistance developed in the leukemia. Furthermore, when ponatinib is used in combination with K562 / Pon1 cells; Ponatinib was determined as DRI 3,49. Since the dose of Ponatinib used can be reduced, it is thought that cytotoxicity on normal cells will decrease. Anahtar kelimeler: KML; Pimozide; STAT5