Changes in cell polarity in aml kasumi-3 and KG-1 cell lines and the effects of in vitro target treatment

The definition of molecular pathogenesis of AML has made remarkable progress in recent years. Two major types of genetic events that are vital for leukemic transformation have been described: changes in myeloid transcription factors that govern hematopoietic differentiation and activating mutations of signal transduction intermediates. Molecular events that alter the transcriptional control in hematopoietic progenitor cells alter the composition of signal transduction molecules present for growth factor receptors, while activating mutations in signal transduction molecules change the activity and expression of various transcription factors. These processes are highly dependent on each other and are very important for normal myeloid differentiation. AML is characterized by clonal growth of immature progenitor cells. Increased growth and apoptosis resistance, as well as differentiation inhibition, seem likely to contribute to the constitutive and / or abnormal activation of this pathogenic event and growth factor receptor signaling pathways. Abnormal and structural activation of signal transduction molecules was found in approximately 50% of the primary AML bone marrow samples. The most common of these activating events was observed in RTK FLT3, N-Ras and K-Ras, KIT and other RTKs. For some of these mutations, the ability to activate and transform myeloid cells has been demonstrated in vitro or in the mouse bone marrow. There is little data in the literature related to changes in cell cytoskeleton and polarity in this pathogenesis. Goal: In this study, it was aimed to learn the change of the expression of Planar Cell Polarization pathway proteins in AML KG-1 and Kasumi-3 cells depending on the disease and to investigate the effect of FLT3 inhibitors, Quizartinib, Midostaurin and Lestaurtinib molecules and mesenchymal stem cell co-culture in in vitro target therapy. Materials and Methods: KG-1, Kasumi-3, Bone Marrow Stem Cells and Mesenchymal Stem Cell lines are transplanted into 75 cm 2 flasks as shown in the cell protocol. Cells are incubated at 37 ° C and 5% CO 2 for 1 week. During this time, the media of the cells are changed and the passage is made when necessary. After reaching the required number, the cells are planted in 6-well new flasks. Mesenchymal stem cells are planted in 3 wells at each flask. To three flask added quizartinib, to three Lestaurtinib and to the last three added Midostaurin. The study was performed as 3 repetitions. After 24 h incubation, cells were taken from flasks and mRNA expression was performed. cDNA was obtained from Taqman Reverse Transciptase Reagents from RNA samples. After the cDNA material was checked in gel electrophoresis, mixtures obtained with pre-ordered Tagman ready primers for each gene were loaded onto the ABI7000 RT PCR device using Taqman 2x RT-PCR. The reaction was monitored simultaneously from the computer of the ABI7000 machine. The Ct value was calculated from the Ct values taken from the device and Fold Change values were calculated with the folding formula. Results: Changes in the expression of genes encoding Planar Cell Polarization pathway proteins were observed according to the values obtained. The effect of the molecules used in the target treatment decreased the expression of known genes. Fold Change values decreased by 3 times in the models using mesenchymal stem cell co-culture and FLT3 inhibitors. Discussion and Conclusion: According to our experimental findings, the expression of cell polarity genes in leukemia cell lines increased compared to healthy bone marrow cells. It is known that this negatively affects the prognosis of leukemia. On the other hand, the expression of genes in the models using Quizartinib, Lestaurtinib, Midostaurin and mesenchymal stem cell co-culture was found to be positively influenced by the prognosis of leukemia. These results demonstrate a novel mechanism of action for Quizartinib, Lestaurtinib, Midostaurin from the FLT3 inhibitor molecules used.