Targeted Therapy of AML1/ETO-Positive AML Cells in Experimental Model.
Authors: Zapotocky Michal , Starkova Julia , Mejstrikova Ester , Smetana Karel , Trka Jan 
Published: October 2019

Abstract
In t(8;21) acute myeloid leukaemia (AML), the leukaemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1/ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. It was shown previously that VPA treatment disrupts the AML1/ETO-HDAC1 complex from AML1 promoter thus leading to apoptosis at different cell lines. However, there is still lack of in-depth morphological and immunophenotypical proof of the hypothesized restoration of differentiation after treatment with VPA. Although very little is known about AML1 target genes, it was shown that AML1 protein binds to promotor region of IL-3 and PU.1 and regulates their expression. There are also reports demonstrating possible candidate target genes of AML1 transcription factor (BPI, IGFBP7). We aimed to characterize the differentiation effect of VPA on AML1/ ETO-positive leukaemic cells and to determine the expression pattern of selected genes. Kasumi-1 (AML1/ETO-positive) cell line and MV4-11 (MLL/AF4-positive) cells were treated with VPA (0,5 mM and 1,0 mM concentrations) and examined by flow cytometry, morphological evaluation and qRT-PCR. Paediatric patients’ bone marrow samples (AML1/ ETO-positive) from the time of diagnosis were taken for in vitro experiment. We optimised the method of patients’ samples cultivation using conditioned medium with cytokines (IL6, FLT3 ligand, TPO, SCF) and we treated leukaemic cells with VPA. We examined immunophenotype and cell cycle of these samples after 24 and 48 hours of cultivation. We show that treatment of AML1/ETO-positive myeloid cells with HDACi VPA resulted in decreased expression of early myeloid progenitor antigens (CD33/34/117) and increased expression of antigens typical for differentiated myeloid cells (CD11a/11b). Cell morphology, nucleolar morphology and cytochemistry evaluation indicated the maturation process and decreased proliferation activity. All these phenomenons were not observed in control MLL/AF4-positive myeloid cells. We quantified the level of expression of selected genes (PU.1, C/EBPalpha, BPI, IGFBP7) and we observed the increase of genes expression after VPA treatment in AML1/ETO-positive cells. In VPA treated AML1/ETO-positive cells PU.1 increased expression 6.2 times (p<0.001), C/EBPalpha 3 times (p<0.001), BPI 2.6 times (p<0.001) and IGFBP7 7 times (p<0.001). Different situation occurred in MLL/AF4-positive cell line, where PU.1 conversely decreased expression 2.5 times (p=0.01), IGFBP7 decreased 2.4 times (p=0.01) and the expression of C/EBPalpha and BPI remained unchanged (p=0.32 and p=0.75) after VPA treatement. Samples recovered from patientś bone marrow reacted differently to VPA therapy. The first patient increased the expression of HLA-DR (p=0.04) and CD11b (p=0.02) and at the same time decreased the expression of CD38 (p=0.006) and CD117 (p=0.03) surface markers, which represents the signs of differentiation. The second patient’s sample presented with expression of HLA-DR (p=0.1) unchanged; expression of CD11b (p=0.05), CD38 (p=0.002), CD117 (p=0.04) and CD34 (p=0.02) decreased. The concentration of VPA used in our experiment seems to have strong cytotoxic effect on the other patient’s leukaemic cells, as they passed into the apoptosis, the amount of 10% of persistent blast cells was not sufficient for the analysis. Cell cycle examination confirmed the results of the experiment with cell lines; patientś samples treated with VPA decreased the proliferation and the number of cells undergoing apoptosis increased. Taken together, we provide a valid evidence of differentiation of AML1/ETO-positive cell line, demonstrated by flow cytometry and confirmed morphologically. This process goes hand in hand with the increase of the repressed genes expression as measured by qRT-PCR in contrast with non-CBF leukemic cells. Patients’ data are not completely in line with those experimental findings, as the flow cytometry analysis showed uneven changes in surface markers expression pattern. VPA induces differentiation and apoptosis; therefore it seems to be a promising drug in treatment of AML/ETO-positive paediatric AML. Supported by Grant Agency of Charles University 71/2006.