Decrease of Malignant Potential of t(8;21) Positive Cells after Stable Expression of RUNX-CBFA2T1-Specific Small Interfering RNA.

The reciprocal chromosomal translocation t(8;21) results in the fusion of the DNA-binding domain of the transcription factor RUNX1 (also called AML1 or CBFα) to CBFA2T1 (also called MTG8 or ETO). The chimeric protein RUNX1-CBFA2T1 interferes with hematopoietic gene expression by recruiting histone deacetylases via N-CoR and mSin3. In addition, impairment of the function of transcription factors such as C/EBPα has been described. To further investigate the function of RUNX1-CBFA2T1 in the development of leukemia, the expression of the fusion protein was inhibited by small interfering RNAs (siRNAs). For stable expression of the siRNA in the target cells, a RUNX1-CBFA2T1-specific siRNA-sequence was cloned into a lentivial vector as short-hairpin-RNA (shRNA). Subsequently, the t(8;21) positive Kasumi-1 cell line was infected with an effeciency of >95% as detected by expression of the GFP reporter gene. As a control, an shRNA targeting luciferase mRNA was used. Expression of the anti-RUNX1-CBFA2T1-shRNA in the Kasumi-1 cells led to a marked reduction of mRNA and protein expression of the fusion gene, whereas the expression of the wildtype RUNX1 gene was not affected. The surface expression of the M-CSF-Receptor, which is a known target gene of C/EBPα, was increased in the RUNX1-CBF2T1 depleted cells. Also, Kasumi-1 cells treated with the shRNA displayed a decrease in CD34 surface expression. In parallel, CD34 mRNA expression was reduced to 10%. To analyze, if CD34 downregulation of the Kasumi-1 cells after RUNX1-CBF2T1 depletion correlates with a loss of progenitor status, the clonogenicity of the cells in semisolid medium was investigated. In Kasumi-1 cells treated with the shRNA against RUNX1-CBFA2T1 the number of spontaneous colonies after 14 days of culture was reduced to about 30% in comparison to cells expressing the control shRNA. In conclusion, these experiments show that RUNX1-CBF2T1 expression can be stably suppressed in t(8;21) positive cells by endogenously expressed shRNAs thereby reducing their clonogenic potential.