AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells

Abstract

The translocation t(8;21) yields the leukemic fusion gene AML1/MTG8 and is associated with 10%-15% of all de novo cases of acute myeloid leukemia. We demonstrate the efficient and specific suppression of AML1/MTG8 by small interfering RNAs (siRNAs) in the human leukemic cell lines Kasumi-1 and SKNO-1. siRNAs targeted against the fusion site of the AML1/MTG8 mRNA reduce the levels of AML1/MTG8 without affecting the amount of wild-type AML1. These data argue against a transitive RNA interference mechanism potentially induced by siRNAs in such leukemic cells. Depletion of AML1/MTG8 correlates with an increased susceptibility of both Kasumi-1 and SKNO-1 cells to tumor growth factor ␤ 1 (TGF␤ 1 )/vitamin D 3 -induced differentiation, leading to increased expression of CD11b, macrophage colony-stimulating factor (M-CSF) receptor, and C/EBP␣ (CAAT/enhancer binding protein). Moreover, siRNA-mediated AML1/MTG8 suppression results in changes in cell shape and, in combination with TGF␤ 1 /vitamin D 3 , severely reduces clonogenicity of Kasumi-1 cells. These results suggest an important role for AML1/MTG8 in preventing differentiation, thereby propagating leukemic blast cells. Therefore, siRNAs are promising tools for a functional analysis of AML1/MTG8 and may be used in a molecularly defined therapeutic approach for

The HDAC class I-specific inhibitor entinostat (MS-275) effectively relieves epigenetic silencing of the LAT2 gene mediated by AML1/ETO

“…Electroporation of Kasumi-1 cells with siRNAs was performed as described (Heidenreich et al, 2003;Dunne et al, 2006). Kasumi-1 cells were treated with different concentrations of HDAC inhibitors for 24 h: trichostatin A (Sigma, Taufkirchen, Germany) dissolved in ethanol; suberoylanilide hydroxamic acid (Alexis Biochemicals, San Diego, CA, USA) dissolved in methanol; entinostat (MS-275, SDX-275; kindly provided by Schering AG, now Bayer Schering Pharma, Berlin, Germany) dissolved in dimethyl sulfoxide; mocetinostat (MGCD0103, Selleck Chemicals, Houston, TX, USA) dissolved in dimethyl sulfoxide, valproic acid and phenylbutyrate (Sigma) dissolved in water.…”

“…We next studied the expression of LAT2 in an AML1/ ETO knockdown system (Heidenreich et al, 2003;Dunne et al 2006). Kasumi-1 cells were electroporated with a control siRNA (mock), with siRNA against AML1/ETO (siAGF1) or a mismatched siRNA control (siAGF6).…”

Histone deacetylase inhibitors induce the degradation of the t(8;21) fusion oncoprotein

“…It is unlikely that these effects are due solely to inducing the degradation of the fusion protein, as these compounds also trigger death receptormediated apoptosis (Insinga et al, 2005;Sutheesophon et al, 2005) and inhibit chromosomal condensation before mitosis Johnstone, 2002;Piekarz and Bates, 2004). Indeed, siRNAmediated knockdown of RUNX1-ETO expression had only modest effects on the viability of Kasumi-1 cells over short periods of time, but allowed the cells to be forced to differentiate in response to various agents (Heidenreich et al, 2003;Kasashima et al, 2004). Antiacetylated histone H3 K9 and antiacetylated histone H4 were used to assess histone acetylation.…”

ELA2 is regulated by hematopoietic transcription factors, but not repressed by AML1-ETO

“…To determine the effect of AML1-ETO on hELA2 expression in Kasumi-1 cells, expression of AML1-ETO was knocked down using siRNAs as previously described (Heidenreich et al, 2003). Figure 5b illustrates that effective reduction of AML1-ETO expression in Kasumi-1 cells can be achieved within 48 h. This results in only a modest increase in hELA2 levels (Figure 5c).…”

“…ORNs were 5 0 -CCUCGAAAUCGUACUGAGAAG-3 0 and 3 0 -UUG-GAGGCUUUAGCAUGACUCU-5 0 for AML1-ETO (Heidenreich et al, 2003). ORNs for AML1 were 5 0 -CCUCGAAGACAUCGGCAGAAA-3 0 and 3 0 -UUGGAG-CUUCUGUAGCCGUCUUU-5 0 .…”