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The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.

The lineage-determining transcription factor CCAAT enhancer binding protein α (C/EBPα) is required for myeloid differentiation. Decreased function or expression of C/EBPα is often found in human acute myeloid leukemia. However, the precise impact of C/EBPα deficiency on the maturation arrest in leukemogenesis is not well understood. To address this question, we used a murine transplantation model of a bcr/abl-induced myeloproliferative disease. The expression of bcr/abl in C/EBPα pos fetal liver cells led to a chronic myeloid leukemia-like disease. Surprisingly, bcr/abl-expressing C/EBPα −/− fetal liver cells failed to induce a myeloid disease in transplanted mice, but caused a fatal, transplantable erythroleukemia instead. Accordingly, increased expression of the transcription factors SCL and GATA-1 in hematopoietic precursor cells of C/EBPα −/− fetal livers was found. The mechanism for the lineage shift from myeloid to erythroid leukemia was studied in a bcr/abl-positive cell line. Consistent with findings of the transplant model, expression of C/EBPα and GATA-1 was inversely correlated. Id1, an inhibitor of erythroid differentiation, was identified as a critical direct target of C/EBPα. Down-regulation of Id1 by RNA interference impaired C/EBPα-induced granulocytic differentiation. Taken together, our study provides evidence that myeloid lineage identity of malignant hematopoietic progenitor cells requires the residual expression of C/EBPα.

Background The fusion protein RUNX1-CBFA2T1 associated with t(8;21)-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t(8;21)-positive cell lines. Methods The t(8;21)-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence. Results Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity. Conclusions RUNX1-CBFA2T1 supports the proliferation and expansion of t(8;21)-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches.

The lineage-determining transcription factor C/EBPα is required for myeloid differentiation. Decreased function or expression of C/EBPα is often found in human acute myeloid leukemia. However, the precise impact of C/EBPα deficiency on the maturation arrest in leukemogenesis is not well understood. To address this question, we used a murine transplantation model of a bcr/abl induced myeloproliferative disease. The expression of bcr/abl in C/EBPα+/+ and C/EBPα+/− fetal liver cells lead to a chronic myeloid leukemia-like disease. Surprisingly, bcr/abl expressing C/EBPα−/− fetal liver cells fail to induce a myeloid disease in transplanted mice, but instead cause a fatal, transplantable erythroleukemia. Accordingly, increased expression of SCL and GATA-1 in hematopoietic precursor cells of C/EBPα−/− fetal livers was found. The mechanism for the lineage shift from myeloid to erythroid leukemia was studied in a bcr/abl positive cell line. Consistent with findings of the transplant model, expression of C/EBPα and GATA-1 was inversely correlated. Id1, an inhibitor of erythroid differentiation, was upregulated upon C/EBPα expression. Chromatin immunoprecipitation was done and C/EBPα binding to a 3 prime enhancer of the Id1 gene was observed. Downregulation of Id1 by RNA interference impaired C/EBPα induced granulocytic differentiation. Thus, Id1 is a direct and critical target of C/EBPα. Taken together, our study provides the first evidence that myeloid lineage identity of malignant hematopoietic progenitor cells requires the residual expression of C/EBPα.

The translocation t(15;17) in acute promyelocytic leukemia results in the PML/RARα fusion protein. PML/RARα recruits histone deacetylases (HDAC) and represses target genes of wild-type RARα. Recently, the anticonvulsant drug valproic acid (VPA) has been described as an HDAC inhibitor. In order to evaluate the role of VPA in the treatment of acute promyelocytic leukemia, the effect of VPA on the t(15;17) positive cell line NB4 was examined. To exclude toxicity on non malignant stem cells, CD34pos cells were analyzed. In NB4 cells, VPA led to an increase of acetylated histone H4. Histone acetylation was associated with a dose dependent inhibition of proliferation. Furthermore, inhibition of NB4 clonogenic growth was observed. In contrast, clonogenic growth of CD34pos cells was not affected by the presence of VPA. The VPA induced inhibition of cell growth in malignant cells was not caused by altered apoptosis. Rather an arrest at the G1/G0 phase was observed in the presence of VPA. In agreement with p21 expression of leukemic cell lines not harboring the t(15;17) translocation, VPA induced p21 mRNA expression also in t(15;17) positive cells. However, no induction of p21 was observed in normal CD34pos cells. VPA triggered the differentiation of NB4 cells measured by NBT test and surface expression of CD11b and CD11c. Flow cytometry of colonies from CD34pos cells showed an increased fraction of CD34pos cells in the presence of VPA. Thus, VPA does not induce differentiation in non malignant hematopoiesis. To study whether VPA and ATRA induced differentiation are mediated via identical pathways, the process of differentiation was studied in NB4 cells and ATRA resistant NB4-R2 cells. While differentiation in the presence of ATRA led to induction of C/EBPβ and C/EBPε, no change in the expression of these transcription factors was observed after VPA treatment. Downregulation of c-myc was detected upon ATRA as well as VPA treatment, an additive effect was seen after the combination. Resistance to ATRA did not interfere with effects of VPA on cell differentiation. However, upon ATRA treatment no c-myc downregulation was observed in the NB4-R2 cell line. Taken together, VPA acts as an HDAC inhibitor in t(15;17) positive cells and induces myeloid differentiation by mechanisms distinct from ATRA and independent of ATRA-resistance. Moreover, VPA does not interfere with normal hematopoiesis. Therefore, this substance might be helpful in patients with APL - especially in ATRA-resistant disease.

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.