NHR4 domain mutations of ETO are probably very infrequent in AML1|[ndash]|ETO positive myeloid leukemia cells

In acute myeloid leukemia (AML), molecular analyses of chromosomal translocations have led to the discovery of gene rearrangements, which can result in block of differentiation and uncontrolled cellular growth.1 One such translocation, t(8;21)(q22;q22) generating the fusion protein AML1–ETO, is found in up to 15% of AML patients.2 This fusion protein consists of the N terminus of AML1 and nearly the entire ETO protein with its four Nervy homology regions (NHRs) (Figure 1). The DNA-binding Runt domain of AML1 is believed to be crucial for leukemogenic activity of AML1–ETO and while only little is known about the contribution of NHR3 to malignant transformation, NHR1 and NHR2 (mediating oligomerization with AML1-ETO) seem to be essential in inhibiting cell proliferation and differentiation.3 NHR4 exhibits a zinc-chelating motif and might contribute to leukemogenesis by recruiting corepressors resulting in transcriptional repression of AML1–ETO target genes, but there exists also evidence that NHR4 inhibits leukemia development by recruiting proteins that cause growth arrest in AML1–ETO-positive cells.3, 4 However, although AML1–ETO is believed to significantly contribute to leukemogenesis, it was shown that this chimeric protein by itself is not sufficient to cause leukemia.5 Thus, the search for ‘second hits’ in t(8;21) leukemia has become the focus of intense investigations including SNP genomic arrays analysis.6 It could be demonstrated that the AML1–ETO fusion gene and FLT3 length mutations collaborate in inducing acute leukemia in mice and there is first evidence that the combination of AML1–ETO and JAK2 V617F mutations results in AML.7, 8 Recently, several studies have shown that in core-binding factor AML up to 40% of t(8;21) patients have additional mutations of the KIT gene, while the total incidence of c-KIT mutations in all AML is ~5%.9, 10 Mutations of the NRAS gene have been reported in up to 10% of AML1–ETO cases, whereas other mutations in genes as AML1 and PU.1 occur only rarely in combination with t(8;21).11 In a very recent study, Ahn et al.4 reported that in a mouse transplantation model, the exchange of one single amino acid within the zinc-chelating structure NHR4 domain of AML1–ETO resulted in rapid onset of leukemia as compared with the inability of unmutated AML1–ETO to induce AML. In this study, the leukemogenesis-inhibiting property of functional NHR4 to block the development of AML is at least in part due to the interaction with the DNA/RNA-binding domain protein SON. When investigating AML patients harboring t(8;21), it was further demonstrated that SON is abnormally localized in the cytoplasm and thus is not capable of interacting with NHR4.4 On the basis of these observations, we hypothesized that a somatic mutation of the NHR4 genomic sequence in AML1–ETO patients might be one possible explanation for the disrupted interplay of SON and AML1–ETO. To test this hypothesis, we extracted genomic DNA from one peripheral blood and 25 bone marrow samples of AML patients with t(8;21) from the University Medical Centers Freiburg, Germany and Nijmegen, The Netherlands. All patients had provided ethic board approved informed consent. The blast count in the peripheral blood sample was 35% and in bone marrow samples >70% in the majority of samples (range: 25–90%). In addition, to explore the mutation status in a homogeneous blast population, we investigated two AML cell lines bearing t(8;21), Kasumi-1 and SKNO. We amplified a 268-bp fragment of AML1–ETO encompassing the entire coding region of the NHR4 domain (aa658-707) (Table 1, Figure 1) and applied bidirectional sequencing (GATC Biotech, Konstanz, Germany). However, we identified no mutations within the NHR4 coding genomic sequence of these AML patients or the two cell lines. With the limitations of a cohort of 26 patient samples, we conclude that mutations within the NHR4 domain of AML1–ETO are probably a very infrequent event in patients with t(8;21). Our results also support the idea that in vivo, other mechanisms than NHR4 mutations, for example, a dysfunctional SON protein might be a reason for hampered interaction of AML1–ETO and SON. The authors declare no conflict of interest. This study was in part supported by a grant from the Deutsche Krebshilfe (108397 to BH). We thank Dong-Er Zhang, University of California, San Diego, USA for her very helpful comments with this study.