Identification of a Novel Alternatively Spliced Form of AML1-ETO and Analysis of Its Role in Leukemogenesis.

Abstract

The 8;21(q22;q22) translocation is found in 12% of all AML cases, including 40–50% of AML M2 subtype and a small portion of M0, M1, and M4 subtypes. The translocation brings together the DNA sequence encoding the N-terminus of the AML1 (RUNX1) with nearly all of ETO (MTG8) to generate the AML1-ETO fusion protein. The most commonly known AML1-ETO fusion protein (full length AML1-ETO) is encoded by AML1 exons 1 to 4 and by ETO exons 2 to 11 and has 752 a.a. However, a recently discovered ETO exon 9a can provide a protein translation stop codon after the last amino acid encoded by exon 8. The predicted AML1-ETO protein using exon 9a (AML1-ETO9a) has 575 a.a. and does not include the NHR3 and NHR4 domains at the C-terminus of full length AML1-ETO. To demonstrate the presence of the AML1-ETO9a transcript, we performed RT-PCR using RNA prepared from t(8;21) patient samples. Exon 9a transcript was detected in all patient samples when PCR generated a 595 bp fragment with ETO exon 6 and exon 9 primers and was detected in seven of the 14 samples when PCR generated a 1381 bp fragment with AML1 exon 4 and ETO exon 9a primers, indicating the presence of AML1-ETO9a mRNA in patient samples. The predicted molecular weight of AML1-ETO9a protein with 575 a.a. is 63.8 kDa and a 64 kDa AML1-ETO fusion protein has been reported in both Kasumi-1 cells and t(8;21) leukemic blasts. We compared the effects of AML1-ETO and AML1-ETO9a on proliferation and differentiation of hematopoietic cells using FDCPmix cell line, which is an IL-3 dependent multipotent hematopoietic stem cell line. Both forms of fusion proteins inhibited the myeloid lineage differentiation when cells were cultured in the presence of GM-CSF and G-CSF. However, these fusion proteins showed opposite effects on cell cycle progression. AML1-ETO suppressed cell cycle with prolonged G0/G1; AML1-ETO9a enhanced cell cycle with shortened G0/G1. Expression of these fusion proteins in primary bone marrow cells showed a similar result. Most importantly, only AML1-ETO9a efficiently induced acute myeloid leukemia in mice. Thus, the observation that a shorter form of AML1-ETO is a dominant factor in t(8;21) related leukemogenesis exhibits a new paradigm in the understanding of t(8;21) tumorigenesis.