HOX-Gene Expression and Inhibition of the Non-Canonical NF-KappaB Pathway in t(7;12) Leukemias

The chromosomal translocation t(7;12) was described recently in MLL negative acute myeloid leukemias of infancy associated with a very poor clinical outcome. The rearrangement involves the genes HLXB9 (7q36) and ETV6 (12p13) with a fusion transcript of exon1/HLXB9 and exon3/ETV6. An alternative in frame splicing variant of exon1/HLXB9 to exon2/ETV6 is also detectable. Leukemic bone marrow samples of 42 infants, diagnosed in Germany with AML, were screened for the fusion transcript HLXB9/ETV6 with RT-PCR. Inclusion criteria were diagnosis of AML, age<2 years, no MLL/AF9, MLL/AF4 or MLL/ENL rearrangement in RT-PCR, no chromosomal 11q23 rearrangement and no trisomy 21. Positive samples were cloned and sequenced. A fusion transcript was found in approx. ~17% of patients. Common features of HLXB9/ETV6 positive leukemias in this cohort are poorly differentiated FAB subtypes, trisomy 19 karyotype of the leukemic cells, coexpression of the T-cell markers CD4 and CD7 as well as high expression of the un-rearranged HLXB9 gene. Quantitative PCR showed no upregulation of HOXA9 and MEIS1 in HLXB9/ETV6 positive patients as reported for MLL+ leukemias. The mechanism of HLXB9/ETV6 induced leukemogenesis remains unknown. We show that in vitro retroviral transduction of murine hematopoietic precursor cells with either HLXB9/ETV6 or HLXB9 or ETV6/RUNX1 was not transforming. Furthermore altered activity of the transcription factor family NF-kappaB has been demonstrated for various types of cancer including acute myeloid leukemias. Therefore we analysed the impact of the HLXB9/ETV6 fusion on the NF-kappaB signaling pathway in a 293T cell-system. HLXB9/ETV6 and HLXB9 showed inhibition of the NF-kappaB dependent transcription signal in a reporter assay, whereas the typical ALL-fusion ETV6/RUNX1 showed no influence. Analysis of nuclear extracts by western blot confirmed, that the inhibition of the NF-kappaB signal is due to a reduced nuclear translocation of the non-canonical NF-kappaB components p52 and RelB, whereas the canonical pathway remains not influenced.

Conclusion: The HLXB9/ETV6 fusion transcript can be found in 17% of infants with MLL-negative AML in a German cohort. HLXB9/ETV6 positive leukemias show no increased expression on HOXA9 and MEIS1, but coexpression of T-cell markers and inhibition of the non-canonical NF-kappaB pathway on protein level. HLXB9/ETV6 did not induce malignant transformation in murine hematopoietic precursor cells.