C/EBP Suppression by Interruption of CUGBP1 Resulting from a Complex Rearrangement of MLL.

CCAAT/enhancer binding proteins (C/EBPs) are transcription factors that play a role in myeloid differentiation and may be dysregulated in myeloid leukemias as targets of BCR/ABL, ETO, and FLT3 mutations. CUG triplet repeat binding protein-1 (CUGBP1) is a translational regulator of C/EBP-β that increases the translation of full length C/EBP-β (LAP) and a dominant negative isoform, LIP. Translocations involving the human mixed-lineage leukemia gene (MLL) on chromosome 11 are leukemogenic events that result in a characteristic profile of HOX gene expression and confer a poor prognosis. Expression of AF1q, a gene involved in normal hematopoiesis, correlates with increased risk in acute myeloid leukemia (AML) and myelodysplastic syndrome and is fused with MLL in translocation t(1;11)(q21;q23). Here we describe a complex rearrangement of chromosome 1 and 11 involving translocation t(1;11)(q21;q23) and a separate event disrupting the MLL gene by either insertion or translocation of the 3′ region into the p arm of chromosome 11 (p11). Both activation of HOX genes and inactivation of C/EBP result from this MLL rearrangement. In blasts obtained from an infant with newly diagnosed AML, G-band karyotyping and fluorescence in situ hybridization (FISH) for chromosome 1 and 11 telomeres revealed an apparent reciprocal translocation. FISH for MLL, however, identified the 3′ end of MLL on 11p rather than the expected 1q. Array comparative genomic hybridization (aCGH, SpectralChip 2600), consisting of BACs that provide an average resolution of 1.0 MB across the genome, revealed no genomic losses or gains. Genomic long distance inverse polymerase chain reaction (LDI-PCR) for the 5′ portion of MLL identified fusion of MLL with AF1q at introns 10/11 and 1/2, respectively. LDI-PCR for the 3′ portion of MLL identified a novel fusion with CUGBP1, located on 11p and consistent with the 3′ FISH probe for MLL. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the predicted MLL-AF1q fusion transcript but did not identify a CUGBP1-MLL product. To confirm that MLL-AF1q was an activating fusion product, we assessed HOXA9 expression in four AML samples by RT-PCR and found HOXA9 in this AML sample to be highly elevated. We then hypothesized that interruption of the CUGBP1 gene could lead to an arrest in differentiation by a decrease in C/EBP expression. We found CUGBP1 expression by quantitative RT-PCR to be markedly reduced in this sample compared to all other AML samples tested. Western blot analyses confirmed the low expression of CUGBP1 and demonstrated marked reduction in C/EBP-β expression. These findings suggest a complex rearrangement event that caused (a) MLL activation by fusion with AF1q and (b) C/EBP suppression by loss of transcriptional enhancement from CUGBP1. This rearrangement could have resulted from two sequential events:

1. independent translocation and deletion/insertion events or

2. a single crossover event involving four recombination sites.

The sequences obtained by LDI-PCR showed complete conservation of the genomic MLL sequence, suggesting the latter mechanism. This is the first report to suggest a role for CUGBP1 in leukemogenesis.