POU1F1 is a novel fusion partner of NUP98 in acute myeloid leukemia with t(3;11)(p11;p15)

Abstract

Background: NUP98 gene rearrangements have been reported in acute myeloid leukemia, giving rise to fusion proteins that seem to function as aberrant transcription factors, and are thought to be associated with poor prognosis.

Findings: A patient with treatment-related acute myeloid leukemia presented a t(3;11)(p11;p15) as the only cytogenetic abnormality. FISH and molecular genetic analyses identified a class 1 homeobox gene, POU1F1, located on chromosome 3p11, as the fusion partner of NUP98. In addition, we have found that the patient harbored an FLT3-ITD mutation, which most likely collaborated with the NUP98-POU1F1 fusion gene in malignant transformation.

Conclusions: We have identified POU1F1 as the NUP98 fusion partner in therapy-related AML with a t(3;11)(p11;p15). This is the first POU family member identified as a fusion partner in human cancer.

Figure 1

Cytogenetic and fluorescence in situ hybridization studies of the AML patient. The bone marrow sample was cultured for 24 h following standard procedures. Chromosome banding analysis of the bone marrow was performed after Leichmann’s staining and the karyotype was described according to ISCN [4]. A) Bone marrow karyotype obtained by GTL banding technique exhibiting a t(3;11)(p11;p15) as the sole chromosome abnormality, suggesting the involvement of the NUP98 gene located at 11p15. Derivative chromosomes 3 and 11 are marked with arrows. BAC clones for NUP98 and POU1F1 were selected using the UCSC Human Genome Browser and obtained from the BACPAC Resource Center. B) Dual color, dual fusion FISH analysis performed on metaphases using BAC probes for NUP98 (CTD-3082 N2, RP11-161I4) labeled in green and for POU1F1 (CTD-2372C6) labeled in red, showed the presence of fusion signals on der(3) and der(11) and isolated red and green signals on normal chromosomes 3 and 11, respectively.

Figure 2

Characterization of the NUP98-POU1F1 fusion. A) RT-PCR analysis with one antisense primer on POU1F1 exon 5 and three sense primers on NUP98 exons 9, 10, and 11 (lanes 3, 5, and 7) showed the presence of PCR fragments of 362, 242, and 101 bp, respectively, suggestive of a fusion between NUP98 exon 11 and POU1F1 exon 5. Additional RT-PCR analysis with several NUP98 and POU1F1 primer combinations gave additional support to this hypothesis since no amplification was observed (lanes 2, 4, 6, 8, and 9). Lane 10: RNA integrity check (370 bp B2M gene fragment). Lanes 1 and 11: 100 bp molecular marker. B) Sequence analysis followed by a BLAST search confirmed that NUP98 exon 11 was fused in-frame with nucleotide 730 of the POU1F1 transcript (arrow). C) Schematic representation of the NUP98, POU1F1, and the NUP98-POU1F1 fusion proteins showing the relevant domains of the partner and chimeric proteins. D) Genomic breakpoint analysis with the NUP98_Fint11H sense primer in combination with the POU1F1_Rint4B and POU1F1_Rint4A antisense primers gave origin to amplification products of 603 and 412 bp, respectively (lanes 1 and 2). Lane 3: 100 bp molecular marker. E) Sequencing of the amplification products showed that the breakpoint was located 7490 bp downstream of NUP98 exon 11 and 129 bp downstream of the start of POU1F1 exon 4 (arrow). F) Schematic representation of the genomic DNA breakpoint (arrow) and nucleotide sequence of the genomic breakpoint of the translocation t(3;11) and corresponding normal chromosomes 3 and 11.