8q24 amplified segments involve novel fusion genes between NSMCE2 and long noncoding RNAs in acute myelogenous leukemia

The pathogenetic roles of 8q24 amplified segments in leukemic cells with double minute chromosomes remain to be verified. Through comprehensive molecular analyses of 8q24 amplicons in leukemic cells from an acute myelogenous leukemia (AML) patient and AML-derived cell line HL60 cells, we identified two novel fusion genes between NSMCE2 and long noncoding RNAs (lncRNAs), namely, PVT1-NSMCE2 and BF104016-NSMCE2. Our study suggests that 8q24 amplicons are associated with the emergence of aberrant chimeric genes between NSMCE2 and oncogenic lncRNAs, and also implicate that the chimeric genes involving lncRNAs potentially possess as-yet-unknown oncogenic functional roles. Electronic supplementary material The online version of this article (doi:10.1186/s13045-014-0068-2) contains supplementary material, which is available to authorized users.

Consequently, three common amplicons were identified between 8q24.13-21 in the patient and the HL60 cells; i.e., the regions covering NSMCE2 (8q24.13), PVT1 (8q24.21) and CCDC26 (8q24.21) (Figures 1c and 2b). Further investigation revealed three fusion transcripts between PVT1 exon 1a and NSMCE2 exon 3 in the patient (Figure 1d and e), and a fusion gene between exon 6 of NSMCE2 and exon 1 of BF104016, a noncoding RNA sharing the sequence of CCDC26 exon 4 (Additional file 3: Figure S1) (Additional file 4: Table S2), in the HL60 cells (Figure 2c-e). Both the NSMCE2 and PVT1 genes were amplified and located in a micronucleus in the patient (Figure 1f-i), and the genomic junction of 5'-PVT1-NSMCE2-3' was located within intron 1 of PVT1 and at 5' upstream of exon 1 of NSMCE2 (Figure 1j and k) (Additional file 5: Figure S3). In the HL60 cells, amplification of 3'NSMCE2 and 5'CCDC26 was colocalized on der(13)hsr(8), ins(2;8) and dmins (Figure 2e-h) (Additional file 5: Figure S3). Aberrant NSMCE2 transcripts were higher than normal NSMCE2 transcripts in the patient and the HL60 cells, while NSMCE2 protein expression did not correlate with normal or abnormal NSMCE2 transcripts among the leukemic patient cells or the HL60 cells, suggesting the presence of regulatory mechanisms other than transcription (Additional file 6: Figure S2).   The present findings are consistent with previous studies demonstrating that segmental genome amplification of 8q24 contains recurrent PVT1 fusion genes, which might be generated by chromothripsis [2,3]. Both lncRNAs, PVT1 and CCDC26, harbor retroviral integration sites and are transcribed into multiple splice forms [4][5][6]. PVT1 overexpression is induced by MYC or p53, contributing to suppression of apoptosis [7][8][9], whereas PVT1 produces six annotated microRNAs that have been implicated in oncogenesis [3,10,11]. The chimeric transcripts involving PVT1 may also regulate the expression of as-yet unspecified target genes through "enhancer-like functions" [12]. CCDC26 amplification has been also identified as a recurrent abnormality that is associated with the response to retinoic acid-induced differentiation in AML [1,11,[13][14][15][16]. This study is the first to identify NSMCE2-associated fusion genes in AML [17][18][19]. Knockdown of NSMCE2 induces chromosomal instability and increases the frequency of chromosomal breakage and loss [20]. We speculate that NSMCE2 gene rearrangement may potentially influence its function. Collectively, our study identified novel PVT1-NSMCE2 and CCDC26-NSMCE2 fusion genes that may play functional roles in leukemia.

Additional files
Additional file 1: Supplementary material information.
Additional file 2: Table S1. CNAG analysis of the region between the MTDH and LRRC6 genes on 8q24 in patient 1 with marker chromosomes. Results show the genomic size of the eight amplified segments that were selected based on the existence of known genes within them and their approximate positions.
Additional file 4: Table S2. Sequences of the primers used in this study.
Additional file 5: Figure S3 Additional file 6: Figure S2. Expression of NSMCE2 in patient 1 and AML-derived cell lines. (a) NSMCE2 mRNA levels measured by RQ-PCR (n=3, mean ± SD). Theoretically, the NSMCE2 7-8 primer/probe can amplify both normal and aberrant NSMCE2 transcripts, while the NSMCE2 2-3 primer/probe set which can amplify only normal NSMCE2 transcript. NSMCE2 mRNA levels were normalized to β-actin and are relative to the control mRNA extracted from normal BM cells. NSMCE2 mRNA levels amplified by the NSMCE2 7-8 primer/probe set are higher than those amplified by the NSMCE2 2-3 primer/probe set in patient 1, HL60 and KG1 cells.