Recurrent fusion of MYB and NFIB transcription factor genes in carcinomas of the breast and head and neck

Abstract

The transcription factor gene MYB was identified recently as an oncogene that is rearranged/duplicated in some human leukemias. Here we describe a new mechanism of activation of MYB in human cancer involving gene fusion. We show that the t(6;9)(q22-23;p23-24) translocation in adenoid cystic carcinomas (ACC) of the breast and head and neck consistently results in fusions encoding chimeric transcripts predominantly consisting of MYB exon 14 linked to the last coding exon(s) of NFIB. The minimal common part of MYB deleted as the result of fusion was exon 15 including the 3'-UTR, which contains several highly conserved target sites for miR-15a/16 and miR-150 microRNAs. These microRNAs recently were shown to regulate MYB expression negatively. We suggest that deletion of these target sites may disrupt repression of MYB leading to overexpression of MYB-NFIB transcripts and protein and to activation of critical MYB targets, including genes associated with apoptosis, cell cycle control, cell growth/angiogenesis, and cell adhesion. Forced overexpression of miR-15a/16 and miR-150 in primary fusion-positive ACC cells did not significantly alter the expression of MYB as compared with leukemic cells with MYB activation/duplication. Our data indicate that the MYB-NFIB fusion is a hallmark of ACC and that deregulation of the expression of MYB and its target genes is a key oncogenic event in the pathogenesis of ACC. Our findings also suggest that the gain-of-function activity resulting from the MYB-NFIB fusion is a candidate therapeutic target.

Fig. 1.

The t(6;9) translocation in ACC results in a MYB-NFIB fusion. (A) Partial SKY-karyotype showing the reciprocal t(6;9) translocation in ACC6. DAPI-banded chromosomes 6 and 9 are shown on the left. (B) Dual-color FISH analysis of a t(6;9)-positive tumor (ACC6) using the NFIB-specific YAC-912E9 (green signals) and BAC clones RP11–104D9 and RP11–349J5 containing the 5′ part of MYB and its flanking sequences (red signals). Chromosome 6 is identified by a red alpha-satellite probe and chromosome 9 by a red telomere probe on the q-arm. Note the presence of the MYB-NFIB fusion gene on the der (6) marker (fused red/green signals marked by arrow). (C) Schematic illustration depicting the MYB and NFIB genes as well as the MYB-NFIB fusion gene (coding exons are shown in darker red and blue) and the resulting fusion protein. Translocation breakpoints are shown by vertical arrows, and miRNA binding sites for miR-15a/16 and miR-150 in the 3′ UTR of MYB are indicated. DBD, DNA binding domain; NRD, negative regulatory domain; TAD, transactivation domain. (D) RT-PCR analyses of MYB-NFIB fusion transcripts using primers located in MYB exons 5/6 and NFIB exon 9 (ACC4, -7, -6) and in MYB exon 14 and NFIB exon 9 (ACC1, -2, -3, -5, - 8–11). Also shown are size markers (M), non-ACC tumors (MEC and PLGA), and negative control. (E) Partial chromatogram showing the MYB-NFIB junction (vertical lines) and the nucleic acid sequence of the chimeric transcript with an in-frame fusion of MYB exon 14 to NFIB exon 9.

Fig. 2.

The t(6;9) translocation in ACC results in overexpression of MYB-NFIB mRNA and protein. (A) Q-PCR analyses of the expression of MYB in ACC1–11 (dark blue bars) and in non-ACC tumors (PLGA, MEC, and Ca-ex-PA) (light blue bars) compared with normal salivary gland (NSG) and breast (NBT) tissues (white bars). (B) Immunostaining of the MYB-NFIB fusion protein in a primary t(6;9)-positive cribriform type of ACC (ACC2). Note the strong nuclear staining of tumor cells and the absence of staining in stromal cells.

Fig. 3.

Forced overexpression of miR-15a, miR-16, and miR-150 in T-ALL and primary ACC cells. (A) Q-PCR analyses of MYB mRNA expression in MOLT-4 cells with duplication/activation of MYB and in MYB-NFIB–expressing primary ACC2 and -6 cells after transfection with a pool of premiR-15a, premiR-16, and premiR-150 oligos. The data represent the average of at least 3 independent experiments. T, transfected; UT, untransfected. (B) Transfection of primary ACC6 cells with a Cy3-labeled premiR negative control shows a transfection efficiency of more than 95%. BF, bright field.