Development and Characterization of MYB-NFIB Fusion Expression in Adenoid Cystic Carcinoma

Abstract

Adenoid cystic carcinoma (ACC) is the second most common cancer type arising from the salivary gland. The frequent occurrence of chromosome t(6;9) translocation leading to the fusion of MYB and NFIB transcription factor genes is considered a genetic hallmark of ACC. This inter-chromosomal rearrangement may encode multiple variants of functional MYB-NFIB fusion in ACC. However, the lack of an ACC model that harbors the t(6;9) translocation has limited studies on defining the potential function and implication of chimeric MYB-NFIB protein in ACC. This report aims to establish a MYB-NFIB fusion protein expressing system in ACC cells for in vitro and in vivo studies. RNA-seq data from MYB-NFIB translocation positive ACC patients' tumors and MYB-NFIB fusion transcript in ACC patient-derived xenografts (ACCX) was analyzed to identify MYB breakpoints and their frequency of occurrence. Based on the MYB breakpoint identified, variants of MYB-NFIB fusion expression system were developed in a MYB-NFIB deficient ACC cell lines. Analysis confirmed MYB-NFIB fusion protein expression in ACC cells and ACCXs. Furthermore, recombinant MYB-NFIB fusion displayed sustained protein stability and impacted transcriptional activities of interferon-associated genes set as compared to a wild type MYB. In vivo tumor formation analysis indicated the capacity of MYB-NFIB fusion cells to grow as implanted tumors, although there were no fusion-mediated growth advantages. This expression system may be useful not only in studies to determine the functional aspects of MYB-NFIB fusion but also in evaluating effective drug response in vitro and in vivo settings.

Keywords: 9) translocation; MYB; MYB-NFIB fusion; adenoid cystic carcinoma; chromosomal t(6.

Figure 1

Schematic of MYB locus showing breakpoint locations. RNA-seq data from the indicated cases of ACC patient tumors was analyzed by combined NeoFuse and Arriba tool to identify MYB breakpoints. * Indicates cases where MYB breakpoint was also detected at exon 7.

Figure 2

Expression of MYB and MYB-NFIB fusion in ACCXs. (A). RNA extracted from the indicated ACCXs were evaluated for expression of MYB-NFIB fusion or MYB^wt by RT-PCR. HA-tagged MYB^wt or HA-MYB-NFIB plasmid cDNA were included for reference control. GAPDH was used as input control. (B). Protein lysates from ACCXs were analyzed using an antibody that recognizes an N-terminal epitope of MYB. Arrows indicate smaller MYB bands. They correlate with the presence of MYB-NFIB product detected in “A” and likely represent MYB-NFIB fusion protein. (C). Protein lysates as in “B” were analyzed using an antibody that recognizes a C-terminal epitope of MYB. Note that the smaller MYB band from “B” is undetectable.

Figure 3

Expression and characterization of MYB-NFIB fusion. (A), Sequencing chromatogram showing MYB-NFIB fusion junctions as derived from three ACCXs. Two variants of MYB-NFIB fusion were detected in ACCX29 (ii and iii) and ACCX20M1 (iv and v) with its second form (iii and v) representing a variant lacking 9 nucleotides on MYB exon 8. * Note that in MYB-NFIB fusion from ACCX11 (i), MYB region represent isoform 2 as sequencing revealed the lack of exon 10 normally present in the larger isoform 1. (B), Schematic representation of dox-inducible FLAG-tagged MYB^wt and MYB-NFIB fusion constructs. Striped and shaded box represents the position of NFIB and FLAG-tag, respectively. DBD: DNA binding domain; TAD transactivation domain; NRD: negative regulatory domain. (C), Immunoblotting analysis of MYB^wt and MYB-NFIB fusion expression in ACC-01 and HACC-2A cells following dox induction. Complementary antibodies (anti-FLAG and N-terminal epitope anti-MYB) were used to verify the expression. (D), ACC-01 cells expressing the various MYB-NFIB fusion variants were verified by immunoblotting with anti-FLAG. In A, B and D, the ”s” represent the absence of 9 nucleotides.

Figure 4

Differential protein stability of MYB^wt and MYB-NFIB fusion. (A,B), Stable ACC-01 cells harboring FLAG-tagged MYB^wt or MYB-NFIB fusion were dox-induced for 24 h, then re-cultured in media without dox for the indicated time period. Cell lysates were prepared and analyzed by Western blotting followed by densitometry to determine half-life of MYB^wt and MYB-NFIB fusion. (C,D), Cells as above induced with dox for 24 h were treated with cycloheximide (CHX; 100 ug/mL) for the indicated time period. Cell lysates were prepared and subjected to immunoblotting and densitometric analysis. (E,F), HACC-2A cells expressing dox-induced FLAG-tagged MYB^wt or MYB-NFIB fusion were incubated with CHX and analyzed as above.

Figure 5

Correlation of MYB-NFIB fusion expression with gene enrichment associated in interferon (IFN) signaling pathway. (A), Reactome pathway enrichment analysis of 215 annotated genes (FDR < 0.05) found in the MYB^wt versus MYB-NFIB fusion differential gene expression analysis. The x-axis indicates the proportion of genes from this set contained in each pathway’s gene set. Twenty-two IFN pathway-associated genes were found in all. (B), Heat-map representation of the normalized and centered gene expression data of the 22 IFN signaling pathway-associated genes. Samples and genes were ordered using the hierarchical clustering with complete linkage of their Euclidean distances. Note: one MYB^wt sample was excluded from the overall analysis because its RNA sequencing read counts were too low to be included in the HiSeq4000 input. (C), Validation of the upregulated genes by qPCR. RNA extracted from parental control, MYB^wt or MYB-NFIB fusion cells incubated in dox-media for 24 h were analyzed for the expression of the various gene targets. (D), qPCR analysis of the target genes in HACC-2A cells expressing MYB^wt or MYB-NFIB fusion. (E), RNA extracted from the indicated ACCXs were evaluated for the expression of representative gene targets as shown.

Figure 6

In vivo tumor growth analysis of MYB-NFIB fusion expressing ACC-01 cells. (A), Mice were implanted subcutaneously with parental or stable cell lines of FLAG-tagged MYB^wt or MYB-NFIB fusion constructs as indicated (n = 6–8 each group). Mice were fed with dox containing drinking water. Tumor growth was then assessed up to 11 weeks. Palpable development of tumor at the end of 8 weeks was considered positive tumor growth. (B), Protein extracts from two representative tumors of each group were analyzed for MYB^wt or MYB-NFIB fusion expression by immunoblotting.