Chromosome 11q23 aberrations activating FOXR1 in B-cell lymphoma

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Citation: Blood Cancer Journal (2016) 6, e433; doi:http://dx.doi.org/10.1038/bcj.2016.43

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LETTER TO THE EDITOR

Chromosome 11q23 aberrations activating FOXR1 in B-cell lymphoma

Blood Cancer Journal (2016) 6, e433; doi:http://dx.doi.org/10.1038/bcj.2016.43

Web End =10.1038/bcj.2016.43 ; published online 10 June 2016

Recurrent chromosome 11q23 abnormalities, including focal gains and losses have been described in mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL) and in a subset of high-grade B-cell lymphomas lacking MYC rearrangements.13 We describe a novel fusion of FOXR1 forkhead box gene, located at 11q23, with a neighboring gene in B-cell lymphoma.

RNAseq and sequencing of cloned PCR products revealed fusion transcripts of 5 Ribosomal Protein S25 (RPS25) with FOXR1 in the DLBCL cell line U-2932, both genes located at the amplied chromosomal region 11q23 (Figure 1a, Supplementary Figure S1A). Genomic cloning localized the breakpoint to intron 2/3 of RPS25 and to the promoter region of FOXR1 (bp 3532).

Cell line U-2932 comprises two distinct clones traceable to subclones present in the patients tumor.4 These differences also affected 11q32, FOXR1 and RPS25 being tetraploid in subclone R1, and triploid in subclone R2 (3n) (Supplementary Figure S1A). In accordance with the genomic data, the RPS25/FOXR1 fusion was detected in subclone R1 but not in R2 (Figure 1b). RPS25/FOXR1 was also veried in the patients DNA, which collectively suggested that the fusion had occurred at some later stages of tumor development (Figure 1b).

Physiological FOXR1 (formerly FOXN5) expression is restricted to the early stages of embryogenesis.5,6 Ectopic expression as result of 11q23 intrachromosomal deletion-fusion has hitherto been described in neuroblastoma only.7 In-frame fusions with the 5

MLL or PAFAH1B2 genes led to overexpression of FOXR1.7 In

accordance with the notion that a constitutively expressed 5 gene (RPS25) might be responsible for the ectopic expression of FOXR1 in B-cell lymphoma also, FOXR1 levels were 1000 higher in the fusion-positive than in the fusion-negative U-2932 subclone. Expression array analyses showed that the RPS25/FOXR1-positive U-2932 subclone had the highest FOXR1 expression level of 55 B lymphoma cell lines tested, three log-scales higher than average (Figure 2a). Quantitative PCR analysis conducted to verify the expression arrays included 17 additional B lymphoma cell lines, revealing that the primary effusion lymphoma cell line CRO-AP3

RPS25 and FOXR1 in cell lines

FOXR1 in cell lines

1.6

1.2

log2 fold change

relative expression (2^-Ct)

U-2932 R1

0.8

0.4

RPS25 exon 2

FOXR1 exon 2

0.0

NAMALWA

CARNAVAL

OCI-LY7

CRO-AP3

HT

U-2932

RPS25

FOXR1

FOXR1 (237613_at) expression in tumor

1 kb marker

DLBCL CLL

DLBCL

NTC

506 bp

298 bp

RPS25/FOXR1

4n 2n

FOXR1

Figure 2. FOXR1 expression in B lymphoma. (a) According to expression array analysis, RPS25 is constitutively expressed in 55 B lymphoma cell lines, FOXR1 is highest in the RPS25/FOXR1-positive U-2932 subclone (red dot). (b) Quantitative reverse-transcriptase PCR-veried ectopic expression in U-2932 subclone R1 and in the PEL cell line CRO-AP3. Cell lines NAMALWA (Burkitts lymphoma), CARNAVAL, OCI-LY7 and HT (all DLBCL) do not express FOXR1. (c) Reanalysis of previously published normalized expression proling data showing ectopic FOXR1 expression in primary DLBCL and chronic lymphocytic leukemia (processed data from GEO).810

Red dots indicate FOXR1-high outliers.

chr. 11q23.3

Figure 1. FOXR1 aberration in B lymphoma cell lines. (a) RPS25 exon 2 / FOXR1 exon 2 fusion expressed in U-2932 subclone R1. Two additional transcripts targeting RPS25 exon 2 with FOXR1 5

sequences were also detected. (b) RPS25/FOXR1 fusion in patients DNA and in one of two subclones of patient-derived cell line. Cell lines OCI-LY3 and HL-60 were used as negative controls. Size of PCR product: 294 bp. (c) FOXR1 was amplied (4n) in CRO-AP3 according to Cytoscan HD Array analysis (Affymetrix, Santa Clara, CA, USA). NTC, nil template control.

Letter to the Editor

2

expressed FOXR1 at a level similar to the DLBCL cell line U-2932 (Figure 2b). High-density genomic array analysis demonstrated copy-number transition from 3n to 4n in CRO-AP3, occurring 5 of

FOXR1 (Figure 1c, Supplementary Figure S1B). Quantitative genomic PCR localized the site of amplication to the rst 170 bases of exon 1. 5-RACE, performed to identify potential 5-mRNA partners in the two FOXR1 expressing cell lines, conrmed RPS25 as fusion partner of FOXR1 in U-2932. In CRO-AP3, the 5-RACE PCR product terminated inside the amplied region of FOXR1 exon 1, upstream of the open reading frame. These results suggested that in CRO-AP3, FOXR1 overexpression was the result of gene amplication without fusion mRNA formation. Fluorescence in situ hybridization using a fosmid clone covering FOXR1 (G248P85736G6) yielded wild-type signals only restricted to chromosome 11 (not shown), leaving the putative 5 regulatory gene elusive.

Santo et al.7 reported that FOXR1 acts as negative regulator of forkhead box factor-mediated transcription and suggested a possible role in tumorigenesis. We found ectopic expression of FOXR1 in 2/72 (2.8%) B lymphoma cell lines. Both cell lines showed amplication of the FOXR1 gene. In one cell line, FOXR1 was fused to a constitutively expressed gene on 11q23, suggesting that the interstitial deletion was responsible for activation of FOXR1. Bioinformatic analyses document that the aberrant expression of FOXR1 is rare, but recurrent in B-cell lymphoma (Figure 2c).810

In conclusion, we show for the rst time that FOXR1 fusions, described as candidate oncogenes in neuroblastoma, also occur in B-cell lymphoma. Cell lines U-2932 and CRO-AP3 are presented as models for the functional analysis of FOXR1-mediated cellular events.

CONFLICT OF INTEREST

The authors declare no conict of interest.

2Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala University Hospital, Uppsala, Sweden and

3Genome Analytics Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany

E-mail: [email protected]

REFERENCES

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Amplication at 11q23 targets protein kinase SIK2 in diffuse large B-cell lymphoma. Leuk Lymphoma 2010; 51: 881891.

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A recurrent 11q aberration pattern characterizes a subset of MYC-negative high grade B-cell lymphomas resembling Burkitt lymphoma. Blood 2014; 123: 11871198.

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U-2932: two clones in one cell line, a tool for the study of clonal evolution.Leukemia 2013; 27: 11551164.

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7 Santo EE, Ebus ME, Koster J, Schulte JH, Lakeman A, van Sluis P et al. Oncogenic activation of FOXR1 by 11q23 intrachromosomal deletion-fusions in neuroblastoma. Oncogene 2012; 31: 15711581.

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9 Linton K, Howarth C, Wappett M, Newton G, Lachel C, Iqbal J et al. Microarray gene expression analysis of xed archival tissue permits molecular classication and identication of potential therapeutic targets in diffuse large B-cell lymphoma. J Mol Diagn 2012; 14: 223232.

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The Author(s) 2016

Supplementary Information accompanies this paper on Blood Cancer Journal website (http://www.nature.com/bcj)

ACKNOWLEDGEMENTS

We thank Wilhelm Sander-Stiftung for nancial support.

C Pommerenke1, V Hauer1, M Zaborski1, RAF MacLeod1, S Nagel1, RM Amini2, M Berglund2, R Geffers3,

HG Drexler1 and H Quentmeier1

1Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany;

Blood Cancer Journal