Non-IG Aberrations of FOXP1 in B-Cell Malignancies Lead to an Aberrant Expression of N-Truncated Isoforms of FOXP1 Leila Rouhigharabaei 1. , Julio Finalet Ferreiro 1. , Thomas Tousseyn 2 , Jo-Anne van der Krogt 1 , Natalie Put 1 , Eugenia Haralambieva 3 , Carlos Graux 4 , Brigitte Maes 5 , Carmen Vicente 1,6 , Peter Vandenberghe 1 , Jan Cools 1,6 , Iwona Wlodarska 1 * 1 Center for Human Genetics, KU Leuven, Leuven, Belgium, 2 Translational Cell and Tissue Research KU Leuven, Department of Pathology UZ Leuven, Leuven, Belgium, 3 Department of Pathology, University of Wu ¨ rzburg, Wu ¨ rzburg, Germany, 4 Mont-Godinne University Hospital, Yvoir, Belgium, 5 Virga Jesse Hospital, Hasselt, Belgium, 6 Center for the Biology of Disease, VIB, Leuven, Belgium Abstract The transcription factor FOXP1 is implicated in the pathogenesis of B-cell lymphomas through chromosomal translocations involving either immunoglobulin heavy chain (IGH) locus or non-IG sequences. The former translocation, t(3;14)(p13;q32), results in dysregulated expression of FOXP1 juxtaposed with strong regulatory elements of IGH. Thus far, molecular consequences of rare non-IG aberrations of FOXP1 remain undetermined. Here, using molecular cytogenetics and molecular biology studies, we comprehensively analyzed four lymphoma cases with non-IG rearrangements of FOXP1 and compared these with cases harboring t(3;14)(p13;q32)/IGH-FOXP1 and FOXP1-expressing lymphomas with no apparent structural aberrations of the gene. Our study revealed that non-IG rearrangements of FOXP1 are usually acquired during clinical course of various lymphoma subtypes, including diffuse large B cell lymphoma, marginal zone lymphoma and chronic lymphocytic leukemia, and correlate with a poor prognosis. Importantly, these aberrations constantly target the coding region of FOXP1, promiscuously fusing with coding and non-coding gene sequences at various reciprocal breakpoints (2q36, 10q24 and 3q11). The non-IG rearrangements of FOXP1, however, do not generate functional chimeric genes but commonly disrupt the full-length FOXP1 transcript leading to an aberrant expression of N-truncated FOXP1 isoforms (FOXP1 NT) , as shown by QRT-PCR and Western blot analysis. In contrast, t(3;14)(p13;q32)/IGH-FOXP1 affects the 59 untranslated region of FOXP1 and results in overexpress the full-length FOXP1 protein (FOXP1 FL ). RNA-sequencing of a few lymphoma cases expressing FOXP1 NT and FOXP1 FL detected neither FOXP1-related fusions nor FOXP1 mutations. Further bioinformatic analysis of RNA- sequencing data retrieved a set of genes, which may comprise direct or non-direct targets of FOXP1 NT , potentially implicated in disease progression. In summary, our findings point to a dual mechanism through which FOXP1 is implicated in B-cell lymphomagenesis. We hypothesize that the primary t(3;14)(p13;q32)/IGH-FOXP1 activates expression of the FOXP1 FL protein with potent oncogenic activity, whereas the secondary non-IG rearrangements of FOXP1 promote expression of the FOXP1 NT proteins, likely driving progression of disease. Citation: Rouhigharabaei L, Finalet Ferreiro J, Tousseyn T, van der Krogt J-A, Put N, et al. (2014) Non-IG Aberrations of FOXP1 in B-Cell Malignancies Lead to an Aberrant Expression of N-Truncated Isoforms of FOXP1. PLoS ONE 9(1): e85851. doi:10.1371/journal.pone.0085851 Editor: Jose Angel Martinez Climent, University of Navarra, Center for Applied Medical Research, Spain Received July 24, 2013; Accepted November 13, 2013; Published January 9, 2014 Copyright: ß 2014 Rouhigharabaei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the concerted action grant from the K.U.Leuven no. 3M040406 (www.Kuleuven.be). PV is a senior clinical investigator of the FWO-Vlaanderen (www.FWO.be). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: iwona.wlodarska@uzleuven.be . These authors contributed equally to this work. Introduction The FOXP1 (Forkhead box P1) gene located at 3p13 (previously assigned to 3p14.1) codes for a transcriptional regulator belonging to the FOX transcription factor family which is implicated in a wide range of biological processes [1,2]. Multiple alternative splicing variants of FOXP1 have been annotated (www. ensem- ble.org). The FOXP1 protein is widely expressed in human tissues. It harbors an N-terminal poly-Gln region, C2H2-type zinc finger and leucine zipper motifs, and a C-terminal DNA binding forkhead or winged helix domain [3]. FOXP1 is an essential factor in cardiac, lung, neural, monocyte and lymphocyte development and maturation, stem cell biology and in immune responses [4–14]. Multiple lines of evidence indicate that FOXP1 plays an important role in tumorigenesis [15]. Initial studies of Banham et al. [16] suggested that FOXP1 acts as a tumor suppressor in epithelial malignancies recurrently characterized by D3p13p14/FOXP1 and loss or decreased expression of the FOXP1 protein. Very recent work of Krohn et al. [17] on prostate cancers supports this concept. Interestingly, subsequent studies postulated an oncogenic role of FOXP1 in lymphoma, particularly in an activated B-cell subtype of diffuse large B-cell lymphoma (ABC- DLBCL) with a poor clinical outcome [18–23], and extranodal marginal zone lymphoma (MZL), where a strong expression of FOXP1 protein correlates with a risk of a high grade transforma- tion [24–27]. Further investigations showed that FOXP1-positive PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e85851