Non-IG Aberrations of FOXP1 in B-Cell Malignancies Result in an Aberrant Expression of N-Truncated FOXP1 Isoforms.

Abstract 2411

Forkhead Box P1 (FOXP1) is a member of the FOX family of transcription factors which play critical roles in immune responses, organ development and tumorigenesis. Multiple lines of evidence suggest that FOXP1 functions as an oncogene in B-cell neoplasms, being overexpressed in a variety of lymphomas and targeted by rare but recurrent translocations in marginal zone lymphoma (MZL) and diffuse large B-cell lymphoma (DLBCL). The most common translocation is t(3;14)(p13;q32) which brings FOXP1 under a transcriptional control of the IGH enhancers. Several cases with non-IG rearrangements of FOXP1 have been reported but thus far, not studied in details. Of note, Brown et al. (2008) demonstrated that FOXP1+ ABC-DLBCLs lacking FOXP1 rearrangements selectively express smaller N-truncated FOXP1 isoforms and subsequently hypothesized that the oncogenic role of FOXP1 is only exhibited by smaller isoforms.

To unravel molecular consequences of non-IG aberrations of FOXP1 in lymphoma, we performed extensive FISH and molecular investigations of five cases harboring the rearrangements (index cases), complemented by studies of t(3;14)+ lymphoma and FOXP1+ and FOXP1-DLBCL/MZL. The index cases were presented with MZL, transforming MZL, CLL in Richter transformation and gastric DLBCL. The identified non-IG aberrations included t(2;3)(q36;p13), t(3;10)(p13;q24), inv(3)(p13q11) and an unknown IGH/IGK/IGL-negative translocation. One case displayed a microdeletion of FOXP1 spanning the region between exon 3 and 7, as shown by FISH and aCGH. The aberrations constituted a part of complex karyotypes and were subclonal in 3 cases. All tumors displayed a nuclear expression of the FOXP1 protein. FISH showed that the 3p13 breakpoints of index cases clustered within the coding region of FOXP1, contrasting with the 5' end breakpoints of a classical t(3;14)/IGH-FOXP1. Further FISH analysis of 3 available cases mapped the reciprocal breakpoints within AP1S3 (2q36) located in an opposite transcriptional orientation to FOXP1, in the 10q24 region harboring 3 genes (TMEM180->, C10orf95<-, ACTRIA<-), and in a gene poor region at 3q11, respectively. The applied 5'Race PCR additionally identified the PLEKHG1/FOXP1 fusion in one case of FOXP1+DLBCL. The fusion, however, occurred out of the reading frame of FOXP1 and was not functional. The pattern of FOXP1 expression was initially investigated using qRT-PCR. Both t(3;14)+cases, one FOXP1+DLBCL and non-malignant lymph nodes revealed an upregulation of all coding exons analyzed (6–18), while the index cases and the remaining FOXP1+DLBCLs displayed a significant upregulation of exons 7–18. Further RNA-sequencing of two index cases, 6 FOXP1+DLBCLs and 2 FOXP1-DLBCLs confirmed a general pattern of FOXP1 detected by qRT-PCR, and additionally identified several expressed FOXP1 isoforms. Their composition and a relative abundance differed in particular cases, but the most frequently expressed was the full length transcript and 3 N-truncated isoforms annotated by Ensembl as FOXP1–004, −203 and −009. The highest expression of the full length transcript was observed in cases with t(3;14) and FOXP1+GC-DLBCL; in the remaining cases a selective expression of N-truncated isoforms was noted. Results of transcriptomic studies were validated by Western analysis.

Altogether, we showed that non-IG aberrations of FOXP1 are recurrent in B-cell lymphoma. These rearrangements are heterogeneous (reciprocal translocations, inversion, internal deletion) but constantly affect the coding part of the gene. They do not generate chimeric genes but result in an aberrant expression of N-truncated FOXP1 isoforms. Molecular mechanisms underlying these aberrations are largely unknown. Our findings suggest a dual role of FOXP1 in the pathogenesis of lymphoma: the full length FOXP1 upregulated by a primary IGH-mediated t(3;14) is a potent transforming factor implicated in development of lymphoma, while the N-truncated FOXP1 isoforms generated and activated by secondary non-IG aberrations, play a role in lymphoma progression. Of note, the latter variants are also overexpressed in ABC-DLBCL. Further studies of the wild type FOXP1 protein and N-truncated isoforms are required to demonstrate their oncogenic potential in vitro and in vivo.