FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas.

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco-Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients

Premature termination codon mutations in the von Willebrand factor gene are associated with allele-specific and position-dependent mRNA decay

Background. The FOXP1 forkhead transcription factor is targeted by rare but recurrent translocations in B-NHL, particularly marginal zone lymphoma (MZL) and diffuse large B-cell lymphoma (DLBCL). The most common is IGH-mediated t(3;14)(p14;q32) resulting in deregulation of FOXP1 transcription by regulatory sequences of IGH. Several translocations of FOXP1 involving non-IG loci have been described, but remain uncharacterized at the molecular level. Aims. This study aimed at genetic and molecular characterization of non-IG FOXP1 aberrations identified in 4 lymphoma cases. Methods. FISH with a panel of BAC probes was applied to map the affected breakpoints at 3p14/FOXP1 and 2q36, 3q11-q13 and 10q24. Expression of FOXP1 mRNA was analyzed by quantitative RT-PCR (QRT-PCR) with primers specific for exons 5-6, 7-8, 11-12, 14-15 and 17-18. Expression of FOXP1 protein was demonstrated by immunohistochemistry (IHC) with the JC12 antibody. Results. All 4 cases showed an aberrant expression of FOXP1 protein by IHC. Three of them displayed various 3p14 aberrations including t(2;3)(q36;p14) (C1) (MZL), inv(3)(p14;q11) (C2) (MZL) and t(3;10)(p14;q24) (C3) (CLL in Richter transformation). In one case of DLBCL (C4) a non-IG t(FOXP1) was detected by interphase FISH. In contrast to lymphomas with t(3;14) showing the 3p14 breakpoints in the 5’end of FOXP1, all cases with non-IG FOXP1 rearrangements displayed breakpoints in the 3’end of the gene. The reciprocal breakpoints were mapped within AP1S3 at 2q36 (C1), in a region at 3q11 lacking known genes (C2) and close to NFKB2 at 10q24 (C3). In C1, AP1S3 has an opposite transcriptional orientation to FOXP1, which precludes the role of its 5’ promoter in deregulation of FOXP1. Also the mechanism of FOXP1 overexpression in C2 with inv(3) affecting no known gene locus at 3q11 remains unknown. To check the hypothesis that non-IG translocations of FOXP1 result in an aberrant expression of variant FOXP1 transcripts, we performed exon-specific QRT-PCR analysis of two available cases (C2 and C4). As controls, we analyzed 2 cases with t(3;14) and 6 cases of DLBCL/MZL expressing FOXP1 but lacking FOXP1 rearrangements. These studies showed that both cases with t(3;14) expressed all analyzed coding exons (6-17) while cases with non-IGH translocations of FOXP1, as well as FOXP1+ DLBCL/MZL cases expressed sequences coded by exons 7-17, but not of exon 6. Conclusions. Our study demonstrates that the 3p14 breakpoints of non-IG FOXP1 rearrangements in lymphoma are clustered in the 3’end of the gene. Consequently, these aberrations result in expression of variant FOXP1 transcripts, likely encoding N-terminally truncated proteins, similar to potentially oncogenic smaller FOXP1 isoforms recently identified in ABC-DLBCL cell lines and primary DLBCLs. Which transcriptional regulatory elements are engaged to aberrantly express variant FOXP1 transcripts in these peculiar non-IG FOXP1 aberrations warrants further studies

Non-IG aberrations of FOXP1 in B-cell malignancies lead to an aberrant expression of N-truncated isoforms of FOXP1

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 (FOXP1NT), as shown by QRT-PCR and Western blot analysis. In contrast, t(3;14)(p13;q32)/IGH-FOXP1 affects the 5′ untranslated region of FOXP1 and results in overexpress the full-length FOXP1 protein (FOXP1FL). RNA-sequencing of a few lymphoma cases expressing FOXP1NT and FOXP1FL 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 FOXP1NT, 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 FOXP1FL protein with potent oncogenic activity, whereas the secondary non-IG rearrangements of FOXP1 promote expression of the FOXP1NT proteins, likely driving progression of disease