FOXP1 truncated isoforms differentially regulate target genes in diffuse large B cell lymphoma.

Introduction: FOXP1 is frequently overexpressed in DLBCL independently to copy number variations and translocations (Goatly et. al. 2008). The prognostic significance of FOXP1 in DLBCL is controversial with some studies describing FOXP1 as a poor prognostic marker (Banham et. al. 2005) and others found no significance (Hans et. al. 2004). Truncated FOXP1 isoforms were identified in DLBCL and are associated with poor outcomes (Brown et. al. 2008). Over 10 isoforms are known, where some exhibit COOH- or NH 2 - truncations and absence of regulatory domains. Since truncated isoforms lack regulatory domains, we suggest that isoforms may aberrantly regulate FOXP1 target genes. We aim to determine FOXP1 function in DLBCL and specifically its target genes, and also investigate how FOXP1 isoforms may contribute to a more aggressive phenotype. Methods: Expression constructs of FOXP1 isoforms (isoforms 1, 2, 3 and 8) were kindly provided by Dr. Philip Brown and Dr. Alison Banham. A stable BJAB cell line expressing FOXP1 isoform 1 was established. Gene expression of BJAB cell lines and 4 DLBCL tumors was analysed using Illumina HT12v4 microarray. Genes with >2-fold changes in expression were investigated. VisANT analysis and GSEA was performed on these genes and candidates were validated by qRT-PCR. Promoter analysis using MatInspector identified forkhead-binding sites in candidate genes, and dual luciferase reporter gene assays were performed to ascertain promoter regulation by FOXP1 isoforms. Results: The overexpression of FOXP1 isoform 1 in BJAB cell line resulted in the differential expression of 271 genes >2-fold. A similar comparison of high/low FOXP1 expressing DLBCL patients identified 2472 genes with >2-fold level. Comparison of gene lists for both the cell line and patient samples revealed all 271 genes differentially expressed in the cell line overlapped with the patient genes, indicating these results are translatable to FOXP1 function in DLBCL tumors. The 271 differentially expressed genes were investigated by pathway analysis, though no canonical pathways were significant. Alternatively, an integrative analysis of biological network information and microarray data showed critical interactions that were not revealed by pathway analysis, and identified genes that are promising FOXP1 targets. The promoter regions of 5 genes were investigated using reporter gene assays, and FOXP1 isoforms 3 and 8 were significantly stronger repressors compared to isoforms 1 and 2. For example, protein O-fucosyltransferase 1 (POFUT1) was downregulated in the presence of isoform 1, however isoform 3 and 8 downregulated POFUT1 expression >2-fold. This gene has not previously been associated with DLBCL, though POFUT1 is implicated in notch signaling and lymphoid homeostasis (Yao et. al. 2011). We are currently investigating the other novel FOXP1 target genes, and how truncated isoforms are involved in the differential regulation of these genes. Conclusion: We have identified 271 genes that are differentially expressed by FOXP1 expression. These are novel genes for DLBCL pathogenesis, and are differentially regulated by FOXP1 isoforms, indicating truncated isoforms may have aberrant functions and may contribute to a more pathogenic phenotype. Citation Format: Emily Camilleri, Carlos Aya-Bonilla, Jamie Nourse, Philip J. Brown, Alison H. Banham, Paula Marlton, Maher K. Gandhi, Lyn R. Griffiths. FOXP1 truncated isoforms differentially regulate target genes in diffuse large B cell lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B38.