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Reciprocal Expression of the Endocytic Protein HIP1R and Its Repressor FOXP1 Predicts Outcome in R-CHOP-Treated Diffuse Large B-Cell Lymphoma Patients

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Reciprocal Expression of the Endocytic Protein HIP1R and Its Repressor FOXP1 Predicts Outcome in R-CHOP-Treated Diffuse Large B-Cell Lymphoma Patients Leukemia (2014) 28, 362–372 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Reciprocal expression of the endocytic protein HIP1R and its repressor FOXP1 predicts outcome in R-CHOP-treated diffuse large B-cell lymphoma patients KK Wong1,2, DM Gascoyne1, PJ Brown1, EJ Soilleux1, C Snell1, H Chen3, L Lyne1, CH Lawrie1,4,5, RD Gascoyne6, LM Pedersen7, MB Møller8, K Pulford1, D Murphy3,9, TM Green8 and AH Banham1 We previously identified autoantibodies to the endocytic-associated protein Huntingtin-interacting protein 1-related (HIP1R) in diffuse large B-cell lymphoma (DLBCL) patients. HIP1R regulates internalization of cell surface receptors via endocytosis, a process relevant to many therapeutic strategies including CD20 targeting with rituximab. In this study, we characterized HIP1R expression patterns, investigated a mechanism of transcriptional regulation and its clinical relevance in DLBCL patients treated with immunochemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone, R-CHOP). HIP1R was preferentially expressed in germinal center B-cell-like DLBCL (Po0.0001) and inversely correlated with the activated B-cell-like DLBCL (ABC-DLBCL) associated transcription factor, Forkhead box P1 (FOXP1). HIP1R was confirmed as a direct FOXP1 target gene in ABC-DLBCL by FOXP1-targeted silencing and chromatin immunoprecipitation. Lower HIP1R protein expression (p10% tumoral positivity) significantly correlated with inferior overall survival (OS, P ¼ 0.0003) and progression-free survival (PFS, P ¼ 0.0148) in R-CHOP-treated DLBCL patients (n ¼ 157). Reciprocal expression with X70% FOXP1 positivity defined FOXP1hi/HIP1Rlo patients with particularly poor outcome (OS, P ¼ 0.0001; PFS, P ¼ 0.0016). In an independent R-CHOP-treated DLBCL (n ¼ 233) microarray data set, patients with transcript expression in lower quartile HIP1R and FOXP1hi/HIP1Rlo subgroups exhibited worse OS, P ¼ 0.0044 and P ¼ 0.0004, respectively. HIP1R repression by FOXP1 is strongly associated with poor outcome, thus further understanding of FOXP1-HIP1R and/or endocytic signaling pathways might give rise to novel therapeutic options for DLBCL. Leukemia (2014) 28, 362–372; doi:10.1038/leu.2013.224 Keywords: diffuse large B-cell lymphoma; HIP1R; FOXP1; rituximab; endocytosis; survival INTRODUCTION response rate when treated with bortezomib, a proteasome Diffuse large B-cell lymphoma (DLBCL) is the most common inhibitor that targets the NF-kB pathway, in combination with subtype of non-Hodgkin’s lymphoma and exhibits an aggressive DA-EPOCH (dose-adjusted etoposide, prednisone, vincristine, 8 9 clinical course. Addition of rituximab to conventional CHOP cyclophosphamide and doxorubicin) or R-CHOP regimens. (cyclophosphamide, doxorubicin, vincristine and prednisone) Thus, DLBCL subtyping into ABC- or GCB-DLBCL subgroups chemotherapy regimen has improved DLBCL patient outcome.1,2 represents a predictive marker for selecting patients for However, 20–40% of DLBCL patients receiving R-CHOP survive less personalized targeted therapy, in addition to a prognostic than 5 years,1,3 and stratification of high-risk patients is important marker for response to R-CHOP therapy. for evaluating molecularly targeted therapies. There are a number of marker proteins whose expression either Gene expression profiling (GEP) identified clinically and independently, or in combination, can be used to help routinely biologically distinct DLBCL subtypes, a good prognosis germinal identify the cell-of-origin (COO) in DLBCL, and predict patients’ 10–13 center B-cell-like DLBCL (GCB-DLBCL) subtype and a poor survival in response to R-CHOP therapy. Our laboratory has prognosis activated B-cell-like DLBCL (ABC-DLBCL) subtype.4 been particularly interested in studying Forkhead box P1 (FOXP1), In DLBCL patients treated with R-CHOP regimen, the 5-year a member of the forkhead box family of transcription factors14 overall survival (OS) of patients with GCB-DLBCL remains whose expression independently correlates with a poor response significantly better than ABC-DLBCL.5,6 to CHOP and R-CHOP therapies in DLBCL.13,15 FOXP1 has been Biological and genetic studies have investigated fundamental included in several subtyping algorithms as a marker of ABC- pathogenic mechanisms within DLBCL subtypes that might be DLBCL with a poor response to R-CHOP.12,13,16 FOXP1 is also amenable to therapeutic targeting. Indeed, an IkB kinase inhibitor targeted by recurrent genetic abnormalities in DLBCL,17,18 and 7 was toxic to ABC-DLBCL cells in vitro via inhibition of NF-kB. activation-induced short isoforms (FOXP1S) expressed in both Moreover, non-GCB-DLBCL patients achieved a significantly higher non-malignant peripheral blood B cells and in ABC-DLBCL cells 1NDCLS, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK; 2Department of Immunology, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia; 3Centre for Human Proteomics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; 4Biodonostia Research Institute, San Sebastian, Spain; 5IKERBASQUE, Basque Foundation for Science, Bilbao, Spain; 6Department of Pathology and Experimental Therapeutics, Centre for Lymphoid Cancer, BC Cancer Agency and BC Cancer Research Centre, Vancouver, Canada; 7Department of Haematology, Roskilde Hospital, Roskilde, Denmark; 8Department of Pathology, Odense University Hospital, Odense, Denmark and 9School of Biological Sciences, Dublin Institute of Technology, Dublin 8, Ireland. Correspondence: Professor AH Banham, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Level 4 Academic Block, John Radcliffe Hospital, University of Oxford, Headington, Oxfordshire OX3 9DU, UK. E-mail: [email protected] Received 16 May 2013; revised 18 July 2013; accepted 19 July 2013; accepted article preview online 25 July 2013; advance online publication, 13 August 2013 HIP1R and FOXP1 expression predict DLBCL outcome KK Wong et al 363 have a potentially important biological role.19 However, Antibodies and plasmid constructs although target genes regulated by FOXP1 have been Details of the anti-HIP1R (clone 44, BD Transduction Laboratories, San Jose, identified in the lung,20 striatal neuronal cells21 and GCB- CA, USA; anti-HIP1R-44 monoclonal antibody, mAb) and anti-HIP1 22 DLBCL, the functional role of FOXP1S in ABC-DLBCL remains (clone 4B10, Abcam, Cambridge, UK; anti-HIP1-4B10 mAb) monoclonal undefined. antibodies and other antibodies including clone names and working Huntingtin-interacting protein 1-related (HIP1R) came to dilutions, for immunohistochemical labeling or western blotting, are provided in Supplementary Table 1. The full-length HIP1R complementary our attention because of its frequent immunological DNA (3.2 kb) was generated by PCR amplification as described in the recognition by DLBCL patients when screening protein Supplementary Methods. The full-length HIP1 expression construct was arrays with DLBCL patients’ sera to identify novel autoanti- obtained from OriGene Technologies Inc. (SC108827; Rockville, MD, USA). gens in the disease.23 HIP1R maps to 12q24.31 and was originally identified on the basis of sequence homology Transfection and recombinant protein expression with its only known mammalian relative, HIP1.24 Although HIP1R itself has not been implicated in lymphomagenesis, HIP1R or HIP1 expression in mammalian cells was achieved by transfecting the plasmid into HEK293T cells. Briefly, plasmid DNA was obtained using HIP1 has been reported as an autoantigen in lymphoid the Qiagen Spin Miniprep Kit (Qiagen, Manchester, UK). pcDNA4/HisMax/ malignancies and has the capacity to transform lymphoid HIP1R, pCMV6-XL4/HIP1 (SC108827; OriGene Technologies Inc., Rockville, 25 cells in vivo. Human B-cell neoplasms frequently expressed MD, USA) or pcDNA4/HisMax vector alone (Invitrogen, Paisley, UK) were elevated levels of HIP1 compared with normal lymph nodes, transfected into HEK293T cells using FuGENE6 transfection reagent leading the authors to propose that the HIP1 pathway could according to the manufacturer’s protocols (Roche, Mannheim, Germany). be explored for diagnostic and therapeutic potential.25 Validation of successful recombinant protein expression is described in HIP1R functions as an endocytic protein in clathrin- Supplementary Methods. mediated endocytosis26 and vesicle trafficking at the trans-Golgi network for lysosome biogenesis.27,28 HIP1R regu- Immunohistochemistry lates trafficking of receptors, including mannose phosphate Immunohistochemical labeling of DLBCL TMAs and reactive tonsils using receptor29 and receptor tyrosine kinases.30 Moreover, clathrin- anti-HIP1R-44 or anti-FOXP1 (clone JC12)14 mAbs was performed, after mediated endocytosis is required for internalization of cell dewaxing and heat-mediated antigen retrieval in 50 mM Tris/EDTA surface receptors including the B-cell receptor that attenuates pH 9.0, using the EnVision kit according to manufacturer’s instructions B-cell receptor signaling,31 suggesting that HIP1R could (DakoCytomation, Glostrup, Denmark). Antigen retrieval for CD10, BCL6, potentially regulate B-cell receptor signaling via its roles in MUM1 and GCET1 was performed in Cell Conditioning 1 (Ventana Medical Systems, Tucson, AZ, USA) buffer. The staining pattern for HIP1R was endocytosis. cytoplasmic, whereas FOXP1 staining showed a well-defined nuclear pattern Although HIP1R has not been previously implicated in (Figure 4a). The HIP1R
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