IL10 Receptor Is a Novel Therapeutic Target in Dlbcls

Leukemia (2015) 29, 1684–1694 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE IL10 receptor is a novel therapeutic target in DLBCLs W Béguelin1, S Sawh1, N Chambwe2,3,4, FC Chan5, Y Jiang1, J-W Choo1, DW Scott5, A Chalmers1, H Geng6, L Tsikitas1,WTam7, G Bhagat8, RD Gascoyne5,9 and R Shaknovich1,8 Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease with marked genomic instability and variable response to conventional R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. More clinically aggressive cases of DLBCLs have high level of circulating interleukin 10 (IL10) cytokine and evidence of activated intracellular STAT3 (signal transducer and activator of transcription 3) signaling. We investigated the role of IL10 and its surface receptor in supporting the neoplastic phenotype of DLBCLs. We determined that IL10RA gene is amplified in 21% and IL10RB gene in 10% of primary DLBCLs. Gene expression of IL10, IL10RA and IL10RB was markedly elevated in DLBCLs. We hypothesized that DLBCLs depend for their proliferation and survival on IL10-STAT3 signaling and that blocking the IL10 receptor (IL10R) would induce cell death. We used anti-IL10R blocking antibody, which resulted in a dose-dependent cell death in all tested activated B-cell-like subtype of DLBCL cell lines and primary DLBCLs. Response of germinal center B-cell-like subtype of DLBCL cell lines to anti-IL10R antibody varied from sensitive to resistant. Cells underwent cell cycle arrest, followed by induction of apoptosis. Cell death depended on inhibition of STAT3 and, to a lesser extent, STAT1 signaling. Anti-IL10R treatment resulted in interruption of IL10-IL10R autostimulatory loop. We thus propose that IL10R is a novel therapeutic target in DLBCLs. Leukemia (2015) 29, 1684–1694; doi:10.1038/leu.2015.57 INTRODUCTION autostimulatory loop via STAT3 signaling.5,13 IL10 can promote the Diffuse large B-cell lymphomas (DLBCLs) are a group of aggressive proliferation of normal B cells that are activated through BCR or 13,14 lymphomas and the most common form of non-Hodgkin CD40. IL10 is also necessary for the development and lymphoma.1 DLBCLs arise from germinal center (GC) and post- maintenance of B-cell lymphomas arising spontaneously in the 14 GC B cells and show marked genetic and epigenetic heterogeneity New Zealand black strain of mice. More recently, it has been and differences in clinical outcomes.2,3 Gene expression profiling shown that serum IL10 levels correlate with IL10 secreted by the 15 identified two main molecular subgroups of DLBCLs: activated primary DLBCL in patients. Most importantly, IL10 serum level is B-cell-like (ABC) and GC B-cell-like (GCB). The ABC subtype of a biomarker of clinical outcome in patients with primary DLBCLs: DLBCLs are more aggressive and are thought to arise from late GC higher IL10 in the serum predicts worse outcomes as shown by or preplasma cells and are characterized by low expression of Gupta et al.16 IL10 is a class 2 cytokine, which is a homodimer, and BCL6 and higher expression of plasma cell-associated transcription is produced by monocytes, type 2 T helper cells (TH2) CD4+CD25+ factors such as XBP1 and PRDM1, marked activation of nuclear Foxp3+ regulatory T cells and subsets of activated T and factor-κB (NF-κB) signaling and STAT3 (signal transducer and B cells.17,18 The main function of IL10 in normal immunity is activator of transcription 3) signaling, as compared with GCB immunosuppressive. This is mediated by the IL10 receptor (IL10R), subtype.2,4,5 Normal lymphocytes and lymphoid malignancies which consists of two subunits: IL10Rα and IL10Rβ.19,20 IL10 depend on cytokine stimulation for growth and survival via both suppresses inflammation by downregulating TH1 cytokines and autocrine and paracrine mechanisms. Cytokines such as major histocompatibility complex class II antigens, and by interleukin 4 (IL4), IL6, IL10 and IL21 have an important role in suppressing synthesis of proinflammatory cytokines such as the differentiation of B cells and in B-cell microenvironment IFN-γ, IL2, IL3 and tumor necrosis factor-α by macrophages and interaction.6–9 Hematologic tumors are known to 'hijack' normal T cells; it also regulates proliferation and survival of B cells. Binding biological processes, which confer a growth advantage. The best to cytokine-specific receptors results in autophosphorylation of studied example is activation of IL6 signaling in multiple the IL10Rα subunit, which in turn leads to the activation of either myeloma, a malignancy arising from a terminal stage of B-cell IL10Rα-associated Janus kinase 1 (JAK1) or IL10Rα-associated TYK2 differentiation.10–12 kinase.18 Both JAK1 and TYK2 activation in turn leads to IL10 signaling is known to have a crucial role in all DLBCLs, but downstream activation of a family of STAT (signal transducer particularly ABC DLBCLs, where IL10 has been shown to be and activator of transcription) proteins. IL10 has been shown to produced at higher levels.5 IL10 is a direct target of NF-κB preferentially signal through STAT3 and, to a much lesser extent, signaling, which is activated in ABC DLBCLs, and contributes to the through STAT1, STAT5 and STAT6.4 After phosphorylation on 1Department of Medicine, Division of Hematology and Oncology, Weill Cornell Medical College, New York, NY, USA; 2The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, New York, NY, USA; 3Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA; 4Tri-Instituitional Training Program in Computational Biology and Medicine, Weill Cornell Medical College, New York, NY, USA; 5Centre for Lymphoid Cancer, British Columbia Cancer Agency, Vancouver, BC, Canada; 6Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA; 7Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA; 8Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA and 9Department of Pathology, University of British Columbia, Vancouver, BC, Canada. Correspondence: Dr R Shaknovich, Department of Pathology and Department of Medicine, Division of Hematology and Oncology, Weill Cornell Medical College, 1300 York Avenue, Building C, Room 620C, New York, NY 10065, USA. E-mail: [email protected] Received 8 July 2014; revised 16 February 2015; accepted 19 February 2015; accepted article preview online 3 March 2015; advance online publication, 7 April 2015 Therapeutic targeting of IL10 receptor in DLBCLs W Béguelin et al 1685 Tyr705 and, to a lesser extent, on Ser737, STAT3 is translocated DLBCL patient samples to the nucleus, where it affects the expression of downstream Leftover, patient-deidentified tissue samples removed for diagnosis from genes containing consensus 'GAS' (gamma-activated site) DNA five patients with de novo DLBCL were obtained from New York motifs.18,21 The STAT3 gene promoter itself contains a GAS motif, Presbyterian Hospital. The use of human tissue was approved by the and thus its activation initiates an autostimulatory loop resulting research ethics board of New York Presbyterian Hospital and the Weill in increased amount of unphosphorylated-STAT3. Recently, it Cornell Medical Center. has been shown that not only phosphorylated-STAT3, but also unphosphorylated-STAT3 can translocate to the nucleus and Primary DLBCL cell culture 22,23 activate downstream genes. Irradiated HK cells were plated in Dulbecco's modification of Eagle's The importance of IL10-JAK1/2-STAT3 axis in DLBCL survival, medium with 10% fetal bovine serum and incubated for 24 h at 37 °C. The proliferation and treatment has been previously appreciated and media were then aspirated and patient DLBCL cells were resuspended in targeted therapeutically. Gupta et al.16 documented higher levels advanced RPMI with 10% human serum at 3 million cells per ml and plated of serum IL10 in patients with more aggressive disease, showing directly on attached HK cells. that IL10 alone, among cytokines, activates JAK2 and STAT3 in patient samples ex vivo, and showed that a JAK2-specific inhibitor Survival analysis can interrupt autocrine IL10 secretion and JAK2/STAT3 activation. Kaplan–Meier analysis was performed using the R Survival Package 4 5 dichotomizing the patients into high and low expression groups based Ding et al. and Lam et al. also demonstrated that STAT3 24 activation preferentially promotes the survival of ABC DLBCLs, and on a median threshold. We divided patients (n = 49; Shaknovich et al. ) into high and low expressers, based on whether they had IL10 and that the use of JAK inhibitors leads to apoptosis. Although prior IL10R levels above or below the median expression, respectively. A log- studies focused on IL10 secretion and signaling as a result of rank test was performed to assess survival differences between the two STAT3 activation in DLBCLs, attempting to target the signaling groups. pathways downstream of JAKs, we discovered that an IL10 autoregulatory loop is also activated upstream in DLBCLs with marked upregulation of IL10R expression, and consequent RESULTS activation of STAT3 signaling. We identified frequent upregulation IL10RA and IL10RB are markedly overexpressed and genomically of the IL10R via genomic amplification of IL10RA and IL10RB amplified in primary DLBCLs subunits in DLBCLs. We used this observation for therapeutic To understand the role of IL10 signaling in DLBCLs, we measured targeting and demonstrated that blocking IL10R results in the gene expression levels of IL10, IL10RA and IL10RB interruption of IL10 signaling. This leads to apoptosis in DLBCL using Affymetrix HG U133 plus 2.0 array in 52 primary DLBCLs.24 cell lines and in primary DLBCL cases treated ex vivo. We determined that all three genes were significantly overexpressed as compared with normal germinal center B cells (NGCBs) (all t-test, Po0.01) (Figure 1a).

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