Abstract LB-235: IL10 autoregulatory loop in DLBCLs: New biomarker and a therapeutic target

Abstract

Abstract Diffuse Large B cell Lymphoma (DLBCL) is a common aggressive lymphoma that represents 30-40% of newly diagnosed cases of non-Hodgkin Lymphomas, but accounts for up to 80% of lymphoma-related mortality. It is biologically and clinically heterogeneous disease with variable response to conventional R-CHOP chemotherapy. R-CHOP remains the standard first line therapy after decades of investigation, but is associated with frequent lack of response. It has been reported that more clinically aggressive cases of DLBCLs have constitutive activation of NF-kB and STAT3 and have higher level of circulating IL10 cytokine in patient's peripheral. We further investigated the role of IL10 and its surface receptor in supporting the neoplastic phenotype of DLBCL. We measured and analyzed copy number changes using SNP array on a subset of 91 primary DLBCLs and identified broad regions of genomic amplification and deletion in this cohort using the GISTIC algorithm and determined that Il10RA is amplified in 17% and IL10RB in 8 % of primary DLBCLs. Gene expression for all 3 genes is markedly elevated, as determined using Affymetrix HG U133 plus 2.0 array data on 59 primary DLBCLs: up to 3 fold for IL10RA, and more than 10 fold for IL10RB and IL10 cytokine as compared to normal Germinal center B cells (NGCB)(all t-test, p<0.01). We also confirmed that IL10R protein expression is elevated using Tissue microarray (TMA) with primary DLBCLs (independent cohort, n=80). Immunohostochemistry on TMA revealed that up to 73% of DLBCLs have overexpression of IL10R several fold above the expression level in normal B cells. ABC DLBCLs tend to have higher level of expression for all 3 genes as compared to GCB DLBCLs. We thus hypothesized that DLBCLs are dependent on the feed-forward autostimulatory loop that starts from autocrine IL10 stimulation through overexpressed receptor leading to cell proliferation and that bliocking the receptor will lead to cell death. We tested the effect of blocking the receptor using anti-IL10R Ab in a panel of 12 cell lines and 5 primary DLBCLs cultured ex-vivo. The Ab effect was dose-dependent and cell death ensued after 1-3 days of treatment. Within 3 days of treatment with 1 ug/ml most cell lines had reduced viability by more than 50%, and after treatment with 10 ug/ml all cell lines were more than 90% dead. On day 3 massive induction of apoptosis was detected in all DLBCLs using standard approaches: measuring Annexin V/ DAPI by flow cytometry and PARP-1 cleavage by western blot. We determined that blocking IL10R results in specific inhibition of signaling through JAK1/2 and loss of phosphorylation at STAT3Y705 immediately after treatment and inhibition of signaling through MAPK and phosphorylation of STAT3S727 at later treatment time points. The inhibition of signaling is sustained for days with only one drug treatment leading to induction of apoptosis. We observed downregulation of the known transcriptional targets of STAT3 that are crucial for maintaining cell cycle and proliferation like CCND1, CCND2, CMYC, JUNB. Anti-IL10R treatment resulted in significant downregulation of IL10 and IL10RA transcription, thus leading to interruption of IL10-IL10R autostimulatory loop. We thus propose that IL10R is a novel therapeutic target in DLBCLs that allows easy detection and targeting. Our findings warranty further animal studies and development of humanized antibody for clinical use in patients. Citation Format: Wendy Beguelin, Seema Sawh, Nyasha Chambwe, Huimin Geng, Yanwen Jiang, Pilar M. Dominguez, Wayne Tam, Rita Shaknovich. IL10 autoregulatory loop in DLBCLs: New biomarker and a therapeutic target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-235. doi:10.1158/1538-7445.AM2014-LB-235