Gene regulation and suppression of type I interferon signaling by STAT3 in diffuse large B cell lymphoma Li Lua,b,1, Fen Zhua,b,1, Meili Zhangc,d,1, Yangguang Lia,b, Amanda C. Drennana,b, Shuichi Kimparaa,b, Ian Rumballa,b, Christopher Selzera,b, Hunter Camerona,b, Ashley Kellicuta,b, Amanda Kelma,b, Fangyu Wanga,b, Thomas A. Waldmannc,2, and Lixin Ruia,b,2 aDepartment of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792; bCarbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792; cLymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and dLaboratory Animal Science Program, Leidos Biomedical Research, Inc., Frederick, MD 21702 Contributed by Thomas A. Waldmann, December 7, 2017 (sent for review August 28, 2017; reviewed by Vu N. Ngo and Demin Wang) STAT3 is constitutively activated in many cancers and regulates cell lines and control GCB DLBCL cell lines that lack STAT3 gene expression to promote cancer cell survival, proliferation, activation, a recent study has identified a total of 10,337 STAT3- invasion, and migration. In diffuse large B cell lymphoma (DLBCL), binding regions corresponding to 8,531 genes, of which 1,545 activation of STAT3 and its kinase JAK1 is caused by autocrine genes are differently expressed between the two subtypes and production of IL-6 and IL-10 in the activated B cell–like subtype largely associated with ABC DLBCL biology (14). However, (ABC). However, the gene regulatory mechanisms underlying the biological significance and functional association of these STAT3 pathogenesis of this aggressive lymphoma by STAT3 are not well targets in ABC DLBCL cells remain to be studied. characterized. Here we performed genome-wide analysis and Here, we use genetic and pharmacological inhibition of STAT3 in identified 2,251 STAT3 direct target genes, which involve B cell ABC DLBCL cells and identify genes that are directly regulated by activation, survival, proliferation, differentiation, and migration. STAT3. These STAT3 target genes are involved in multiple sig- Whole-transcriptome profiling revealed that STAT3 acts as both naling pathways, which promote cancer cell survival and pro- a transcriptional activator and a suppressor, with a comparable liferation. In addition, STAT3 negatively regulates lethal type I number of up- and down-regulated genes. STAT3 regulates multi- signaling by inhibiting expression of IRF7, IRF9, STAT1,and ple oncogenic signaling pathways, including NF-κB, a cell-cycle STAT2, key transcription factors for IFNβ production and signaling. checkpoint, PI3K/AKT/mTORC1, and STAT3 itself. In addition, STAT3 negatively regulates the lethal type I IFN signaling pathway Results by inhibiting expression of IRF7, IRF9, STAT1, and STAT2. Inhibition Genome-Wide Analysis Identifies STAT3 Transcriptional Target Genes of STAT3 activity by ruxolitinib synergizes with the type I IFN in- in ABC DLBCL Cells. To identify STAT3 target genes genome-wide, ducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro we performed STAT3 ChIP-seq in the ABC DLBCL cell lines and in a xenograft mouse model. Therefore, this study provides a TMD8 and OCI-Ly10, in which high levels of STAT3 phosphor- mechanistic rationale for clinical trials to evaluate ruxolitinib or a ylation were detected by immunoblot analysis (Fig. 1A). Since specific JAK1 inhibitor combined with lenalidomide in ABC DLBCL. STAT3 activity was sufficiently inhibited by the JAK1/JAK2 in- hibitor AZD1480 (Fig. 1A) and this inhibitor was used for H3Y41- STAT3 | interferon | diffuse large B cell lymphoma P ChIP-seq analysis (3), AZD1480-treated cells served as a control for STAT3 ChIP-seq experiments. Using the model-based analysis iffuse large B cell lymphoma (DLBCL), the most common Dnon-Hodgkin lymphoma, includes two main molecular sub- Significance types: an activated B cell–like (ABC) and a germinal center B cell– like (GCB) (1, 2). ABC DLBCL is more aggressive, with autocrine We demonstrate that STAT3 is a critical transcriptional regulator signaling from the cytokines IL-6 and IL-10 that constitutively ac- of the activated B cell–like subtype of diffuse large B cell lym- – tivates JAK1 (3) and STAT3 (4 7) to promote cell survival. ABC phoma (ABC DLBCL), the most common, aggressive, non-Hodgkin DLBCL cells have high NF-κB activity (8) due to genetic alterations lymphoma. By genome-wide assessment, we have identified tar- in the Toll-like receptor (TLR) and B cell receptor signaling get genes of STAT3. Gene regulation by STAT3 in ABC DLBCL ac- pathways (9–11). Somatic mutations of MYD88 (mainly L265P), a centuates survival signaling pathways while dampening the lethal key signaling adaptor in TLR signaling, engage the NF-κB pathway type I interferon pathway. Knowledge of these STAT3-regulated and induce production of IL-6, IL-10, and IFNβ (11). In contrast to genes has led to our demonstration that a small-molecule in- IL-6 and IL-10, IFNβ is proapoptotic, and its basal level is low to hibitor in the JAK1-STAT3 signaling pathway synergizes with the undetectable in ABC DLBCL cells (12). IFNβ production can be type I interferon inducer lenalidomide, suggesting a new thera- prevented by the transcription factors IRF4 and SPIB through re- peutic strategy for ABC DLBCL, a subtype that is particularly dif- pression of IRF7, a transcription factor for IFNβ expression (12). ficult to treat and has poor prognosis. Recently, we revealed that, in addition to STAT3 activation, JAK1 is present in the nucleus and directly phosphorylates his- Author contributions: L.R. designed research; L.L., F.Z., M.Z., Y.L., A.C.D., S.K., I.R., C.S., tone H3 on tyrosine 41 (H3Y41) to induce expression of nearly H.C., A. Kellicut, A. Kelm, and F.W. performed research; T.A.W. and L.R. supervised re- κ search; L.L., F.Z., M.Z., Y.L., S.K., and L.R. analyzed data; and L.L., F.Z., M.Z., T.A.W., and 3,000 genes, including the NF- B pathway genes MYD88 and L.R. wrote the paper. IRF4 (3, 13). These findings suggest a positive feedback loop Reviewers: V.N.N., City of Hope National Medical Center; and D.W., BloodCenter between the cytokine and NF-κB signaling pathways that pro- of Wisconsin. motes the malignant phenotype of ABC DLBCL cells. This The authors declare no conflict of interest. epigenetic gene regulatory mechanism by JAK1 is distinct from Published under the PNAS license. the canonical JAK-STAT signaling pathway, given that more 1L.L., F.Z., and M.Z. contributed equally to this work. than 90% of these genes do not bear a STAT motif in their 2To whom correspondence may be addressed. Email: [email protected] or lrui@ promoter region (3). Using STAT3 chromatin immunoprecipi- medicine.wisc.edu. tation followed by DNA sequencing (ChIP-seq) and whole- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genome transcriptome (RNA-seq) analysis in ABC DLBCL 1073/pnas.1715118115/-/DCSupplemental. E498–E505 | PNAS | Published online January 2, 2018 www.pnas.org/cgi/doi/10.1073/pnas.1715118115 Downloaded by guest on September 28, 2021 OCI-Ly10 PNAS PLUS A B TMD8 DMSO AZD1480 High DMSO AZD1480 High AZD1480 TMD8 (2 M): 0 15' 30' 1h 2h 4h 6h 0 pSTAT3 STAT3 Low Low -actin AZD1480 OCI-Ly10 (2 M): 0 15' 30' 1h 2h 4h 6h pSTAT3 STAT3 -actin Density heatmap of 22,856 peaks Density heatmap of 11,487 peaks -5K Apex +5K -5K Apex +5K C STAT3 motif enrichment ((TMD8)TMD8) STAT3 motif enrichment (OCI-Ly10) 2 2 Probability Probability Probability 1 1 bits E-valueE 3.6e-21 E-value 9.5e-8 bits 0 0 3 2 7 8 1 9 6 4 5 11 3 2 7 8 9 1 4 6 10 5 11 10 Position of Best Site in Sequence Position of Best Site in Sequence D E B cell activation 7.03E-05 OCI-Ly10 Lymphocyte proliferation 3.87E-03 TMD8 MEDICAL SCIENCES (6,058) (4,746) Lymphocyte differentiation 1.27E-02 Intracellular signal transduction 5.81E-12 Apoptotic process 8.49E-04 Regulation of cell migration 4.81E-02 Cell cycle 5.00E-03 2,495 2,251 3,807 Cellular response to stress 5.29E-04 Response to external stimulus 3.97E-03 Regulation of metabolic process 5.38E-06 01234 Enrichment Score Fig. 1. Genome-wide analysis reveals STAT3 transcriptional targets in ABC DLBCL. (A) Immunoblot analysis of phospho-STAT3 and total STAT3 protein in TMD8 and OCI-Ly10 cells treated with 2 μM AZD1480. (B) Heat maps of STAT3 ChIP-seq in TMD8 and OCI-Ly10 cells after 4 h of treatment with either DMSO or 2 μM AZD1480. STAT3 peak summits were centered within 5 kb of the flanking sequence on either side. Darker color indicates a higher density of reads. STAT3 peaks were ranked by signal intensity at the peak center, and the same order is used to display the AZD1480-treated sample. (C) The CentriMo plots show the distribution of known the STAT3 motif in the ChIP-seq peak summit regions (P < 0.001). De novo motif discovery from STAT3 ChIP-seq peaks shows identical sequence logs to the known STAT consensus motif (15). (D) Venn diagram shows 2,251 STAT3-binding genes that are shared in TMD8 and OCI- Ly10 cells. (E) Gene ontology analysis of the 2,251 STAT3-binding genes (P < 0.05). of ChIP-seq (MACS) for peak calling (16), we identified a total of OCI-Ly10 cells compared with the AZD1480-treated control sam- 11,487 STAT3-binding sites (peaks) in TMD8 cells and 22,856 in ple (Fig.