regulation and suppression of type I 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 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 , 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 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 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 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 (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 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 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

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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 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 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) 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. 1B and Dataset S1). Specificity of these STAT3-binding

Lu et al. PNAS | Published online January 2, 2018 | E499 Downloaded by guest on September 28, 2021 A TMD8(4,746) OCI-Ly10 (6,058) B TMD8 OCI-Ly10 shRNA shSTAT3 shRNA shSTAT3 shSTAT3 shSTAT3 Ctrl #10 #16 Ctrl #10 #16 Ctrl #10 #16 Ctrl #10 #16 1 TNFAIP8

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Fig. 2. Whole-transcriptome profiling reveals signaling pathways regulated by STAT3 in ABC DLBCL. (A) Heat maps show expression changes of STAT3-binding genes in TMD8 and OCI-Ly10 cells after 2 d of knockdown of STAT3 by two shRNAs. (B) Heat maps show down-regulation of IL6-JAK-STAT3 and NF-κB pathway genes by two STAT3 shRNAs. STAT3-binding genes are shown in red. (C) STAT3 is recruited to regulatory regions of indicated genes, as shown by read density tracks (DMSO controls in red, AZD1480-treated samples in green). (D) Quantitative PCR analysis of SOCS3 and IL-10 mRNA levels (normalized to GAPDH mRNA levels) in TMD8 and OCI-LY10 cells after 1 d (only for SOCS3 in TMD8) or 2 d of knockdown of STAT3 by shRNAs. Error bars represent mean ± SD of triplicates (*P < 0.05, **P < 0.01, ***P < 0.001).

sites was confirmed by the MEME motif enrichment analysis (15), Based on genomic loci of these peaks, we mapped near a protein- with a similar distribution pattern of STAT3 motifs between the two coding gene within a window extending from −15 kb 5′ of the cell lines (Fig. 1C). transcriptional start site to the 3′ end of any annotated transcript

E500 | www.pnas.org/cgi/doi/10.1073/pnas.1715118115 Lu et al. Downloaded by guest on September 28, 2021 PNAS PLUS A +10K 0 -10K +3K +2K +1K 0 15 15 30

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B Type 1 IFN pathway C _TMD8 OCI-Ly10_ SUDHL7_ TMD8 OCI-Ly10 shSTAT3 + + + Dox shRNA shSTAT3 shRNA shSTAT3 pSTAT3 Ctrl #10 #16 Ctrl #10 #16 STAT3 STAT1 1 DDX60 0.5 pSTAT1 RTP4 0 USP18 STAT1 GBP4 -0.5 pSTAT2 CASP8 -1 IFI44 STAT2 EIF2AK2 EPSTI1 IRF7 OASL NUB1 IRF9 IRF1 -actin SP110 MX1 IRF9 D STAT3-C (OCI-Ly10) STAT3-C (HBL1) ADAR CD47 PARP14 STAT2 pSTAT3 MEDICAL SCIENCES TDRD7 TRAFD1 pSTAT1 IRF2 IFITM2 STAT1 CD74 IRF7 IL15 CNP IRF9 CXCL11 -actin CXCL10

Fig. 3. STAT3 suppresses type I IFN signaling in ABC DLBCL. (A) STAT3 is recruited to regulatory regions of STAT1, STAT2, IRF7, and IRF9, as shown by read density tracks (DMSO controls in red, AZD1480-treated samples in green). (B) Heat maps show expression changes of type I IFN pathway genes in TMD8 and OCI-Ly10 cells after 2 d of knockdown of STAT3 by two shRNAs. STAT3-binding genes are shown in red. (C) Immunoblot analysis of the indicated in TMD8, OCI-Ly10, and the control cell line SUDHL7 after 2 d of knockdown of STAT3 by shRNA. (D) Immunoblot analysis of the indicated proteins in TMD8 and HBL1 cells after 2 or 4 d of retroviral expression of constitutively activated STAT3 (STAT3-C).

associated with the gene, as for our previous study (3). We identi- cell-cycle progression, stress response, cell migration, and metab- fied 4,746 potential STAT3 target genes in TMD8 cells and 6,058 in olism (Fig. 1E), suggesting an important role for STAT3 in the OCI-Ly10 cells, with an overlap of 2,251 genes between the two cell pathogenesis of ABC DLBCL. lines (Fig. 1D and Dataset S1). Considering these overlapped genes as common STAT3 targets in ABC DLBCL, we performed Whole-Transcriptome Profiling Reveals That STAT3 Acts as both a PANTHER gene ontology analysis (17). The results revealed that Transcriptional Activator and a Repressor in ABC DLBCL. To determine these common target genes were enriched for biological pro- genes that are directly regulated by STAT3, we performed the cesses that include B cell activation, proliferation, differentiation, whole-transcriptome analysis by RNA-seq in the above TMD8 and

Lu et al. PNAS | Published online January 2, 2018 | E501 Downloaded by guest on September 28, 2021 SUDHL7 A TMD8 OCI-Ly10 45 ** ** 40 40 ** 30 * 35 35 * * 25 30 Control 30 * * 25 shSTAT3 25 20 20 Lena 20 ** 15 15 shSTAT3 15 10 10 10 +Lena 5 Live cells (x 1,000) 5 5 0 0 0 Day 0 Day 3 Day 0 Day 3 Day 0 Day 3

4 M Lena plus B TMD8 OCI-Ly10 E *** DMSO ctrl 4 M Lena 4 M Ruxolitinib 4 M Ruxolitinib 25 *** 3 * *** * 34 34 29 *** ** S:34 20 * 2 TMD8 15 G1:46 47 48 55 G2/M:9 8 8 7 10 1

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Vehicle control D 1.2 1.2 Lenalidomide Synergism: CI<1 Synergism: CI<1 3 Ruxolitinib ...... 1 1 Combination 0.8 0.8 ** * ** 0.6 0.6

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Fig. 4. Synergism between STAT3 inhibition and lenalidomide in growth inhibition of ABC DLBCL. (A) STAT3 shRNA enhanced lenalidomide-mediated toxicity in TMD8 and OCI-Ly10 cells but not in control SUDHL7 cells. The 4,000 cells per well from each cell line were plated on a 96-well plate and induced with doxycycline for expression of STAT3 shRNA. No doxycycline cultures served as a control. Total live cells were counted with trypan blue exclusion assay after 3 d of shRNA induction in the presence of 4 μM lenalidomide or DMSO. Data are presented as mean ± SD of triplicates (*P < 0.05, **P < 0.01). (B) Quantitative PCR analysis of IFNβ mRNA levels (normalized to GAPDH mRNA levels) in TMD8 and OCI-Ly10 cells after 3 d of knockdown of STAT3 by shRNAs in the presence of 4 μM lenalidomide or DMSO. Error bars represent mean ± SD of triplicates (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Immunoblot analysis of phospho-STAT3 and β-actin loading control in TMD8 and OCI-Ly10 cells after 30 min of treatment with the indicated concentrations of ruxolitinib. (D) Synergism between ruxolitinib and lenalidomide in cell killing in TMD8 and OCI-Ly10 cells. Cells were treated with ruxolitinib and lenalidomide for5d before trypan blue dye exclusion viability assay. Combination index (CI) was calculated with CompuSyn software. (E) Cell-cycle analysis of TMD8 and OCI- Ly10 cells after 5 d of ruxolitinib and lenalidomide. Cells were pulsed with 10 μM BrdU for 3 h before flow cytometric analysis. (F) The combination therapy of lenalidomide with ruxolitinib significantly inhibits OCI-Ly10 tumor growth in vivo. Female NOD/SCID mice were injected s.c. with 1 × 107 OCI-Ly10 cells. Ten days later, lenalidomide was given i.p. at a dose of 15 mg/kg/d for 14 d, and ruxolitinib was continuously administered by an s.c. miniosmotic pump at a dose of 60 mg/kg/d for 14 d. Shown is a photograph of tumors from each group at day 15 (Top) and average tumor volumes during the therapeutic course for each group (Bottom). Error bars show the SD of eight mice/group (*P < 0.05, **P < 0.01, lenalidomide or ruxolitinib group compared with vehicle control group; ***P < 0.001, combination group compared with lenalidomide or ruxolitinib group).

OCI-Ly10 cell lines. We knocked down STAT3 by two shRNAs 6,058) in OCI-Ly10 cells changed their expression when STAT3 from our previous study (7). As shown in Fig. 2A, 53% (2,495/ was knocked down. Of note, the number of down-regulated genes 4,746) of the STAT3 target genes in TMD8 cells and 68% (4,146/ was comparable to that of up-regulated genes, suggesting that

E502 | www.pnas.org/cgi/doi/10.1073/pnas.1715118115 Lu et al. Downloaded by guest on September 28, 2021 IL-6/IL-10 IFN PNAS PLUS

MYD88 (L265P) P TYK2 P JAK1 NF- B IRF7

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STAT3P STAT1 P P P IRF7 IRF9 P STAT3 STAT3 IRF9 STAT2 Genes ISRE IL6/JAK1/STAT3, PI3K, NF- B signaling cell cycle progression... Cell survival Cell death proliferation

Fig. 5. Schematic illustration of gene regulation by STAT3 in ABC DLBCL. JAK1 and STAT3 are activated by autocrine IL-6/IL-10 due to the MYD88 L265P mutation. STAT3 regulates expression of genes that involve many oncogenic pathways, including NF-κB, PI3K, cell cycle, and itself, signaling to promote cancer cell survival and proliferation while suppressing proapoptotic type I IFN signaling.

STAT3 functions as both a transcriptional activator and a re- analysis confirmed that protein levels of STAT1, STAT2, IRF7, pressor in ABC DLBCL cells. and IRF9 were all increased by STAT3 shRNA in these two Next, we performed gene set enrichment analysis (GSEA) to ABC DLBCL cell lines but not in the control GCB cell line identify the signaling pathways in which these up-regulated and SUDHL7 (Fig. 3C). In addition, phosphorylation of STAT1 and down-regulated genes are involved. The results revealed signif- STAT2 was remarkably increased in the STAT3 shRNA ex- icant enrichment in gene signature of multiple signaling path- pressing ABC DLBCL cells (Fig. 3C). To further validate these ways (Fig. S1), including the PI3K/AKT/mTORC1 (Fig. S2), results, we used the constitutively activated form of STAT3 /G2M cell-cycle checkpoint (Fig. S3), IL6-JAK-STAT3, NF- (STAT3-C) with activating mutations (A661C and N663C) in the κB, and type I IFN signaling pathways. Consistent with previous SH2 domain (21). Retroviral expression of STAT3-C in OCI- studies (4, 5, 14), STAT3 is involved in the positive feedback Ly10 and HBL1 ABC DLBCL cells reduced IRF7 and IRF9 regulation of the IL-6/IL-10 signaling and shows crosstalk with expression and completely removed STAT1 phosphorylation NF-κB signaling pathways in ABC DLBCL cells (Fig. 2 B–D). (Fig. 3D). Taken together, these data suggest that STAT3 ac- We verified SOCS3, a negative regulator of JAK-STAT signaling tivity blocks the type I IFN signaling pathway by inhibiting ex- (18), as a target gene of STAT3 (Fig. 2D). More significantly, pression of multiple essential signaling components, including 16 of the genes down-regulated by STAT3 shRNAs are direct STAT1, STAT2, IRF7, and IRF9. STAT3 targets, including TNFAIP8, TRAF1, CD44, CD69, BCL3 FOS SGK1 NF-κB2 STAT3 B C , , , , and itself (Fig. 2 and ). Synergism Between STAT3 Inhibition and Lenalidomide in Growth MEDICAL SCIENCES Inhibition of ABC DLBCL. Lenalidomide, an active agent in ABC STAT3 Suppresses Type I IFN Signaling in ABC DLBCL. In ABC DLBCL, DLBCL, induces type I IFN response by down-regulation of IRF4 production of the proapoptotic cytokine IFNβ can result from and SPIB, which otherwise inhibit IRF7 expression (12, 22). Given oncogenic MYD88 mutations (11). This type I IFN signaling is partial inhibition of IRF4 expression by lenalidomide (12) and that suppressed by the transcription factors IRF4 and SPIB, which IRF4 is not a direct target of STAT3, we hypothesized that in- repress IRF7 expression to prevent IFNβ transcription and TYK2 hibition of STAT3 activity augments IFNβ production and syner- activation (12). Our recent work demonstrated that IRF4 and gizes with lenalidomide in killing ABC DLBCL cells. To test this SPIB are epigenetic targets of JAK1 due to H3Y41 phosphory- hypothesis, we performed an in vitro survival assay in TMD8 and lation, but IRF4 expression is not regulated by STAT3 (3). These OCI-Ly10 cells when STAT3 shRNA was induced for expression in findings along with the above RNA-seq analysis prompted us to the presence of lenalidomide. We used the GCB DLBCL cell line investigate whether STAT3 directly targets critical genes in the SUDHL7 as a control. As shown in Fig. 4A, after 3 d of culture, IFNβ signaling pathway. It is known that, in response to IFNβ, both STAT3 shRNA and lenalidomide significantly reduced cell STAT1 and STAT2 are phosphorylated, together with IRF9, to viability in the two ABC DLBCL cell lines but not in the control. form the tripartite transcription factor IFN-stimulated factor gene Of note, expression of STAT3 shRNA increased lenalidomide- 3 (ISGF3), which binds to distinct IFN-stimulated elements of mediated cytotoxicity in these ABC DLBCL cultures (Fig. 4A). genomic DNA for gene transcription (19, 20). A reduction in viable cells was mainly due to inhibition of cell Indeed, STAT3 ChIP-seq data displayed peaks in the pro- proliferation (Fig. S3B) although a slight increase in was motor region, near the transcription start sites of STAT2, IRF7, observed (Fig. S4). Quantitative PCR analysis confirmed that IFNβ and IRF9, and a peak in the enhancer region of STAT1. These expression was significantly increased in cells that expressed peaks were significantly reduced after AZD1480 treatment, STAT3 shRNA and were treated with lenalidomide, consistent suggesting that they are direct targets of STAT3 (Fig. 3A). In- with the above survival assay demonstrating a cytotoxic synergism creased expression of STAT1, STAT2, and IRF9 was observed between STAT3 shRNA and lenalidomide (Fig. 4B). when STAT3 was knocked down by two different shRNAs in To further examine the above synergistic effect, we used both TMD8 and OCI-Ly10 cell lines (Fig. 3B). Immunoblot ruxolitinib, a clinically used JAK1 and JAK2 inhibitor (23), to

Lu et al. PNAS | Published online January 2, 2018 | E503 Downloaded by guest on September 28, 2021 inhibit STAT3 activity. Immunoblot analysis confirmed dose- The multilayer suppression of IFNβ signaling by STAT3 is one dependent inhibition of STAT3 phosphorylation in both of the major mechanisms by which autocrine IL-6/IL-10 signal- TMD8 and OCI-Ly10 cell lines (Fig. 4C). As expected, we ing prevents cancer cell death. In addition, this cytokine sig- observed a synergism between ruxolitinib and lenalidomide in naling inhibits IFNβ production through the JAK1-mediated killing these cells (Fig. 4D). Cell-cycle analysis revealed that a epigenetic mechanism; that is, JAK1 targets histone H3 to in- combination of the two drugs increased G1 phase population duce expression of IRF4 and SPIB, which form a transcription (Fig. 4E), suggesting inhibition of cell proliferation. More complex to inhibit IRF7 expression (3, 12). importantly, our xenograft analysis in the OCI-Ly10 cell line The type I IFN signaling pathway has emerged as an effective demonstrated that cotreatment of ruxolitinib and lenalido- therapeutic target in DLBCL. Recent clinical trials have revealed mide caused nearly complete tumor growth inhibition during that lenalidomide treatment alone or combined with immu- the period of treatment, but the single drug treatment nochemotherapy achieved promising efficacy in DLBCL (37–39). achieved only partial inhibition (Fig. 4F). Thus, these data However, remission after a single lenalidomide treatment lasted suggest that the ruxolitinib and lenalidomide combination is a for only 6 mo (37), suggesting that combinations of targeted agents potential therapeutic strategy for ABC DLBCL cases. that inhibit distinct survival pathways will be necessary. Our findings demonstrated that STAT3 regulates expression of genes Discussion that involve multiple oncogenic pathways and suppresses genes in Deregulation of the JAK-STAT signaling pathway, such as con- the type I IFN signaling pathway. Inhibition of STAT3 activity by stitutive activation of STAT3, plays a pathogenic role in many ruxolitinib synergizes with lenalidomide in growth inhibition of – hematologic malignancies (24 26). In DLBCL, STAT3 is acti- ABC DLBCL cells in vitro and in a xenograft mouse model. vated in the ABC subtype by IL-6/IL-10 and JAK1 to promote Therefore, this study provides a mechanistic rationale for clinical – cancer cell survival (3 7). STAT3 activity is also associated with a trials of ruxolitinib or a specific JAK1 inhibitor and lenalidomide poor prognosis in DLBCL (27, 28). Here, we conducted genome- in ABC DLBCL. wide assessment and established a working model of STAT3 in the pathogenesis of ABC DLBCL (Fig. 5). STAT3 acts as both a Materials and Methods transcriptional activator and a suppressor. Gene set enrichment Full details of the methods used and data analysis are presented in SI Ma- analysis revealed that genes regulated by STAT3 are involved in terials and Methods. several oncogenic signaling pathways, including NF-κB, PI3K/ AKT/mTORC1, cell-cycle checkpoint, and STAT3 itself. Notably, Cell Lines and Culture. All doxycycline-inducible human DLBCL cell lines that STAT3 suppresses expression of STAT1, STAT2, IRF7,andIRF9, express the bacterial tetracycline repressor were engineered as described all of which are critical transcription factors in the type I IFN previously (40). Doxycycline (20 ng/mL) was used to induce the expression of pathway. Thus, gene regulation by STAT3 in ABC DLBCL ac- genes or shRNAs of interest. All cultures were routinely tested for mycoplasma centuates the survival signaling pathways while dampening the contamination. lethal type I IFN pathway. Crosstalk between the NF-κB and IL-6/IL-10/JAK1/STAT3 ChIP-Seq Analysis. ChIP-enriched DNA samples were used to create adapter- ligated libraries for massively parallel sequencing with the Ovation Ultralow signaling pathways is an oncogenic process in ABC DLBCL (8, 29). ’ Through histone H3 phosphorylation but independent of STAT3, Library System V2 (NuGen Technologies) following the manufacturer s pro- tocol. ChIP-Seq data are available at https://www.ncbi.nlm.nih.gov/geo/ JAK1 up-regulates expression of IRF4 and MYD88, which is re- (accession no. GSE106844). quired for cancer cell survival (3). The present study revealed that κ many other genes in the NF- B pathway are STAT3 targets, in- RNA-Seq Analysis. Total RNA was extracted using RNeasy plus mini (Qia- cluding NF-κB2 and TRAF1. Interestingly, NF-κB2 signaling is gen) according to the manufacturer’s protocol. RNA-seq libraries were pre- associated with MYD88 mutations and promotes development pared by using the Illumina TruSeq stranded mRNA LT sample preparation of the ABC subtype (30). In a transgenic mouse model, TRAF1 is kit (Illumina). Sequencing was performed on Illumina Hiseq 2500 at 50-bp involved in lymphomagenesis mediated by constitutively activated length. RNA-seq data are available at https://www.ncbi.nlm.nih.gov/geo/ NF-κB2 (31). These findings suggest a role for the noncanonical (accession no. GSE106844). NF-κB pathway in the pathogenesis of ABC DLBCL. Disruption of oncogenic loops between the NF-κB and JAK1/STAT3 signaling Xenografts. The xenograft tumor model of human ABC DLBCL lymphoma was pathways by their small-molecule inhibitors produces the syner- established by s.c. injection of OCI-Ly10 cells into nonobese diabetic/severe gistic cytotoxicity in ABC DLBCL (3, 4, 32). combined immunodeficiency (NOD/SCID) mice (Jackson Labs). The tumor growth was monitored by measuring tumor size in two orthogonal dimen- Several biochemical studies have found a phenomenon of sions. All animal experiments were approved by the National Cancer In- STAT3-mediated suppression of IFN antiviral responses in im- – stitute Animal Care and Use Committee (NCI ACUC) and were performed in mune cells (33 36). In ABC DLBCL, the type I IFN signaling accordance with NCI ACUC guidelines. pathway, which can be activated by the MYD88 L265P mutation, is proapoptotic to the cancer cells (11) (Fig. 5). Our integrated ACKNOWLEDGMENTS. This research was supported by the University of Wis- genomic analysis elucidates the molecular mechanisms of STAT3 consin, Madison, Start-up Funds and KL2 Scholar Award (UL1TR0000427 and in suppression of this lethal pathway in ABC DLBCL: active STAT3 KL2TR000428); the National Cancer Institute (Grant 1R01 CA187299) (to L.R.); β the University of Wisconsin, Madison, T32 Hematology Training Award (T32 prevents the cancer cells from producing IFN through inhibition of HL07899) (to A.C.D.); the University of Wisconsin Forward Lymphoma Fund IRF7 expression and also suppresses transcription of STAT1, (L.L. and S.K.); and the Intramural Research Program of the National Cancer STAT2,andIRF9 (ISGF3 complex) to block IFNβ signaling. Institute (T.A.W.).

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