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. 2008 Apr 1;111(7):3701-13.
doi: 10.1182/blood-2007-09-111948. Epub 2007 Dec 26.

Cooperative signaling through the signal transducer and activator of transcription 3 and nuclear factor-{kappa}B pathways in subtypes of diffuse large B-cell lymphoma

Lloyd T Lam  1 , George Wright, R Eric Davis, Georg Lenz, Pedro Farinha, Lenny Dang, John W Chan, Andreas Rosenwald, Randy D Gascoyne, Louis M Staudt
Affiliations

Cooperative signaling through the signal transducer and activator of transcription 3 and nuclear factor-{kappa}B pathways in subtypes of diffuse large B-cell lymphoma

Lloyd T Lam et al. Blood. .

Abstract

The activated B cell-like (ABC) subgroup of diffuse large B-cell lymphoma (DLBCL) is characterized by constitutive activation of the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we showed that the NF-kappaB pathway induced the expression of the cytokines interleukin (IL)-6 and IL-10 in ABC DLBCL cell lines, which also have high levels of total and phosphorylated signal transducer and activator of transcription (STAT) 3 protein, suggesting autocrine signaling. Using RNA interference for STAT3, we defined a gene expression signature of IL-6 and IL-10 signaling through STAT3. Based on this signature, we constructed a molecular predictor of STAT3 signaling that defined a subset of ABC DLBCL tumors with high expression of STAT3, IL-6, and/or IL-10 and their downstream targets. Although the STAT3-high and STAT3-low subsets had equivalent expression of genes that distinguish ABC DLBCL from germinal center B cell-like DLBCL, STAT3-high ABC DLBCLs had higher expression of signatures that reflected NF-kappaB activity, proliferation, and glycolysis. A small-molecule inhibitor of Janus kinase signaling, which blocked STAT3 signature expression, was toxic only for ABC DLBCL lines and synergized with an inhibitor of NF-kappaB signaling. These findings suggest that the biological interplay between the STAT3 and NF-kappaB pathways may be exploited for the treatments of a subset of ABC DLBCLs.

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Figures

Figure 1
Figure 1
IL-6 and IL-10 are targets of the NF-κB pathway. (A,B) Gene expression patterns of lymphoma cell line OCI-Ly3 and OCI-Ly10 induced to express IkBsr with doxycycline for the indicated times. Each column is a single experiment comparing 2 cDNA populations; the treated sample was labeled red (Cy5) and the untreated sample was labeled green (Cy3). Each row represents data from a single cDNA microarray spot. The red-to-green (Cy5/Cy3) ratio reflects hybridization to that spot, a measure of relative gene expression; intensity reflects the magnitude of the difference between the samples according to the ratio color scale. Red indicates Cy5/Cy3 ratios greater than 1, green indicates Cy5/Cy3 ratios less than 1, black indicates no significant change in gene expression, and gray indicates that the spot did not meet data selection criteria. These ratios were depicted according to the color scale shown at the bottom. The criteria for selecting these genes are described in “Methods.” (C) Expression of IκBsr inhibits IL-6 secretion. OCI-Ly3 cells were induced to express IκBsr with doxycycline. Cells were washed before adding doxycycline. ELISA was used to determine the amount of IL-6 secreted in the medium. (D) Expression of IκBsr inhibits IL-10 secretion. OCI-Ly10 cells were induced to express IκBsr with doxycycline. Cells were washed before adding doxycycline. ELISA was used to determine the amount of IL-10 secreted in the medium. (E) Expression of IL-6 and IL-10 in ABC and GCB DLBCL cell lines was measured using the Quantikine colorimetric sandwich ELISA kit. (F) Steady-state levels of total and phosphorylated STAT3 in ABC and GCB DLBCL cell lines. Cell lysate were separated on Western blotting and immunoblotted with antibodies specific for p-STAT3 (Tyr705), pSTAT3 (Ser727), STAT3, or AKT (control). A vertical line has been inserted to indicate a repositioned gel lane. (G) Relative STAT3 mRNA levels in ABC DLBCL and GCB DLBCL cell lines as assessed by gene-expression profiling on Affymetrix U133 plus 2.0 arrays (probe ID_1123163).
Figure 2
Figure 2
Identification of IL-10, IL-6, and STAT3 target genes in ABC DLBCL cells. (A) Gene-expression patterns of lymphoma cell line OCI-Ly10 treated with IL-6 for 0.5, 1, 3, 6, and 24 hours. Genes indicated with an asterisks are also IL-10 targets. (B) Gene-expression patterns of lymphoma cell line OCI-Ly3 treated with IL-10 for 1, 3, 6, 24, 48, and 96 hours. Genes marked with an asterisk are also IL-6 targets. (C) Comparison of the genes in response to IL-6 or IL-10 treatment. (D) Western blotting showing down-regulation of total STAT3 and phospho-STAT3 protein expression in OCI-Ly10 cell transfected with STAT3 siRNA. (E) Gene-expression patterns of lymphoma cell line OCI-Ly10 transient transfected with STAT3 siRNA pool for 8, 24, and 48 hours.
Figure 3
Figure 3
STAT3 expression in primary lymphoma tumors. (A) Mean STAT3 mRNA levels in tumor samples as assessed by gene-expression profiling. PMBL, primary mediastinal B-cell lymphoma; FL, follicular lymphoma; SLL, small lymphocytic lymphoma; MALT, mucosa-associated lymphoid tissue; FH, follicular hyperplasia; MCL, mantle cell lymphoma; MM, multiple myeloma; BL, Burkitt lymphoma. (B) Histogram of STAT3 mRNA levels in individual ABC DLBCL tumor biopsies. (C) Immunostaining with a p-STAT3 (Tyr705)–specific antibody showing dense brown staining in ABC DLBCL cases. (D) Immunostaining with an antibody recognizing total STAT3 showing dense brown staining in STAT3-high ABC DLBCL cases.
Figure 4
Figure 4
Differential STAT3 signature expression in subsets of ABC DLBCLs. (A) The expression levels of STAT3 and STAT3 target genes in the subgroup predictor in 92 ABC DLBCL tumor samples. The probabilities that the ABC DLBCL samples belong to the STAT3-high and STAT3-low subgroups based on a Bayesian predictor (see “Methods”) are graphed at the top, and the cases are arranged accordingly. The cases that belong to either the STAT3-high or STAT3-low ABC DLBCL subgroups with more than 90% likelihood are indicated. (B) Mean STAT3 mRNA, IL-6 mRNA, and IL-10 mRNA levels among STAT3-high ABC DLBCL, STAT3-low ABC DLBCL, and GCB DLBCL. (C) Expression of STAT3 target genes and ABC DLBCL signature genes in STAT3-high ABC DLBCL, STAT3-low ABC DLBCL, and GCB DLBCL. (D) Average expression of STAT3 target genes and ABC DLBCL signature genes in STAT3-high ABC DLBCL, STAT3-low ABC DLBCL, and GCB DLBCL.
Figure 5
Figure 5
Gene set enrichment analysis of signatures in the STAT3-high and STAT3-low subgroups of ABC DLBCL patients. (A) Expression of IL-6 target genes, IL-10 target genes, and NF-κB target genes in the STAT3-high and STAT3-low subgroups of patients with ABC DLBCL. (B) Average expression of IL-6 target genes, IL-10 target genes, and NF-κB target genes in the STAT3-high and STAT3-low subgroups of patients with ABC DLBCL. (C) Average expression of proliferation and glycolysis genes in the STAT3-high and STAT3-low subgroups of patients with ABC DLBCL.
Figure 6
Figure 6
Toxicity of JAK inhibition for ABC DLBCL cell lines and synergism with NF-κB pathway inhibition. (A) ABC DLBCL (OCI-Ly3, OCI-Ly10, HBL1, and SUDHL2) and GCB DLBCL (OCI-Ly7, HT, HS602) were treated with 0 to 50 μmol/L JAK inhibitor for 2 days and assigned for viability by MTT assays. The cell numbers at each drug dose are expressed as the percentage of cell numbers obtained with untreated cells cultured in parallel. (B) OCI-Ly10 cells were treated with JAK inhibitor (0-10 μmol/L) or U0126 (0-12.5 μmol/L) for 4 hours. Cell lysate was made for the measurement of phospho-STAT3 (Tyr705). (C) Gene-expression patterns of lymphoma cell line OCI-Ly10 treated with 5 μmol/L JAK inhibitor for 3 and 6 hours. (D) JAK inhibitor treatment diminishes IL-6 and IL-10 secretion. OCI-Ly3 and OCI-Ly10 cells were washed before adding JAK inhibitor. Shown are relative cytokine units from ELISAs for IL-6 (OCI-Ly3) and IL-10 (OCI-Ly10) using supernatants of cells that were washed with fresh media immediately before addition of the JAK inhibitor. (E) OCI-Ly3, OCI-Ly10, and HBL1 cells were treated with both JAK inhibitor and MLN120B for 3 days and assigned for viability by MTT assays. The cell numbers at each drug dose are expressed as the percentage of cell numbers obtained with untreated cells cultured in parallel.
Figure 7
Figure 7
Model depicting the molecular cross-talk between the STAT3 and NF-κB pathways in ABC DLBCL. See “Discussion” for details.
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