The effect of the STAT3 ^M206K mutation on STAT3 translocation to the nucleus, transactivation activity, and cell proliferation.

<p>(<b>A</b>) STAT3 phosphorylation (n = 3) was assessed in both nucleus and cytosol in stably transfected HEK-293T cells by mutant STAT3α^M206K and WT STAT3α plasmids. HDAC2 was used as a marker for nuclear protein, and GAPDH was used as a marker for cytoplasmic protein. (<b>B</b>) The transactivation activity of WT STAT3α^WT and mutant STAT3α^M206K was evaluated by luciferase reporter assay in Ly3 cells. Briefly, Ly3 cells were transiently transfected with plasmids STAT3α^WT and STAT3α^M206K along with a STAT3 luciferase reporter followed by IL-10 treatment for 6 hours and cells were used for luciferase assay. Bars represent mean ± SD from 3 replicates. Data was repeated three times (**P = 0.0042; ***P<0.001). The data were analyzed by the two-tailed unpaired Student's t test. (<b>C</b>) The effect of WT STAT3α^WT and mutant STAT3α^M206K on Ly3 cells proliferation by H³-thymidine incorporation assay. Bars represent mean ± SD from 4 replicates. The experiment was repeated three times (*P = 0.013). The data were analyzed by the two-tailed unpaired Student's t test.</p

A Novel Missense (M206K) STAT3 Mutation in Diffuse Large B Cell Lymphoma Deregulates STAT3 Signaling

<div><p>Persistent STAT3 activation has been found in activated B-cell like diffuse large B cell tumors (DLBCL). To investigate whether genetic mutations play a role in aberrant STAT3 signaling in DLBCL, we bi-directionally sequenced all 24 exons of the STAT3 gene in DLBCL tumors (n = 40). We identified 2 novel point mutations in 2 separate (2/40; 5%) patients at exon 7 and 24. Point mutation 2552G>A was a silent mutation in the stop codon. Another heterozygous mutation 857T>A encoded a methionine substitution by lysine at codon 206 (M206K) in the coiled-coil domain of STAT3. We performed site directed mutagenesis to mutate wild type (WT) STAT3α and STAT3β at codon 206 and constructed stable cell lines by lentiviral transfection of STAT3α^WT, STAT3α^M206K, STAT3β^WT and STAT3β^M206K plasmids. The mutation was found to increase STAT3 phosphorylation in STAT3α mutant cell lines with no effect on the STAT3β mutant cell line. Transcriptional activation was also increased in the STAT3α mutant cells compared with STAT3α WT cells as detected by a luciferase reporter assay. Moreover, STAT3α^M206K mutant cells were resistant to JAK2 pathway inhibition compared to STAT3α WT cells. These results indicate that missense mutations in STAT3 increase signaling through the JAK/STAT pathway. JAK2 inhibitors may be useful in the patient with this STAT3 mutation as well as those with pathway activation by other mechanisms.</p></div

Identification of STAT3 mutations in DLBCL tumors.

<p>(<b>A</b>) Data summarizing STAT3 mutations in DLBCL tumors (n = 40) (<b>B</b>) Chromatograms of STAT3 DNA sequence showing 857T>A heterozygous mutation. (<b>C</b>) Modular structure of STAT3 showing that Methionine 206 is located in the coiled-coiled domain of STAT3. Alignment of part of STAT3 protein sequence from various species showing that methionine 206 is a conserved amino acid.</p

The effect of STAT3 (M206K) mutation on STAT3 signaling.

<p>(<b>A</b>) Western blot (n = 4) analysis shows overexpression of STAT3 in stably transfected HEK-293T cells by STAT3α^WT, STAT3β^WT, STAT3α^M206K and STAT3β^M206K plasmids. (<b>B</b>–<b>C</b>) STAT3 phosphorylation was assessed in STAT3α^WT, STAT3β^WT, STAT3α^M206K and STAT3β^M206K stably transfected HEK-293T cells by western blotting (n = 3) (<b>B</b>) and transiently transfected Ly3 cells (n = 2) (<b>C</b>)<b>.</b> (<b>D</b>) STAT3 phosphorylation (n = 2) was assessed in response to IL-10 in HEK-293T cells stably transfected with STAT3α^WT, STAT3β^WT, STAT3α^M206K and STAT3β^M206K plasmids. DHL2 cells were used as a positive control for IL-10 treatment.</p

The effect of STAT3α M206K mutation on the sensitivity of DLBCL cells to JAK2 inhibitor TG101348.

<p>(<b>A–B</b>) The effect of JAK2 inhibitor TG101348 on STAT3 dephosphorylation was assessed in WT STAT3α^WT and mutant STAT3α^M206K in stably transfected HEK-293T cells (<b>A</b>) and transiently transfected Ly3 cells (<b>B</b>). Data were repeated three times. (<b>C</b>) The effect of the overexpression of WT STAT3α^WT and mutant STAT3α^M206K on cell proliferation in the presence or absence of JAK2 inhibitor TG101348. Bars represent mean ± SD from 4 replicates. The experiment was repeated two times (**P = 0.0023). The data were analyzed by the two-tailed unpaired Student's t test.</p

Identification 2552G>A mutation in STAT3 in DLBCL tumors.

<p>Chromatograms of part of the patient STAT3 DNA sequence show 2552G>A mutation.</p

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

<div><p>While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (T_reg) cells by enhancing expression of Foxp3 in CD4⁺ T cells and suppressed effector helper T (T_H) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.</p> </div

Effect of TGF-β on the differentiation of intratumoral T cells in B-cell NHL.

<p>(A) Representative histograms (n = 3) showing proliferation measured by CFSE staining of CD4⁺ or CD8⁺ T cells treated with different doses of TGF-β. Proliferative capacity was expressed by calculating the number of CFSE^dim cells. (B) Representative dot plots showing the expression of Foxp3, IFN-γ or IL-17 in CD4⁺, CD4⁺CD45RA⁺ or CD4⁺CD45RO⁺ T cells treated with or without TGF-β. (C) Summary of the numbers of T_reg (CD4⁺Foxp3⁺), T_H1 (CD4⁺IFN-γ⁺) or T_H17 (CD4⁺IL-17⁺) cells induced by TGF-β. (D) Representative dot plots (n = 3) showing the expression of IFN-γ in CD4⁺ T cells treated with or without TGF-β or IL-12 or IL-23 alone or in combination. (E) Representative dot plots (n = 10) showing the expression of IL-17 in CD4⁺ T cells treated with or without IL-1β plus IL-6. (F) Representative dot plots (n = 6) showing the expression of IL-17 in CD4⁺ T cells treated with or without TGF-β in the presence of IL-1β plus IL-6.</p

Soluble TGF-β expression in B-cell NHL.

<p>(A) TGF-β serum levels measured by ELISA in untreated FL patients (mean: 27.67+/−13.2, n = 10) and healthy donors (median: 30.76+/−9.47 pg/ml, n = 10; p = 0.56). (B) TGF-β levels in culture supernatants of freshly-isolated malignant B (B, n = 11) and intratumoral T cells (T, n = 5) from B-cell NHL treated with (A) or without (R) PMA/Ion measured by ELISA. (C) A representative graph showing TGF-β levels in culture supernatants of B and T cell lines treated with (Stim) or without (Unstim) PMA/Ion measured by ELISA (n = 3).</p

Effects of IL-2 signaling on TGF-β-mediated regulation of T cell differentiation in B-cell NHL.

<p>(A) Representative dot plots (n = 6) showing the expression of IFN-γ and IL-17 in CD4⁺ T cells treated with or without αIL-2 or αIL-2Rα or β in the presence or absence of TGF-β plus IL-6 and IL-23. (B) Representative dot plots (n = 3) showing the expression of Foxp3 and CD25 in CD4⁺ T cells treated with or without TGF-β, αIL-2 or αIL-2Rα or β alone or in combination. (C) Dot plots from a representative sample (n = 5) showing the expression of IFN-γ, IL-17 and IL-2 in CD4⁺ T cells treated with or without TGF-β or αIL-2.</p