Autocrine release of interleukin-9 promotes Jak3-dependent survival of ALK+ anaplastic large-cell lymphoma cells by Lin Qiu, Raymond Lai, Quan Lin, Esther Lau, David M. Thomazy, Daniel Calame, Richard J. Ford, Larry W. Kwak, Robert A. Kirken, and Hesham M. Amin Blood Volume 108(7):2407-2415 October 1, 2006 ©2006 by American Society of Hematology

ALK+ ALCL-cell lines express IL-9Rα. ALK+ ALCL-cell lines express IL-9Rα. (A) RT-PCR studies show the presence of IL-9Rα mRNA in all ALK+ ALCL cells. The Hodgkin lymphoma-cell line L1236 was used as positive control and the colon carcinoma HT29-cell line as negative control. β-Actin shows equal loading. (B) Immunohistochemical staining of paraffin-embedded tissue sections from cell blocks confirmed the expression of IL-9Rα in all the ALK+ ALCL-cell lines. Similar to RT-PCR studies, L1236 and HT29 cells were used as positive and negative controls, respectively (original magnification × 200). (C) Confocal microscopy after immunofluorescence staining of cytospin slides with antibodies directed against IL-9Rα (bottom panel) or control IgG (top panel) demonstrates the expression of IL-9Rα in the ALK+ ALCL-cell lines, Karpas 299, SU-DHL-1, and SUP-M2 (original magnification × 600). (D) Immunoprecipitation and Western blotting show the physical association between γc and pJak3 in Karpas 299 cells. As shown in the right panel, immunoprecipitation was first performed on the cell lysate using anti-γc antibody followed by Western blotting using anti-pJak3 antibody. The left panel demonstrates a simultaneous control study where the lysate was analyzed only by Western blotting using an anti-pJak3 antibody. pJak3 bands are present at the expected molecular weight of 118 kDa in the 2 panels. Similar findings were noted when anti-Jak3 antibody was used instead of anti-pJak3 antibody (data not shown). Lin Qiu et al. Blood 2006;108:2407-2415 ©2006 by American Society of Hematology

ALK+ ALCL-cell lines express IL-9. ALK+ ALCL-cell lines express IL-9. (A) RT-PCR studies demonstrate the presence of IL-9 mRNA in ALK+ ALCL cells. L1236 and HT29 cells were used as positive and negative controls, respectively. β-Actin demonstrates equal loading. (B) Immunohistochemical staining of the ALK+ ALCL-cell lines confirmed the expression of IL-9. L1236 and HT29 were used as positive and negative controls, respectively (original magnification × 200). (C) Confocal microscopy and immunofluorescence staining using specific antibody (bottom panel) or IgG (top panel) show the expression of IL-9 in ALK+ ALCL-cell lines (original magnification × 600). Lin Qiu et al. Blood 2006;108:2407-2415 ©2006 by American Society of Hematology

Expression of IL-9Rα and IL-9 in a reactive lymph node and in primary ALK+ ALCL tumors from patients. Expression of IL-9Rα and IL-9 in a reactive lymph node and in primary ALK+ ALCL tumors from patients. (A) IL-9Rα is strongly expressed in a significant number of cells within the germinal center (left side) and the mantle zone of a reactive lymph node. The frequency and intensity of expression of IL-9Rα are relatively diminished in the marginal zone and interfollicular areas (original magnification × 200). (B) IL-9 is strongly expressed in scattered small lymphoid cells and mast cells in a reactive lymph node. Most of these cells are localized in the interfollicular areas as well as around and within the lymph node sinuses (original magnification × 200). (C-D) An example of a lymph node showing sclerotic tissue with dense infiltration by ALK+ ALCL cells that are strongly positive for IL-9Rα (C) and IL-9 (D; original magnification × 200). (E-F) Another example of a lymph node involved by ALK+ ALCL demonstrating large neoplastic cells that are positive for IL-9Rα (E) and IL-9 (F). The large neoplastic cells are confined to the lymph node sinuses, a characteristic morphologic feature of ALK+ ALCL (original magnification × 200). In all of the tissue samples, the staining for both IL-9Rα and IL-9 was membranous and cytoplasmic, whereas the nuclei were negative for the 2 proteins. Lin Qiu et al. Blood 2006;108:2407-2415 ©2006 by American Society of Hematology

Autocrine release of IL-9 by ALK+ ALCL cells. Autocrine release of IL-9 by ALK+ ALCL cells. Western blot studies show the presence of IL-9 protein in cell lysates from Karpas 299, SU-DHL-1, and SUP-M2 cells. Anti–IL-9–neutralizing antibody had no significant effect on IL-9 levels in the cell lysates. β-Actin shows equal loading of the proteins. Also, these studies show the presence of IL-9 in the culture medium from the same cells after being maintained in FBS-free medium for 12 hours. The anti–IL-9–neutralizing antibody (80 μg/mL) effectively bound to IL-9, as demonstrated by the lack of IL-9 in the culture medium after protein complexes were eluted. The negative control cells HT29 demonstrate lack of IL-9 in the cell lysate and the cell-culture medium. The experiment was repeated 2 times with consistent results. Lin Qiu et al. Blood 2006;108:2407-2415 ©2006 by American Society of Hematology

Effects of specific blockade of IL-9 on Jak3, Stat3, and ALK Effects of specific blockade of IL-9 on Jak3, Stat3, and ALK. (A) Western blot studies show that increasing concentrations of anti–IL-9–neutralizing antibody (80 and 160 μg/mL) decreases pJak3 in the 3 ALK+ ALCL-cell lines. Effects of specific blockade of IL-9 on Jak3, Stat3, and ALK. (A) Western blot studies show that increasing concentrations of anti–IL-9–neutralizing antibody (80 and 160 μg/mL) decreases pJak3 in the 3 ALK+ ALCL-cell lines. The decrease in pStat3 levels in SU-DHL-1 and SUP-M2 cells was more pronounced at a concentration of 160 μg/mL. Despite the slight increase in pStat3 levels in Karpas 299 cells at a concentration of 80 μg/mL, it decreased to the baseline level at 160 μg/mL. Total levels of Jak3 and Stat3 were not affected. β-Actin confirmed equal loading of the proteins. The experiment was performed 3 times with consistent results. (B) Western blot studies did not demonstrate similar changes in pJak3, Jak3, pStat3, and Stat3 levels after treating the negative control cells HT29 with similar concentrations of the anti–IL-9–neutralizing antibody. β-Actin confirmed equal loading of the proteins. (C) After treating the ALK+ ALCL-cell lines with anti–IL-9–neutralizing antibody (160 μg/mL), tyrosine kinase activity of Jak3 and ALK were measured. Normalized data reveal reduction to 60% or less of the control levels of the kinase activity of the 2 enzymes. Control cells were treated with IgG. Shown are averaged data of 2 consistent experiments. Lin Qiu et al. Blood 2006;108:2407-2415 ©2006 by American Society of Hematology

Specific blockade of IL-9 decreases ALK+ ALCL-cell proliferation and colony formation potential. Specific blockade of IL-9 decreases ALK+ ALCL-cell proliferation and colony formation potential. (A) anti–IL-9–neutralizing antibody induces concentration-dependent decrease in ALK+ ALCL-cell proliferation as revealed by the [3H]-thymidine incorporation assay. The decrease in cell proliferation reached approximately 40% of the baseline level at a concentration of 80 μg/mL (P < .001). Similar effects were not detected when the anti–IL-9–neutralizing antibody was preincubated with rhIL-9 before treating Karpas 299 cells, and cell proliferation remained stable with a very slight decrease to 90% of its baseline level at 80 μg/mL concentration of the antibody. Similarly, changes in cell proliferation were not seen in the negative control cells HT29 after treatment with anti–IL-9–neutralizing antibody. When the Hodgkin lymphoma cells L1236 were used as a positive control, a significant and gradual decrease in cell proliferation was observed, which became 69% of the baseline level at a concentration of 80 μg/mL (P < .01). The results are shown as means ± SE of at least 3 consistent experiments. *Statistically significant compared with baseline cells (0) treated with IgG. (B) anti–IL-9–neutralizing antibody (80 μg/mL) induces marked decrease in colony formation of Karpas 299 cells in soft agar. The top panel shows the means ± SD of 3 consistent experiments. Compared to a control level of 60 ± 8 colonies/plate in control cells treated with IgG, cells treated with the anti–IL-9–neutralizing antibody developed only 24 ± 6 colonies/plate. The bottom panel shows examples of the cultured plates. The plate on the right side contains Karpas 299 cells treated with anti–IL-9–neutralizing antibody before being cultured for 2 weeks. The plate on the left side contains control cells treated with IgG under the same experimental conditions. Lin Qiu et al. Blood 2006;108:2407-2415 ©2006 by American Society of Hematology

Specific blockade of IL-9 induces G1 cell-cycle arrest associated with increased p21 and decreased Pim-1 kinase levels in ALK+ ALCL cells. Specific blockade of IL-9 induces G1 cell-cycle arrest associated with increased p21 and decreased Pim-1 kinase levels in ALK+ ALCL cells. (A) Analysis of the cell cycle using flow cytometry and PI/7-ADD staining. The right panel shows histograms of the ALK+ ALCL cells treated with 80 μg/mL anti–IL-9–neutralizing antibody compared with control cells treated with equivalent concentrations of IgG and shown in the left panel. The anti–IL-9–neutralizing antibody induces G1 cell-cycle arrest as demonstrated by the marked decrease of cells in the S phase. The number of the cells in the S phase decreased to 59%, 40%, and 36% of their corresponding baseline levels in Karpas 299, SU-DHL-1, and SUP-M2 cells, respectively. The experiment was repeated twice with consistent findings. (B) Western blot studies showing concentration-dependent increase in p21 levels after treating the ALK+ ALCL cells with 80 and 160 μg/mL anti–IL-9–neutralizing antibody. There was a simultaneous concentration-dependent decrease in Pim-1 levels in Karpas 299 and SUP-M2 cells. Whereas Pim-1 level in SU-DHL-1 cells slightly increased at a concentration of 80 μg/mL anti–IL-9–neutralizing antibody, it decreased to the baseline level at a concentration of 160 μg/mL. Significant changes were not detected in p27 and cyclin D3. β-Actin confirmed equal loading of the proteins. The results represent 1 of 2 consistent experiments. Lin Qiu et al. Blood 2006;108:2407-2415 ©2006 by American Society of Hematology