ALK+ALCLs induce cutaneous, HMGB-1–dependent IL-8/CXCL8 production by keratinocytes through NF-κB activation by Emilie Dejean, Marianne Foisseau, Fréderic Lagarrigue, Laurence Lamant, Naïs Prade, Abdelghafour Marfak, Georges Delsol, Sylvie Giuriato, Fréderique Gaits-Iacovoni, and Fabienne Meggetto Blood Volume 119(20):4698-4707 May 17, 2012 ©2012 by American Society of Hematology

Culture supernatant collected from ALK+ALCL cell lines up-regulates IL-8 secretion and NF-κB activation by keratinocytes via HMGB-1 secretion. Culture supernatant collected from ALK+ALCL cell lines up-regulates IL-8 secretion and NF-κB activation by keratinocytesviaHMGB-1 secretion. Culture supernatants were collected from keratinocytes (HaCaT) or ALK+ALCL (Karpas-299 and Su-DHL-1) cells (A) and HaCaT cells exposed to Karpas-299 and SU-DHL-1 cell line supernatants for 24 hours; and (B) IL-8 production was measured by ELISA. IL-8 mRNA levels were also evaluated by qRT-PCR in Karpas-299 (Karpas), SU-DHL-1 and HaCat (keratinocytes) cell lines (A). (C) Western blotting was performed to monitor Ser536 phosphorylation of NF-κB p65 from keratinocytes exposed to Karpas-299 or SU-DHL-1 cell culture supernatants for 24 hours or those exposed to DMEM as a control. qRT-PCR (D) and Western blotting (E) were performed to determine IL-8 mRNA levels and NF-κB activation in keratinocytes treated by the rhHMGB-1 cytokine or not (PBS). Results are expressed as the mean ± SD of 3 independent experiments done in triplicate. *P < .05 and **P < .01 by Student t test. Emilie Dejean et al. Blood 2012;119:4698-4707 ©2012 by American Society of Hematology

ALK+ALCL cells secrete MMP-9, which enhances IL-8 mRNA expression by keratinocytes through PAR-2 and NF-κB activation. ALK+ALCL cells secrete MMP-9, which enhances IL-8 mRNA expression by keratinocytes through PAR-2 and NF-κB activation. (A) IL-8 mRNA levels were evaluated by qRT-PCR on PAR-2– or control siRNA–transfected HaCaT cells cultured in DMEM supplemented with (+) or without (−) rhMMP-9. (B) HaCaT cells exposed to culture supernatant from Karpas-299 (Karpas) and SU-DHL-1 cells supplemented with 10μM (+) or without (−) MMP inhibitor (GM6001) for 24 hours were collected and IL-8 mRNA measured by qRT-PCR. Results are expressed as the mean ± SD of 3 independent experiments done in triplicate. ***P < .001 by Student t test. (C) Western blotting was performed with anti–NF-κB p65 and anti–phospho-Ser536 NF-κB p65 Abs to determine NF-κB activation in keratinocytes (HaCaT cell line) under the influence of culture supernatant from Karpas-299 cells supplemented with (+) or without (−) GM6001. β-actin was used as a loading control. Emilie Dejean et al. Blood 2012;119:4698-4707 ©2012 by American Society of Hematology

HMGB-1 induces IL-8 mRNA expression and NF-κB activation via PAR-2 receptors by keratinocytes. HMGB-1 induces IL-8 mRNA expression and NF-κB activation via PAR-2 receptors by keratinocytes. (A) IL-8 mRNA levels were evaluated by qRT-PCR on PAR2- or control siRNA–transfected keratinocytes (HaCaT cell line) exposed to Karpas-299 (Karpas) and SU-DHL-1 cell line (ALK+ALCL) supernatants for 24 hours. The level of IL-8 mRNA was normalized to that from HaCaT cells cultured in DMEM (control medium). (B) Protein lysates from HaCaT cells exposed or not to ALK+ALCL cell line supernatants were subjected to western-blotting with anti–NF-κB p65 and anti–phospho-Ser536 NF-κB p65 Abs. β-actin was used as a loading control. A vertical line has been inserted to indicate a repositioned gel lane. (C-D) The same experiments as in panels A and B were performed on PAR2- or control siRNA–transfected HaCaT cells treated with rhHMGB-1 or not (PBS). IL-8 mRNA levels were normalized to the endogenous housekeeping gene GAPDH. Results are expressed as the fold increase compared with the control PBS condition (expression level = 1), and represent the mean ± SD of 3 independent experiments done in triplicate; *P < .05 by Student t test (C). Lysates from HaCaT cells were subjected to Western blotting with anti–NF-κB p65 and anti–phospho-Ser536 NF-κB p65 Abs. β-actin was used as a loading control (D). Emilie Dejean et al. Blood 2012;119:4698-4707 ©2012 by American Society of Hematology

Pharmacologic inhibition of HMGB-1 impairs ALK+ cell dissemination in vitro and in vivo. Pharmacologic inhibition of HMGB-1 impairs ALK+ cell dissemination in vitro and in vivo. Karpas-299 (Karpas) and SU-DHL-1 cells were incubated or not with 100mM glycyrrhizin. (A) The effect of glycyrrhizin on cell number was measured by following the absorbance at 490nm using the MTS assay. (B) ALK+ALCL cells cultured with supernatant from HaCaT cells previously exposed to Karpas-299 or SU-DHL-1 cell culture supernatants supplemented or not with 100mM glycyrrhizin were subjected to the 3D-Matrigel invasion assay described for Figure 5B. (C-E) Karpas-299 cells were subcutaneously injected into SCID mice. Tumor development progressed for 7 days, followed by 7 days of treatment with glycyrrhizin (one 10mg/kg IP injection/d) or PBS. (C) Tumor volume was measured every 2 days using callipers. (D) Histogram represents the mean ± SD of ALK+ cells infiltrating the lungs. The number of lymphoma cells was assessed by anti-ALK (rabbit monoclonal SP8) staining. (E) Histopathological examination of lung from xenografted NPM–ALK+ Karpas-299 mice shows that HMGB-1 inactivation leads to a decrease in ALK+ cells in the lungs. Mice were treated as described previously: lungs were harvested, fixed, and paraffin embedded for immunohistochemical analysis with anti-ALK (Sp8) to visualize invasion by ALK+ cells (original magnification, 400×). Emilie Dejean et al. Blood 2012;119:4698-4707 ©2012 by American Society of Hematology

Concomitant increase of serum CXCL1/KC and skin hyperplasia are a prerequisite to NPM-ALK+ lymphoma cell epidermotropism. Concomitant increase of serum CXCL1/KC and skin hyperplasia are a prerequisite to NPM-ALK+ lymphoma cell epidermotropism. (A-H) Histological analysis of lymphoma cell dissemination in a conditional mouse transgenic model. Conditional NPM-ALK lymphoma mouse models develop ALK+ B-cell (B220+) lymphomas. Images show normal architecture and negative lymphoma cell staining of spleen (A) and skin 10 (B) and 15 days (D) after birth. Lymphoma cells positive for ALK staining are observed in the spleen 15 (C), 20 (E), and 30 (G) days after birth. Note that the skin presents a hyperplasia of the epidermis and is negative for lymphoma cells staining (B220−; F). In contrast, 30 days after birth, 30% of the animals display B220+ tumoral cells infiltrated below the skin lesions (H; right inset shows magnification of bracket). Original magnification was 400× for the spleen and 100× for the skin, except in panel H, where the original magnification was 50× and 640×, respectively (right inset). Emilie Dejean et al. Blood 2012;119:4698-4707 ©2012 by American Society of Hematology

HMGB-1, IL-8, and IL-8 receptor (CXCR1 and CXCR2) expression by ALK+ALCL patients. HMGB-1, IL-8, and IL-8 receptor (CXCR1 and CXCR2) expression by ALK+ALCL patients. HMGB-1 (A) or CXCR1 and CXCR2 (C) mRNAs levels were evaluated by qRT-PCR on 15 ALK+ALCL biopsies. mRNA amounts were normalized to levels found in reactive lymph nodes. Results are expressed as the mean ± SD of independent experiments done in triplicate. **P < .01 by Student t test. (B,E) HMGB1 and IL-8 secretion was measured using an ELISA assay on sera from 35 ALK+ALCL patients with (n = 20) or without (n = 7) leukemic dissemination and from healthy patients (n = 10). The number of biopsies in each group corresponds to the n value. (D) CXCR1 and CXCR2 immunohistochemical staining of lymph nodes from 2 ALK+ALCL patients. Sections were stained with anti-CXCR1 or anti-CXCR2 Abs, and nuclei were counterstained with hematoxylin. Vessels were positive for both CXCR staining and were used as an internal control (asterisks). Staining intensity varies from tumoral cell to cell. Original magnification, 200×. Emilie Dejean et al. Blood 2012;119:4698-4707 ©2012 by American Society of Hematology

Schematic overview. Schematic overview. Shown is a schematic diagram of the possible relationship among the pro-inflammatory mediator HMGB-1, MMP-9, PAR-2, and the chemokine IL-8 to establish a hyperplastic environment within the skin that might participate in ALK+ lymphoma cell epidermotropism and cutaneous metastasis development. Solid arrows indicate pathways established by our data; dashed arrows are currently supported only by circumstantial evidence and remain to be experimentally validated. Illustration done by Servier Medical Art (http://www.servier.fr/servier-medical-art). Emilie Dejean et al. Blood 2012;119:4698-4707 ©2012 by American Society of Hematology