The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia by Christine Le Roy, Pierre-Antoine Deglesne, Nathalie Chevallier, Taoufik Beitar, Virginie Eclache, Maude Quettier, Marouane Boubaya, Rémi Letestu, Vincent Lévy, Florence Ajchenbaum-Cymbalista, and Nadine Varin-Blank Blood Volume 120(2):356-365 July 12, 2012 ©2012 by American Society of Hematology

Cell surface IgM levels as well as expression and global phosphorylation levels of Syk reflect CLL B-cell responsiveness. Cell surface IgM levels as well as expression and global phosphorylation levels of Syk reflect CLL B-cell responsiveness. Presence of surface IgM (A), CD20 (B), or Syk (C) and Zap70 (D) kinase levels was assayed on normal (control) and CLL B cells using flow cytometry analysis. (A) Cell surface IgM levels were calculated and graphed relative to control isotype labeling for 11 controls, 20 nonresponder (NR) samples, and 20 responder (R) samples. MFI indicates mean fluorescence intensity. (B-D) Cell surface CD20 expression on 20 NR and 20 R patients and intracellular Syk and Zap70 on 22 NR and 24 R cases were evaluated and graphed relative to control B cells. Means (± SEM) and significant P values are indicated (ns denotes not significant). (E) Anti-phosphotyrosine immunoprecipitation ([IP]; α-pY) was performed on cellular extracts of NR (n = 6) and R (n = 6) CLL B cells. Cells were either left unstimulated (−) or stimulated (+) with anti-IgM (α-IgM) antibody. Precipitates were analyzed with anti-Syk or anti-Zap70 antibodies, allowing detection of pSyk and pZap70. Total extracts are analyzed as a control for Syk and Zap70 expression. Fold increase is calculated as a ratio between stimulated and unstimulated levels. Christine Le Roy et al. Blood 2012;120:356-365 ©2012 by American Society of Hematology

BCR triggering promotes PLCγ2 phosphorylation and calcium mobilization in responding CLL B cells. BCR triggering promotes PLCγ2 phosphorylation and calcium mobilization in responding CLL B cells. (A) Phospho-Y759-PLCγ2 levels were analyzed using a specific PE-coupled antibody in CLL B cells (CD20+/CD5+/CD3−) by flow cytometry analysis. Histogram plots show pY759 PLCγ2 MFI in unstimulated (α-IgM−, top) and stimulated (α-IgM+, bottom) B cells from 1 representative patient for each CLL group (NR, left; R, right). Phospho-PLCγ2 was measured for 43 samples after 10 minutes of incubation with anti-IgM antibody in NR (n = 20, black dots) and R (n = 23, black squares) cases, calculated as fold induction over unstimulated cells and graphed. (B) Calcium release curves established in NR (n = 7, left) and R (n = 8, right) cases. After 60-second stabilization, CLL B cells were stimulated with anti-IgM antibody (+ α-IgM) followed at 290 seconds by ionomycin addition (+ ionomycin). Christine Le Roy et al. Blood 2012;120:356-365 ©2012 by American Society of Hematology

NFAT2 is overexpressed and to some extent localized into the nucleus of CLL B cells. NFAT2 is overexpressed and to some extent localized into the nucleus of CLL B cells. (A) Relative NFAT2 transcript levels were assessed by quantitative RT-PCR from freshly isolated normal blood peripheral B and T lymphocytes (normal lymphocytes B and T; n = 6 and n = 3, respectively) and from CLL B cells from NR and R cases (n = 17). NFAT levels were normalized to Abelson expression. (B) Freshly purified CLL B cells (n = 10) were immunostained with anti-NFAT2 antibody. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. One representative case of each CLL group (NR and R) is presented. Images were acquired using confocal laser-scanning microscope (TCS SP2; Leica). Scale bars represent 5 μm. Single optical sections were obtained with high numerical aperture lens (63 × 2.8 NA) to determine the percentage of NFAT2-positive nuclei in NR and R cells and graphed on the right histogram; an average of 90 cells/sample (6 samples) were analyzed. No statistical difference was observed (ns). Christine Le Roy et al. Blood 2012;120:356-365 ©2012 by American Society of Hematology

On BCR ligation, NFAT2 is differentially activated in B cells from both groups of patients. On BCR ligation, NFAT2 is differentially activated in B cells from both groups of patients. (A) Nuclear or cytoplasmic extracts from freshly isolated B cells from R (n = 9) and NR (n = 6) cases and from unstimulated or CD3+/CD28+-stimulated Jurkat cell line (horizontal dashed lines) were analyzed for their NFAT2 DNA-binding ability using an ELISA-derived assay. Competition with a specific oligonucleotide was used to ensure binding specificity (P, probe; n = 3). No statistical difference (ns) was observed between NR and R patient extracts. (B) Nuclear extracts from NR (n = 13) and R (n = 12) cells stimulated or not with an anti-IgM antibody (10 μg/mL/106 cells) for 18 hours were used following the same protocol as described in panel A. Results are presented as fold induction of the NFAT2 DNA-binding ability on stimulation. Christine Le Roy et al. Blood 2012;120:356-365 ©2012 by American Society of Hematology

VIVIT peptide inhibitor blocks the BCR/NFAT-dependent pathway in responding CLL B cells. VIVIT peptide inhibitor blocks the BCR/NFAT-dependent pathway in responding CLL B cells. (A) NFAT2 immunofluorescence staining of CLL B cells from 1 representative R case (n = 3) stimulated or not in the presence or not of NFAT inhibitor 11-R VIVIT (5μM for 24 hours). Scale bar corresponds to 5 μm. (B) Quantitative RT-PCR analysis of CD23 transcript expression levels in freshly isolated B cells from R cases (n = 5) on stimulation with anti-IgM (10 μg/mL) in the presence or not of 11-R VIVIT (5μM). CD23 mRNA expression was normalized on ABL transcript expression in each tested sample, and results were graphed. Cell surface expression of CD23 protein (C) or CD71 protein (D) was determined using flow cytometry analysis on CLL cells from 7 (C) or 8 (D) R cases stimulated with anti-IgM (10 μg/mL) in presence of increasing concentrations (0, 2, and 5μM) of 11-R VIVIT. Levels are indicated as fold increase on stimulation. (E) Fold increase of metabolic activity (% MTS) was determined on 48-hour BCR stimulation for 13 NR and 12 R cases. B lymphocytes were incubated in the presence or not of 11-R VIVIT (5μM). Christine Le Roy et al. Blood 2012;120:356-365 ©2012 by American Society of Hematology

IgM-induced cell survival is relevant for CLL clinical outcome. IgM-induced cell survival is relevant for CLL clinical outcome. Kaplan-Meier plots showing PFS (A) and OS (B) distributions in R and NR patients. Log-rank test was used to compare statistical differences in PFS and survival between both groups (P values are indicated). Christine Le Roy et al. Blood 2012;120:356-365 ©2012 by American Society of Hematology