Surface IgM stimulation induces MEK1/2-dependent MYC expression in chronic lymphocytic leukemia cells by Sergey Krysov, Samantha Dias, Alex Paterson, C. Ian Mockridge, Kathleen N. Potter, Kelly-Ann Smith, Margaret Ashton-Key, Freda K. Stevenson, and Graham Packham Blood Volume 119(1):170-179 January 5, 2012 ©2012 by American Society of Hematology

Induction of MYC protein after sIgM stimulation in CLL samples. Induction of MYC protein after sIgM stimulation in CLL samples. (A,C) CLL samples were incubated with anti-IgM for 3 or 6 hours or for 6 hours with the isotype control antibody (IC). Expression of MYC and MAX protein was analyzed by immunoblotting. Results shown are representative of those obtained from analysis of 18 intracellular Ca2+-responsive (A) and 9 intracellular Ca2+ nonresponsive (C) CLL samples. MAX expression demonstrates equal loading of protein samples. (B,D) Quantitation of the fold increase in MYC protein expression (relative to isotype-control treated cells; C) measured by densitometry analysis of immunoblots at 3 or 6 hours after stimulation with anti-IgM for intracellular Ca2+-responsive (B) and -nonresponsive (D) samples. Graphs show data for individual samples, and any statistically significant differences (Student matched paired t test) between control and anti-IgM–stimulated cells (NS indicates not significant, P > .05). (E) Comparison between MYC induction (> 20% increase compared with control cells) and positive (□) and negative (■) intracellular Ca2+ responses (Fisher exact test) in anti-IgM–treated CLL samples (n = 27). Sergey Krysov et al. Blood 2012;119:170-179 ©2012 by American Society of Hematology

Effect of anti-IgM on expression of MYC target genes. Effect of anti-IgM on expression of MYC target genes. CLL samples (n = 8) were stimulated with anti-IgM for 1 or 6 hours or with isotype control antibody. Expression of ODC1, CDK4, and CCND2 RNA were analyzed by quantitative real-time PCR. Expression values for the isotype control antibody (C) were set to 1.0 for each time point. Graphs show the fold induction for each sample and statistically significant differences are indicated (Student matched paired t test; NS indicates not significant, P > .05). Sergey Krysov et al. Blood 2012;119:170-179 ©2012 by American Society of Hematology

Activation of ERK1/2 phosphorylation in sIgM-stimulated CLL B cells. Activation of ERK1/2 phosphorylation in sIgM-stimulated CLL B cells. (A) CLL samples were stimulated with anti-IgM for up to 60 minutes and ERK1/2 phosphorylation analyzed by flow cytometry. Graphs show the fold increase in ERK1/2 phosphorylation after stimulation with anti-IgM relative to untreated cells for 5 representative samples. (B) Correlation between the maximal percentage of cells showing increased intracellular Ca2+ and maximal fold induction of ERK1/2 phosphorylation after sIgM stimulation. Results of linear regression are shown (n = 37). Sergey Krysov et al. Blood 2012;119:170-179 ©2012 by American Society of Hematology

Effect of U0126 on MYC induction. Effect of U0126 on MYC induction. Cells were stimulated with anti-IgM for 3 hours in the presence of U0126 (10μM) or DMSO as a control. (A) Expression of MYC and phosphorylated and total ERK1/2 was analyzed by immunoblotting. Two representative samples are shown of a total of 5 samples analyzed. Note that intervening lanes were removed for clarity, and the complete blot is shown in supplemental Figure 1D. (B) Quantitation of MYC expression. Graphs show mean (± SEM) MYC expression relative to untreated cells (set to 1.0), derived from 5 independent experiments. The reduction in MYC expression in cells treated with anti-IgM and U0126 was statistically significant compared with cells treated with anti-IgM alone (Student matched pairs t test). Sergey Krysov et al. Blood 2012;119:170-179 ©2012 by American Society of Hematology

Effect of U0126 on CpG-ODN–treated CLL cells. Effect of U0126 on CpG-ODN–treated CLL cells. CLL cells (n = 6) were pretreated with 10μM U0126 or DMSO for 15 minutes, before stimulation with CpG-ODN (7μg/mL) for 3 or 48 hours. (A) Representative analysis of BrdU and 7AAD staining in untreated (top) and CpG-ODN (bottom)–treated CLL cells (48 hours). S-phase and dead cells are gated in the bottom panel. (B) Quantitation of S-phase (48 hours, in the presence or absence of CpG-ODN ± U0126. (C) Immunoblot analysis of MYC, phosphorylated ERK1/2, and β-actin expression at 3 hours. Sergey Krysov et al. Blood 2012;119:170-179 ©2012 by American Society of Hematology

Activation of ERK1/2 phosphorylation and MYC expression in sIgD-stimulated CLL and normal B cells. Activation of ERK1/2 phosphorylation and MYC expression in sIgD-stimulated CLL and normal B cells. (A) CLL samples were stimulated with anti-IgD for up to 60 minutes and analyzed for ERK1/2 phosphorylation by flow cytometry. Graphs show the fold increase in ERK1/2 phosphorylation after stimulation with anti-IgD relative to untreated cells for 4 representative samples. Note that the same samples are shown in Figure 3A (after sIgM stimulation), and direct comparison of sIgM and sIgD responses is shown in supplemental Figure 1B. (B) Comparison between protracted/transient ERK1/2 responses after stimulation of sIgM (□) or sIgD (■; Fisher exact test, P = .0001; n = 37). (C) Quantitation of MYC protein induction after stimulation of sIgM or sIgD, relative to isotype antibody-treated controls (n = 18). Note values for anti-IgM–treated cells are the same as those shown in Figure 1B and are shown again here to allow direct comparison with anti-IgD–treated cells. See supplemental Figure 1C for side-by-side immunoblot analysis of MYC expression in sIgM- or sIgD-stimulated CLL cells. (D) Normal B cells were stimulated with anti-IgM (□) or anti-IgD (■) for up to 60 minutes and ERK1/2 phosphorylation analyzed by flow cytometry. Graph shows mean fold increase in ERK1/2 phosphorylation for CD20+CD27− cells. Data are mean values ± SEM (n = 8). (E) CD19+CD27− B cells isolated from healthy donors were stimulated with anti-IgM or anti-IgD for 3 or 6 hours or for 6 hours with the isotype control. MYC and MAX expression was analyzed by immunoblotting. Results are shown for cells isolated from 2 separate donors. Sergey Krysov et al. Blood 2012;119:170-179 ©2012 by American Society of Hematology

Expression of phosphorylated ERK1/2 and MYC in vivo. Expression of phosphorylated ERK1/2 and MYC in vivo. Immunohistochemical analysis of MYC (A,C) and phospho-ERK1/2 (B,D) in 2 CLL/SLL lymph node biopsy samples. The images show expression within representative PCs comprising larger, less densely stained cells. (E-F) MYC expression in Burkitt lymphoma and phospho-ERK1/2 expression in colon cancer, respectively. Original magnification ×600. Sergey Krysov et al. Blood 2012;119:170-179 ©2012 by American Society of Hematology