Cirmtuzumab Inhibits Wnt5a-Induced Rac1 Activation in Chronic Lymphocytic Leukemiatreated

Cirmtuzumab Inhibits Wnt5a-Induced Rac1 Activation In Chronic Lymphocytic LeukemiaTreated With Ibrutinib

Jian Yu, et al

Supplementary Figure Legends

Supplementary Figure S1

Ibrutinib Inhibits BCR Signaling, But Not Wnt5a/ROR1 Signaling - A) Activated Rac1 was measured in CLL cells incubated with or without Wnt5a or ibrutinib at concentrations of 0, 0.25, 0.5, or 1.0 μM, as indicated on the top of each lane. (B) CLL cells were treated with increasing doses of ibrutinib for 1 hour and then assayed for occupancy of the BTK active site. (C)Anti-μ-induced calcium mobilization in CLL cells after treatment with or without ibrutinib at concentrations indicated in the top right corner. The relative mean fluorescence intensity in intracellular calcium is plotted as a function of time. The arrow labeled “IgM” indicates the time at which the anti-μ was added to the cells. (D)Determination of cell viability by staining with DiOC6and PI. Presented are dot plots of CLL cells from a representative patient defining the relative green (DiOC6) and red (PI) fluorescence intensities of the leukemia cells on the horizontal and vertical axes, respectively. The proportion of viable cells (DiOC6+PI−) was determined after treatment with ibrutinib at concentrations indicated in parentheses. The percentages of the cells that were viable (lower right quadrants) are indicated in each dot plot.

Supplementary Figure S2

CFSE Assay For CLL Proliferation Induced By Wnt5a Without CD154 -(A) Gating strategy for dividing CLL cells. Cells were first gated on size and singularity followed by PI exclusion to identify live cells for further analysis. Live CD5 and CD19 CLL cells were examined for fluorescence after staining with CFSE. The percentages of dividing CLL cells were calculated by computing the proportion of cells that had lower CFSE fluorescence. (B) Fluorescence of CFSE-labeled CLL cells (n=6) co-cultured for 5 days with wild-type HeLa cells without (top panel) or with (lower panel) exogenous Wnt5a in the presence of IL-4/10. The results of one representative CLL sample are shown with the percent of dividing cells indicated in the lower left corner of each histogram.

Supplementary Figure S3

Wnt5a-Induced Rac1 Activation Or Proliferation OfTCL1 Leukemia Cells - (A) Rac1 activation was measured in serum-starved TCL1 leukemia cells, which were treated with Wnt5a for 30 min. Whole-cell lysates were run on parallel gels to examine total Rac1. The numbers below each lane are ratios of band optical densities of activated versus total GTPase, normalized with respect to that of untreated samples. (B) CFSE assay for TCL1 leukemia cell proliferation induced by Wnt5a and/or CD154. Fluorescence of CFSE-labeled TCL1 leukemia cells (n=3) co-cultured for 5 days with wild-type HeLa or HeLaCD154 cells without or with exogenous Wnt5a in the presence of IL-4/10. Mean proportions of dividing CLL cells from TCL1 leukemia cells under conditions indicated at the bottom. Data are shown as mean ± SEM for each group; p-values were calculated using one-way ANOVA with Tukey’s multiple comparisons test.

Supplementary Figure S4

Cell Cycle Analysis Of CLL Cells Treated With Cirmtuzumab Or Ibrutinib, With Or Without Exogenous Of Wnt5a - (A)Leukemia cells were co-cultured on HeLaCD154in the presence of IL-4/10 or Wnt5a, and then treated with cirmtuzumab (10 μg/ml) or ibrutinib (0.5 μM) for 4 days, subjected to cell-cycle analysis following PI staining. One representative leukemia sample is shown. (B)The mean proportions of leukemia cells in S/G2/M phase for all 3 samples tested is presented. Data are shown as mean ± SEM, *P < 0.05; **P < 0.01, as determined by one-way ANOVA with Tukey’s multiple comparisons test.

Supplementary Figure S5

Dose-Dependent Inhibitory Effect Of Cirmtuzumab Or Ibrutinib OnROR1×TCL1 Leukemia Engrafted Mice -(A)Dose-dependent inhibitory effect of ibrutinib in ROR1×TCL1 leukemia engrafted mice.(B) Dose-dependent inhibitory effect of cirmtuzumab in ROR1×TCL1 leukemia engrafted mice. Each symbol represents the number of leukemia cells found in individual mice. Data are shown as mean ± SEM for each group of animals (n=6); *P < 0.05; ***P < 0.001, as calculated using one-way ANOVA with Tukey’s multiple comparisons test.

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