Clonal B cells in Waldenström's macroglobulinemia exhibit functional features of chronic active B-cell receptor signaling

Abstract

Waldenström's macroglobulinemia (WM) is a B-cell non-Hodgkin's lymphoma (B-NHL) characterized by immunoglobulin M (IgM) monoclonal gammopathy and the medullary expansion of clonal lymphoplasmacytic cells. Neoplastic transformation has been partially attributed to hyperactive MYD88 signaling, secondary to the MYD88 L265P mutation, occurring in the majority of WM patients. Nevertheless, the presence of chronic active B-cell receptor (BCR) signaling, a feature of multiple IgM+ B-NHL, remains a subject of speculation in WM. Here, we interrogated the BCR signaling capacity of primary WM cells by utilizing multiparametric phosphoflow cytometry and found heightened basal phosphorylation of BCR-related signaling proteins, and augmented phosphoresponses on surface IgM (sIgM) crosslinking, compared with normal B cells. In support of those findings we observed high sIgM expression and loss of phosphatase activity in WM cells, which could both lead to signaling potentiation in clonal cells. Finally, led by the high-signaling heterogeneity among WM samples, we generated patient-specific phosphosignatures, which subclassified patients into a 'high' and a 'healthy-like' signaling group, with the second corresponding to patients with a more indolent clinical phenotype. These findings support the presence of chronic active BCR signaling in WM while providing a link between differential BCR signaling utilization and distinct clinical WM subgroups.

Figure 1

WM cells exhibit constitutive activation of the BCR pathway. (a) Differences in basal levels of phosphorylation between HD B cells (black) and WM cells (red) expressed in arcsinh scale (HD n=9, WM n=23, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, Mann–Whitney test). (b) representative WM bone marrow sections stained for pSYK (Immunohistochemistry-tyramide signal amplification technology, × 40). (c) Inihbition of basal levels of pPLCγ2, assessed 60 min after in vitro preinhibition with Dasatinib (10 μM), Tamatinib (10 μM) and Ibrutinib (10 μM), illustrated as a ratio from dimethyl sulfoxide (DMSO) treated matched control samples (WM n=5, Repeated measures analysis of variance (ANOVA) with Tukey's multiple comparison test, ^^P<0.01, ^^^P<0.001, ^ represents significant difference from DMSO matched control). (d) Inhibition of basal levels of pPLCγ2 at 6 months of continuous ibrutinb treatment in eight WM patients, as a ratio from pre-ibrutinib phosphorylation levels (WM n=8, Wilcoxon test, *P<0.05, * represents significant difference from pre-ibrutinib treatment matched samples). (e) Viability of WM mononuclear cells assessed after 24 h of treatment with Dasatinib (1 μM), Tamatinib (1 μM) and Ibrutinib (1 μM). Viability is normalized to DMSO treated control. (WM n=8, Repeated measures ANOVA with Tukey's multiple comparison test, ^P<0.05, ^^P<0.01, ^ represents significant difference from DMSO matched controls, *P<0.05, * represents significant difference in matched samples among different inhibitors).

Figure 2

BCR crosslinking elicits augmented phosphoresponses in WM cells. (a) anti-IgM induced fold change in phosphorylation in HD B-cells (black) and WM cells (red) expressed in the arcsinh scale (HD n=9, WM n=23, ***P<0.001, **P<0.01, *P<0.05, Mann–Whitney test). (b) Dose response of anti-IgM induced fold change in pPLCγ2 in HD (black) and WM (red) cells and EC₅₀ values of all individuals (HD n=4, WM n=5, *P<0.05, Mann–Whitney test). (c) Kinetics of anti-IgM induced fold change in pPLCγ2 in HD (black) and WM (red) cells. At 0, fold change is 0 by definition. (HD n=4, WM n=6, **P<0.01, *P<0.05, Mann–Whitney test). (d) Inhibition of anti-IgM induced pPLCγ2. Stimulation was performed for 4 min after a 60 min in vitro preinhibition with Dasatinib (10 μM), Tamatinib (10 μM) and Ibrutinib (10 μM). Here illustrated as a ratio from dimethyl sulfoxide (DMSO) treated matched control samples (WM n=5, repeated measures analysis of variance (ANOVA) with Tukey's multiple comparison test, ^P<0.05, ^^^^P<0.0001, ^ represents significant difference from DMSO matched control, *P<0.05, **P<0.01, * represents significant difference in matched samples among different inhibitors). (e) Inhibition of anti-IgM induced pPLCγ2 at 6 months of continuous ibrutinb treatment, as a ratio from pre-ibrutinib matched anti-IgM induced pPLCγ2 levels (WM n=8, Wilcoxon test). (f) correlation between anti-IgM induced pPLCγ2 ratio and percentage change in intratrabecular (IT) space clonal infiltration post-ibrutinib treatment (Pearson test).

Figure 3

WM cells express higher levels of the receptor and show loss of phosphatase activity compared with HD cells. (a) sIgM overlayed cluster plots (left) for HD IgM+ B-cells (black) and WM (red) cells and bar graphs for all tested individuals (HD n=4, WM n=11, **P<0.01, Mann–Whitney test). (b) Surface μ-chain mRNA level relative expression in IgM+ HD (n=4) B-cells and WM (n=8) cells. Human GAPDH was used as an endogenous control (Mann–Whitney test). (c) Phosphatase score for HD B-cells (black) and WM cells (red) PTP_X=pX (arcsinh fold change)_aIgM+H2O2—pX(arcsinh fold change)_aIgM, where pX is any phosphoprotein (HD n=9, WM n=23, ****P<0.0001, ***P<0.001, **P<0.01, Mann–Whitney test). (d) Surface CD45 median fluorescence intensity (MFI) and surface CD22 MFI in HD (n=4) B-cells and WM (n=11) cells. Intracellular SHP-1 and intracellular SHIP-1 MFI in HD (n=4) B-cells and WM (n=6) cells. MFI is normalized to an FMO (fluorescence minus one) control.

Figure 4

Intra-WM signaling heterogeneity is driven by differential BCR expression and correlates with distinct clinical outcomes. (a) Intra-WM anti-IgM-induced phophoprotein variance (HD, black; WM, red). (b) Inter-phosphoprotein Spearman correlation network analysis (positive correlations, red; negative correlations, blue). (c) Agglomerative clustering analysis for 23 patients interrogated for all six phosphoproteins on IgM crosslinking. (d) Patient status on sample collection (χ² test P<0.0001). (e) Time to second line treatment from diagnosis date to sample collection date (log-rank survival analysis). (f) sIgM levels among groups (HD n=4, WM_high n=6, WM_healthy-like n=5, **P<0.01, analysis of variance (ANOVA) test). (g) Phosphatase score for pPLCγ2 among groups (HD n=9, WM_high n=13, WM_healthy-like n=10, ANOVA test).