For total ERK and NF-KB detection we used primary purified anti-ERK1/2 (polyclonal) and anti- NF-KB, clone D14E12, both from Cell Signaling Technology and subsequent staining with a secondary goat anti-rabbit antibody Pacific-Blue conjugate (ThermoFisher Scientific). In support of those findings we observed high sIgM expression and PLD1 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. == Introduction == B-cell receptor (BCR) signaling governs cellular homeostasis throughout all stages of mature B-cell differentiation. Naive, antigen-inexperienced cells, which constitute the majority of the mature B-cell pool, require low levels of tonic BCR signaling for their survival,1while antigen-induced BCR signaling, in the presence of cytokine and co-receptor signaling, initiates a cascade of B-cell activation, clonal expansion, and subsequent memory and plasma cell formation.2The sequence of intracellular events following BCR engagement in normal B cells has been extensively investigated over the last 20 years. Cross-linking of surface immunoglobulins induces tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs of Ig and Ig by Src family kinases (SFK), which recruit and activate the spleen tyrosine kinase (SYK), which in turn mediates the activation of Bruton’s tyrosine kinase (BTK), the adapter B-cell linker protein (BLNK), and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/ protein kinase B (AKT) axis, among other G-proteins, phosphatases and lipid hydrolases. P276-00 This cascade of proximal events results in the formation of a multi-protein signaling complex, P276-00 known as P276-00 the BCR signalosome, whose ultimate effector is phospholipase C-gamma-2 (PLC2), a fundamental molecule for the activation of downstream protein targets, including extracellular-signal-regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B (Supplementary Figure 1).3,4,5,6,7The presence of aberrant BCR signaling has long been established as a key feature of B-cell lymphomagenesis.8Specifically, the phenomenon of chronic active BCR signaling has been evidenced by skewed immunoglobulin heavy chain variable region (IGHV) segment usage, BCR upregulation and preclustering, signaling molecule mutations and strong BCR-related transcriptome and phosphorylation signatures.8,9Aspects of it have been demonstrated in the context of multiple immunoglobulin M (IgM)+ B-cell non-Hodgkin’s lymphoma subtypes, yet more consistently in activated B-cell like diffuse large B-cell lymphoma10,11and chronic lymphocytic leukemia (CLL).12,13 Waldenstrm’s macroglobulinemia (WM) is an indolent B-cell non-Hodgkin’s lymphoma characterized by the accumulation of IgM-secreting clonal lymphoplasmacytic cells in the bone marrow and extramedullary sites.14After an extensive characterization of the genomic landscape in WM, MYD88 L265P (>90% of cases) and CXCR4-WHIM (warts, hypogammaglobulinemia, Infections, myelokathexis)-like mutations (~27% of cases) have emerged as the pathologic hallmarks of the disease, demonstrating the significance of these two signaling axes in the pathobiology of WM.15,16,17BCR-signaling-associated mutations occur less frequently, and are restricted to the CD79A and CD79B genes, in approximately 15% of WM cases.16,18The strongest evidence for BCR utilization in WM, stems from IGHV studies, which demonstrate P276-00 a high mutational load and skewed repertoire, suggesting pastin vivoactivation of the pathway.19,20,21SYK and BTK inhibition have been shown to have tumoricidal effects in pre-clinical studies focused on WM cell lines,22,23while targeting BTK with ibrutinib in the recently completed clinical trial NCT0161482 generated overall response rates of 90.5% among refractory/relapsed patients.24Nevertheless, considering that both SYK and BTK are elements of multiple signaling pathways, including toll-like receptors (TLR), chemokine receptors, integrins and Fc receptors, the role of BCR signaling and its net contribution in WM remains ill-defined. To comprehend the activity of the BCR network in primary WM cells, we interrogated multiple BCR-related phosphoproteins in a resting andex vivostimulated state, utilizing multiparametric phosphoflow cytometry, which allows the precise quantification of multiple signaling events at a single-cell level.25,26We evaluated aspects of network remodeling in WM cells, compared with physiological BCR signaling,.