Proximal B-cell Receptor Signaling Research Articles

Quantitative modeling of signaling in aggressive B cell lymphoma unveils conserved core network.

B cell receptor (BCR) signaling is required for the survival and maturation of B cells and is deregulated in B cell lymphomas. While proximal BCR signaling is well studied, little is known about the crosstalk of downstream effector pathways, and a comprehensive quantitative network analysis of BCR signaling is missing. Here, we semi-quantitatively modelled BCR signaling in Burkitt lymphoma (BL) cells using systematically perturbed phosphorylation data of BL-2 and BL-41 cells. The models unveiled feedback and crosstalk structures in the BCR signaling network, including a negative crosstalk from p38 to MEK/ERK. The relevance of the crosstalk was verified for BCR and CD40 signaling in different BL cells and confirmed by global phosphoproteomics on ERK itself and known ERK target sites. Compared to the starting network, the trained network for BL-2 cells was better transferable to BL-41 cells. Moreover, the BL-2 network was also suited to model BCR signaling in Diffuse large B cell lymphoma cells lines with aberrant BCR signaling (HBL-1, OCI-LY3), indicating that BCR aberration does not cause a major downstream rewiring.

Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL) are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin ligase with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiologic KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the bric-a-brac tramtrack broad domain of KLHL6 disrupted its localization and heterodimerization and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL. Significance: Oncogenic BCR signaling sustains DLBCL cells. We discovered that Cullin-3-RING E3 ubiquitin ligase substrate-adapter KLHL6 targets BCR heterodimer (CD79A/CD79B) for ubiquitin-mediated degradation. Recurrent somatic mutations in the KLHL6 gene cause corrupt BCR signaling by disrupting surface BCR homeostasis. Loss of KLHL6 expression and mutant-induced phenotypes associate with targetable disease characteristics in B-cell lymphoma. See related commentary by Leveille et al. See related commentary by Corcoran et al.

Abstract 3913: Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy

Abstract Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas. However, the mechanisms by which GCs kill malignant lymphoma cells still largely remains elusive due to the pleiotropic consequences of GC binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. This prompted us to search for critical direct targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To determine effector genes that increase glucocorticoid toxicity, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone. Screens in cell line models of Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor (BCR) signaling pathway (≥2 SD). To identify direct transcriptional GR targets and binding sites genome-wide, we performed the cleavage under targets and release using nuclease (CUT&RUN) assay and integrated this dataset with RNA-seq upon GC treatment. The pairwise comparison analysis (p<0.05) for GR-bound genes across BL and DLBCL cell lines revealed a significant enrichment of negative regulators of BCR/PI3K signaling and BCR-related tumor suppressors (LAPTM5, KLHL14, ARID5B, DDIT4, DAPP1, INPP5D, and CSK). Interestingly, expression of CSK, an inhibitor of all Src-family kinases (SFKs), was repressed while that of other BCR negative regulators were directly activated by binding of GR. Combinatorial deletion of activated genes counteracted the toxicity of GC up to 83.9%. In contrast, depletion of CSK paradoxically increased the cytotoxic effects of GC in BCR-dependent aggressive lymphoma with decreased proximal BCR signaling. To gain further insights on CSK function in aggressive lymphomas, we performed phospho-proteome and ubiquitinome profiling by treating lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi). The CSKi treatment initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-κB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs. Furthermore, small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers. Citation Format: Jaewoo Choi, Michele Ceribelli, James D. Phelan, Björn Häupl, Da Wei Huang, George W. Wright, Tony Hsiao, Vivian Morris, Francesco Ciccarese, Boya Wang, Sean Corcoran, Sebastian Scheich, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Joyce Zhou, Grace Zhang, Jagan Muppidi, Giorgio Ga Inghirami, Thomas Oellerich, Craig J. Thomas, Wyndham H. Wilson, Louis M. Staudt. Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3913.

MYD88L265P augments proximal B-cell receptor signaling in large B-cell lymphomas via an interaction with DOCK8

AbstractDiffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease with at least 5 recognized molecular subtypes. Cluster 5 (C5)/MCD tumors frequently exhibit concurrent alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) pathway members, MYD88L265P and CD79B, and have a less favorable prognosis. In healthy B cells, the synergy between TLR and BCR signaling pathways integrates innate and adaptive immune responses and augments downstream NF-κB activation. In addition, physiologic TLR9 pathway engagement via MYD88, protein tyrosine kinase 2 (PYK2), and dedicator of cytokinesis 8 (DOCK8) increases proximal BCR signaling in healthy murine B cells. Although C5/MCD DLBCLs are selectively sensitive to Bruton tyrosine kinase (BTK) inhibition in in vitro studies and certain clinical trials, the role of mutated MYD88 in proximal BCR signaling remains undefined. Using engineered DLBCL cell line models, we found that concurrent MYD88L265P and CD79B alterations significantly increased the magnitude and duration of proximal BCR signaling, at the level of spleen tyrosine kinase and BTK, and augmented PYK2-dependent DOCK8 phosphorylation. MYD88L265P DLBCLs have significantly increased colocalization of DOCK8 with both MYD88 and the proximal BCR-associated Src kinase, LYN, in comparison with MYD88WT DLBCLs, implicating DOCK8 in MYD88L265P/proximal BCR cross talk. Additionally, DOCK8 depletion selectively decreased proximal BCR signaling, cellular proliferation, and viability of DLBCLs with endogenous MYD88L265P/CD79BY196F alterations and increased the efficacy of BTK blockade in these lymphomas. Therefore, MYD88L265P/DOCK8-enhanced proximal BCR signaling is a likely mechanism for the increased sensitivity of C5/MCD DLBCLs to BTK blockade.

MYD88 L265P Augments Proximal B-Cell Receptor Signaling in Large B-Cell Lymphomas Via an Interaction with DOCK8

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease comprised of five subtypes including a subset of poor-prognosis activated B cell (ABC)-enriched tumors with frequent MYD88L265P mutations, often in association with CD79B alterations (Cluster 5 DLBCLs) (Nat. Med. 2018; 24:679-690). Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) have similar genetic signatures including recurrent MYD88L265P mutations and concurrent CD79B alterations (Blood 2016; 127: 869-81). These findings prompted us to evaluate a potential role for MYD88L265P in proximal B-cell receptor (BCR) signaling, in addition to its defined function as an intermediary in the Toll-Like Receptor (TLR) pathway and downstream NF-kB activation.In previous studies by Jabara et al., wild-type (WT) MYD88 was found to be constitutively associated with the DOCK8 adapter and the PYK2 tyrosine kinase in normal B-cells (Nat. Immunol. 2012; 13:612-20). In this setting, physiologic ligation of TLR9 with CpG oligodeoxynucleotides (CpG) induced PYK2-mediated phosphorylation of DOCK8, recruitment of Src kinases, including LYN, and downstream activation of the proximal BCR pathway member, spleen tyrosine kinase (SYK) (Nat. Immunol. 2012; 13:612-20). We postulated that mutated MYD88L265P might similarly augment proximal BCR signaling in DLBCLs in the absence of physiologic (CpG-induced) TLR9 signaling. Using three DLBCL cell lines (OCI-Ly1, SU-DHL4 and OCI-Ly7) with intact BCR signaling and WT endogenous MYD88 and CD79B, we first established that physiologic CpG activation of TLR signaling induced the phosphorylation of PYK2 and the proximal BCR signaling components, SYK and Bruton's tyrosine kinase (BTK). Thereafter, we genetically engineered these three DLBCL cell lines to express MYD88 L265P or MYD88 WT, alone or in association with CD79B Y196F. In all three cell lines, the co-expression of MYD88 L265P and CD79B Y196F significantly increased magnitude and duration of SYK and BTK phosphorylation following BCR crosslinking. These findings highlight the likely role of MYD88L265P in CD79BY196F-associated proximal BCR signaling in DLBCL.To elucidate the potential role of the DOCK8 adapter in MYD88 L265P-augmented BCR signaling, we first assessed the colocalization of MYD88 WT or MYD88 L265P with DOCK8 in the same three genetically engineered DLBCL cell lines using proximity ligation assays (PLA), which detect protein-protein interactions at less than 40 nm in situ. In each of these cell lines, we detected significantly increased co-localized MYD88 L265P/DOCK8 signals in comparison to MYD88 WT/DOCK8 signals (p<.0001, all). Additionally, there were significantly increased co-localized DOCK8/LYN signals in DLBCL cell lines that expressed MYD88 L265P rather than MYD88 WT (p<.0001, all). These data provide the first direct evidence of an enhanced association between MYD88 L265P, DOCK8 and LYN in BCR-dependent DLBCLs and a basis for enhanced BCR signaling in primary tumors with concurrent MYD88L265P and CD79B genetic alterations.We next analyzed the consequences of MYD88 L265P-associated, DOCK8-dependent increased proximal BCR signaling by depleting DOCK8 in BCR-dependent DLBCL cells with endogenous MYD88L265P/CD79BY196F alterations (HBL1 and TMD8) or endogenous unmutated MYD88 WT/CD79B WT (OCI-Ly1 and SU-DHL4). ShRNA-mediated DOCK8 knockdown (KD) significantly decreased BCR-mediated phosphorylation of SYK and BTK in MYD88L265P/CD79BY196F DLBCL cell lines but not in lines with MYD88 WT/CD79B WT, highlighting the specific role of DOCK8 in MYD88 L265P-associated proximal BCR signaling. Of great interest, DOCK8 KD selectively decreased the proliferation of MYD88L265P/CD79BY196F, but not MYD88WT/CD79BWT, DLBCLs (p<.004, HBL1 and p<.009, TMD8; p = non sig., OCI-Ly1 and SU-DHL4). Additionally, DOCK8 KD significantly increased the efficacy of chemical PI3Kα/δ (copanlisib) and BTK (ibrutinib) inhibition in MYD88L265P/CD79BY196F DLBCLs (HBL1 and TMD8). Taken together, these data identify DOCK8 as an intermediary in MYD88L265P-driven proximal BCR signaling and a possible treatment target in LBCLs with co-occurring MYD88L265P/CD79BY196F mutations. DisclosuresShipp: AstraZeneca: Consultancy, Research Funding; Immunitas Therapeutics: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Research Funding; Bayer: Other: Institution: Research Grant/Funding; Abbvie: Other: Institution: Research Grant/Funding.

Role of NFAT in Chronic Lymphocytic Leukemia and Other B-Cell Malignancies.

In recent years significant progress has been made in the clinical management of chronic lymphocytic leukemia (CLL) as well as other B-cell malignancies; targeting proximal B-cell receptor signaling molecules such as Bruton Tyrosine Kinase (BTK) and Phosphoinositide 3-kinase (PI3Kδ) has emerged as a successful treatment strategy. Unfortunately, a proportion of patients are still not cured with available therapeutic options, thus efforts devoted to studying and identifying new potential druggable targets are warranted. B-cell receptor stimulation triggers a complex cascade of signaling events that eventually drives the activation of downstream transcription factors including Nuclear Factor of Activated T cells (NFAT). In this review, we summarize the literature on the expression and function of NFAT family members in CLL where NFAT is not only overexpressed but also constitutively activated; NFAT controls B-cell anergy and targeting this molecule using specific inhibitors impacts on CLL cell viability. Next, we extend our analysis on other mature B-cell lymphomas where a distinct pattern of expression and activation of NFAT is reported. We discuss the therapeutic potential of strategies aimed at targeting NFAT in B-cell malignancies not overlooking the fact that NFAT may play additional roles regulating the inflammatory microenvironment.

BAFF promotes heightened BCR responsiveness and manifestations of chronic GVHD after allogeneic stem cell transplantation

Patients with chronic graft-versus-host disease (cGVHD) have increased B cell–activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex–mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic μMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)–activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell–depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.

In Vitro-Selected Nanobody-Based Cellular Therapy Targeting CD72 for Treatment of Refractory B-Cell Malignancies

Introduction: B-cell acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr; also known as KMT2Ar) have particularly dismal clinical outcomes. Although CAR T immunotherapies targeting CD19 have shown impressive responses treating MLLr B-ALL and other B cell malignancies, relapse, often with loss of relevant CD19 epitope, remains a major clinical concern. The mixed results of CD19 CAR T as a monotherapy underscores the need to pursue additional immunotherapy targets and novel therapeutic modalities for high-risk patients. Results and Methods: Data with existing CAR-T's suggest that increased target antigen density frequently correlates with increased tumor elimination. Therefore, we aimed to define the cell surface proteomic landscape of B-ALL to identify novel, MLLr-enriched candidates for targeted immunotherapy of this poor-prognosis subtype. As an initial screen, using N-glycoprotein capture and mass spectrometry, we quantified differentially abundant cell surface proteins in MLLr (n= 4) versus non-MLLr (n= 5) B-ALL cell lines (Figure 1). Label-free proteomics (n= 3 replicates) quantified &amp;gt;900 high-confidence membrane proteins (FDR=0.05). Principal component analysis identified unique cell surfaceome signatures between B-ALL subtypes, implying different surface landscapes associated with specific genetic alterations. The MLLr B-ALL "surfaceome" is notably characterized by increased expression of adhesion molecules not identified by RNA-sequencing alone. We focused on CD72 as a novel immunotherapy target given significant enrichment on MLLr B-ALL vs. other B-ALL subtypes, near equivalent antigen density to CD19, undetectable expression on HSPCs, T-cells, and other normal tissues, and reported widespread expression on other mature B-cell malignancies. Analysis of transcriptome and ChIP-seq data suggested increased CD72 expression in MLLr B-ALL is not regulated directly by the MLL-AF4 oncoprotein but instead a function of increased CD72 expression at pro-B-cell stage. Flow cytometry and immunohistochemistry on primary samples confirmed high expression of CD72 both in MLLr B-ALL as well as DLBCL. Recombinant CD72 ECD was panned against a fully in vitro nanobody yeast display library (McMahon et al., Nat Struct Mol Biol(2018)) resulting in isolation of multiple unique, highly-specific CD72 nanobody binders with KD's &amp;lt; 5nM. Nanobodies were incorporated into 2nd generation CAR constructs and transduced into normal donor CD8+ T-cells and assessed in vitro for tumor cell lysis, cytokine release, and exhaustion marker expression. Nanobody clone Nb.D4 outperformed others in lysis of B-ALL and DLBCL cells lines displaying a broad range of CD72 expression, had no activity versus CD72 negative cells, and showed similar efficacy to that found with a clinically-used CD19 CAR. To assess in vivo activity, CD72(Nb.D4) CAR-T's at 1:1 CD4:CD8 ratio were injected at an effector:tumor ratio of 5:1 into tumor-bearing NSG mice (luciferase-labeled SEM or MLLr PDX). In vivo results confirmed strong anti-tumor effect of CD72 nanobody CAR-T's, equivalent to clinical CD19 CAR, and significantly increased survival in mice (Figure 2). A CRISPR interference-generated antigen escape model of CD19 was also effectively eliminated by CD72 CAR-T's. We also introduce "antigen escape profiling", where cell surface proteomics of a CRISPRi CD72-knockdown model demonstrated extensive surfaceome rewiring with potential implications for leukemia cell trafficking and adhesion in the setting of acquired resistance. Given CD72's role as a BCR signaling inhibitory receptor, we are currently examining its influence on proximal B-cell receptor signaling and relationship to combination therapies affecting this pathway. Conclusions:By characterizing the surface proteomic landscape of B-ALL, we develop a resource for the research community and identify CD72 as a promising therapeutic target. We demonstrate that a novel, fully recombinant nanobody library can generate potent cellular therapies, which may be extended to other targets in the future. We anticipate that antigen escape profiling will prove broadly useful for anticipating mechanisms of resistance to novel immunotherapies. CD72 CAR-T's are a promising strategy across a range of B-cell malignancies, particularly those refractory to CD19 therapy. Disclosures Nix: UCSF: Patents &amp; Royalties. Wiita:UCSF: Patents &amp; Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Dedicator of cytokinesis protein 2 couples with lymphoid enhancer–binding factor 1 to regulate expression of CD21 and B-cell differentiation

B-cell receptor (BCR) signaling, combined with CD19 and CD21 signals, imparts specific control of B-cell responses. Dedicator of cytokinesis protein 2 (DOCK2) is critical for the migration and motility of lymphocytes. Although absence of DOCK2 leads to lymphopenia, little is known about the signaling mechanisms and physiologic functions of DOCK2 in B cells. We sought to determine the underlying molecular mechanism of how DOCK2 regulates BCR signaling and peripheral B-cell differentiation. In this study we used genetic models for DOCK2, Wiskott-Aldrich syndrome protein (WASP), and lymphoid enhancer-binding factor 1 deficiency to study their interplay in BCR signaling and B-cell differentiation. We found that the absence of DOCK2 led to downregulation of proximal and distal BCR signaling molecules, including CD19, but upregulation of SH2-containing inositol 5 phosphatase 1, a negative signaling molecule. Interestingly, DOCK2 deficiency reduced CD19 and CD21 expression at the mRNA and/or protein levels and was associated with reduced numbers of marginal zone B cells. Additionally, loss of DOCK2 reduced activation of WASP and accelerated degradation of WASP, resulting into reduced actin accumulation and early activation of B cells. Mechanistically, the absence of DOCK2 upregulates the expression of lymphoid enhancer-binding factor 1. These differences were associated with altered humoral responses in the absence of DOCK2. Overall, our study has provided a novel underlying molecular mechanism of how DOCK2 deficiency regulates surface expression of CD21, which leads to downregulation of CD19-mediated BCR signaling and marginal zone B-cell differentiation.

Anti-CD20 Monoclonal Antibodies Hijack the B-Cell Receptor Signaling Cascade Thereby Activating the NOTCH1 Signaling Pathway

NOTCH1 is a cell surface receptor, regulation of which depends on the integrity and subsequent cleavage of its inhibitory domain. Subtle mechanical forces transmitted after ligand-binding [Wang et al., 2013] or removal of Ca2+-ions [Rand et al., 2000] make the site accessible for cleavage, resulting in release of the transcription factor NICD1. Mutations in NOTCH1 that prolong NICD1 activity have been found in chronic lymphocytic leukemia (CLL) with enrichment in up to 30% of Richter transformation (RT). Clinical trials have revealed that NOTCH1 mutant CLL derives no benefit from the addition of type I anti-CD20 monoclonal antibodies (mAb) such as rituximab. This lack of benefit has not been observed with the type II anti-CD20 mAb obinutuzumab.To understand how anti-CD20 mAbs influence NOTCH1, we studied intracellular signaling events induced by rituximab or obinutuzumab and assessed their implications on NOTCH1 signaling.As a model for this, mass spectrometry-based phosphoproteomic analysis was applied to rituximab or obinutuzumab treated SU-DHL4 B-lymphoma cells (1h and 24h time-points) and >8500 phosphorylation sites measured relative to untreated controls. Activation of kinase pathways was inferred by kinase-substrate enrichment analysis (KSEA). NOTCH1 receptor activity was assessed by western blot for NICD1 and qPCR for HES1 expression (NICD1 target gene). We used rituximab F(ab')2 fragments, trastuzumab as an isotype control and SB2H2 to cross-link the IgG B-cell receptor (BCR). Where applicable, cells were pre-incubated with inhibitors for 48h before mAb exposure.Rituximab or obinutuzumab treatment resulted in strong phosphorylation of all proximal BCR signaling proteins at 1h and 24h, strongly activating MAPK and PLCγ2-induced signaling and remodeling processes in the actin cytoskeleton. Cell cycle promoting proteins were inhibited and in addition, obinutuzumab promoted pro-apoptotic signaling. AKT signaling was inhibited due to SHIP activation following FcγRIIB binding.We hypothesized that BCR-induced Ca2+-flux and/or mechanical forces inflicted by cytoskeletal rearrangements disrupt the integrity of the inhibitory domain of NOTCH1 and promote ligand-independent activation. A strong, immediate increase in NOTCH1 activity was observed after rituximab, rituximab F(ab')2 and SB2H2 treatment. This was preventable by SYK inhibition, which also abrogated Ca2+-flux. Neither Ca2+-flux nor a relevant increase in NICD1 activity were induced by obinutuzumab, clearly supporting our first hypothesis. To test, if cytoskeletal rearrangements also contributed to NOTCH1 activation, we treated cells with a Rac inhibitor and found significantly reduced basal HES1 expression.PI-4,5-P2, which is the substrate for the Pi3-kinase (Pi3K), suppresses Ca2+-flux and cytoskeletal rearrangements. In vitro, the Pi3K inhibitors idelalisib, duvelisib and umbralisib reduced maximum HES1 expression after rituximab exposure as well as basal HES1 expression levels significantly more than BTK inhibition. Thus, Pi3K inhibitors likely act as suppressors of NOTCH1 signaling. Abnormally strong NOTCH1 signaling is one driver of RT and our observations can serve as explanation why idelalisib treated patients might develop RT at lower rates than patients treated with ibrutinib or venetoclax.In summary, we demonstrate clear and direct links between BCR and NOTCH1 signaling and show that anti-CD20 mAbs induce cell cycle arrest and apoptosis after strong BCR signaling. With c-MYC as an established target gene, NICD1 release upon Ca2+-flux and cytoskeletal movements in B-cells generates survival and proliferation signals. BCR-triggered effects on cell fate depend on signal strength and vary between activation/survival and apoptosis, the latter occurring when signal strength exceeds a threshold. CLL cells have low CD20 expression and the threshold for apoptosis induction after anti-CD20 mAb treatment may thus be difficult to reach. NOTCH1 mutations leading to abnormally strong effects of NICD1 would then lead to a prominence of survival over apoptotic signals, thereby promoting the observed resistance to rituximab.We believe that Ca2+-flux inducing type I anti-CD20 mAbs should be avoided in the treatment of NOTCH1 mutant CLL. Furthermore, we hypothesize that patients at increased risk for RT (mutant NOTCH1, c-MYC gain, highly autoreactive BCR) may benefit from Pi3K inhibition. DisclosuresCragg:Boehringer Ingleheim: Consultancy; Bioinvent: Consultancy, Patents & Royalties: Patent licenced to Bioinvent around CD32b blockade. Gribben:TG Therapeutics: Honoraria; NIH: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Kite: Honoraria; Roche: Honoraria; Unum: Equity Ownership; Cancer Research UK: Research Funding; Medical Research Council: Research Funding; Abbvie: Honoraria; Pharmacyclics: Honoraria.

Increased TLR7 Signaling of BCR-Activated B Cells in Chronic Graft-Versus Host Disease (cGVHD)

After hematopoietic allogeneic stem cell transplantation (HCT), B cells reconstitute in the presence of alloantigen in a nucleic acid (NA)-rich environment. Ongoing tissue and cellular damage results in release of endogenous NA that may serve as ligand for endosomal Toll-Like Receptors (TLRs). Alloantibodies and autoantibodies, including NA-binding antibodies, have been associated with the presence of cGVHD manifestations. In patients after HCT, B cells are constitutively activated via key proximal B Cell Receptor (BCR) signaling molecules, Syk and BLNK (Allen JL et al Blood 2014, Flynn R et al Blood 2015) . In mouse models of autoimmunity, TLR7 or TLR9 can operate in conjunction with the BCR to mediate autoantibody production (Suthers A et al. Front Immunol. 2017). Whether TLR7/9-BCR signaling contributes to cGVHD remains unknown.We studied responses by TLR9 and TLR7 in B cells from patients >12 months post-HCT who had active, inactive or no cGVHD at the time of sample acquisition and were not receiving high dose steroid. In contrast to a previous study (She K et al. BBMT 2007) and consistent with findings by others (de Masson A et al Blood 2015), we did not find a consistent increase in B cell response to TLR9 agonist. Instead we found significantly higher TLR7 transcript expression in B cells from patients with active cGVHD (n=7) compared to no/inactive cGVHD patient B cells (n=11, p=0.042). TLR7 over-expression in mice leads to anti-RNA antibody production by immature B cells (Giltiay NV et al JEM 2013). We employed ELISA to measure antibodies to a known RNA-containing autoantigen, Ro-52 in a group of 66 plasma samples. We found that significantly more patients with active cGVHD (n=32) had anti-Ro-52 antibody compared to inactive cGVHD patients (n=22) or patients without cGVHD (n=12) (Fisher exact test for both comparisons = p<0.0001). This suggests potential opposing roles for TLR9 and TLR7 in cGVHD (Sharma et al, J.Immunol. 2015), and allowed us to hypothesize that B cells in cGVHD were aberrantly activated via TLR7.To determine if TLR7 activation was increased in cGVHD B cells, we measured IL-6 production. Without ex vivo stimulation, cGVHD patient B cells produced significantly more IL-6 (p=0.039) (Fig.1A). After TLR7 stimulation with R848, B cells from patients with active cGVHD had significantly increased IL-6 production compared those from patients without (p= 0.008) or those with inactive cGVHD (p=0.017) (Fig.1A). We next investigated the influence of the BCR-activated state in cGVHD on TLR7 responses and signaling. Using flow cytometry, we measured Ki-67 expression as a marker of activation after ex vivo stimulation with both low level surrogate BCR antigen and R848. We found that B cells from active cGVHD patients were significantly more responsive compared to no cGVHD patients (p=0.015) and healthy donors (p=0.045), revealing increased synergistic BCR-TLR7 signaling in cGVHD B cells (Fig.1B). To further investigate this, we employed qPCR to measure levels of the proximal BCR signaling molecule, Lyn, known to negatively regulate anti-NA antibody production (Lamagna et al. J. Immunol. 2014). We found that Lyn expression was significantly decreased in B cells from active cGVHD patients compared to healthy donor (p=0.001), inactive (p=0.042) or no (p=0.007) cGVHD patients (Fig.1C). Lyn can also negatively regulate TLR7/9 activation by directly associating with Interferon Regulatory Factor 5 (IRF5), which is important for pro-inflammatory cytokine production, including IL-6 (Ban et al, J.Immunol. 2016). Consistent with a role for Lyn in TLR7 activation, we found that IRF5 was significantly increased in active cGVHD patient B cells compared to all other groups (healthy donor, p=0.035; inactive cGVHD, p=0.011; no cGVHD, p=0.006) (Fig.1D). Notably, no difference in expression was found in downstream TLR7 transcription factors, IRF3 and IRF7 . Using a mouse model (Zhang C et al Blood 2006), we found significantly higher IRF5 expression in splenic B cells in animals with cGVHD manifestations (p=0.005). Ongoing mouse work will ascertain mechanistic roles for Lyn and IRF5 in TLR7 signaling by cGVHD B cells. Together data support a potentially pathogenic role of TLR7 signaling in cGVHD B cells. Development of agents that block this newly elucidated TLR7-BCR signaling axis in cGVHD is warranted.This work was supported by National Institutes of Health grant R01 HL129061 [Display omitted] DisclosuresRizzieri:Erytech: Research Funding; Shire: Research Funding.

PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma.

Cell line models of the activated B cell-like (ABC) subtype of diffuse large B cell (DLBCL) depend on both NF-κB and phosphatidylinositol 3-kinase (PI3K) signaling pathways for survival, especially those with gain-of-function B cell receptor (BCR) mutations. Here we show that these cells depend specifically on the PI3Kδ isoform, but that PI3K pathway interruption by PI3Kδ inhibitors is short-lived due to feedback activation of the PI3Kα isoform. PI3Kδ and PI3Kα inhibition cooperated in killing ABC DLBCL lines, and genetic knockdown of PI3Kα sensitized cells to PI3Kδ inhibition and prolonged the interruption of PI3K signaling. PI3Kδ inhibition evoked feedback activation of proximal BCR signaling, which increased the association of PI3Kα with BCAP and CD19 and increased overall PI3K activity. These results support the clinical evaluation of dual PI3Kδ and PI3Kα inhibition in patients with ABC DLBCL.

Increased BCR responsiveness in B cells from patients with chronic GVHD

AbstractAlthough B cells have emerged as important contributors to chronic graft-versus-host-disease (cGVHD) pathogenesis, the mechanisms responsible for their sustained activation remain unknown. We previously showed that patients with cGVHD have significantly increased B cell–activating factor (BAFF) levels and that their B cells are activated and resistant to apoptosis. Exogenous BAFF confers a state of immediate responsiveness to antigen stimulation in normal murine B cells. To address this in cGVHD, we studied B-cell receptor (BCR) responsiveness in 48 patients who were >1 year out from allogeneic hematopoietic stem cell transplantation (HSCT). We found that B cells from cGVHD patients had significantly increased proliferative responses to BCR stimulation along with elevated basal levels of the proximal BCR signaling components B cell linker protein (BLNK) and Syk. After initiation of BCR signaling, cGVHD B cells exhibited increased BLNK and Syk phosphorylation compared with B cells from patients without cGVHD. Blocking Syk kinase activity prevented relative post-HSCT BCR hyper-responsiveness of cGVHD B cells. These data suggest that a lowered BCR signaling threshold in cGVHD associates with increased B-cell proliferation and activation in response to antigen. We reveal a mechanism underpinning aberrant B-cell activation in cGVHD and suggest that therapeutic inhibition of the involved kinases may benefit these patients.

BCR Hyper-Responsiveness In B Cells From Patients With Chronic Gvhd Is Blocked With The Syk Inhibitor R406

Our previous findings indicate that the excess BAFF present in patients with chronic GVHD (cGVHD) induces increased metabolic activity and B-cell survival ex vivo (Allen et al., Blood. 2012. 120:2529). Mechanistic work in murine B cells shows that BAFF treatment increases proliferation in response to B-cell receptor (BCR) stimulation (Patke et al., J Exp Med. 2006. 203: 2551). Taken together, these data suggested that B cells in patients with cGVHD might respond more readily to the allo- and neo-autoantigens present post-transplant. We aimed to determine whether B cells from cGVHD patients were hyper-responsive to BCR stimulation. B cells from 13 allo-HSCT patients who were >12 months post-transplant and not receiving high dose steroid were purified. Proliferation was determined by CFSE incorporation. B cells from patients with cGVHD (n = 6) had a significantly increased proliferative response to BCR stimulation with anti-IgM, even with limiting amounts of ligand, compared to patients without cGVHD (n = 7; p = 0.01). This B-cell hyper-proliferation was specific to BCR signaling as proliferation in response to anti-CD40 plus IL-4 was similar between patient phenotypes.To determine potential mechanisms underlying the increased BCR-driven proliferation we performed pathway-focused mRNA expression profiling of 84 genes in highly purified CD27- and antigen-experienced CD27+ B cells from patients with cGVHD. One gene integral to BCR-signaling, BLNK, was increased >5-fold in both cGVHD B cell subsets compared to healthy donors. BLNK is a central adapter protein in B-cell activation. It couples antigen-BCR engagement with SYK activation and is required for BCR-driven proliferation in mice. We examined baseline protein levels of BLNK and SYK in un-manipulated B cells from 10 patients by mean fluorescence intensity (MFI). The MFI of BLNK and SYK were significantly increased in B cells from patients with cGVHD (n = 4) compared to B cells from patients without cGVHD (n = 6; BLNK: p = 0.009. SYK: p = 0.009). We next determined that such elevated baseline levels of BLNK and SYK in B cells from patients with cGVHD might amplify BCR signaling. Specifically, stimulation with anti-IgM resulted in increased phosphorylation of BLNK and SYK in cGVHD B cell subsets. Antigen-experienced CD27+ B cells from patients with cGVHD (n = 5) had significantly increased BCR-driven phosphorylation of BLNK (pY84: p < 0.05) and SYK (pY348: p < 0.005) compared to those B cells from patients without cGVHD (n = 6). CD27- B cells from patients with cGVHD (n = 6) also had significantly increased pBLNK compared to non-cGVHD B cells (n = 7; p < 0.0005). Of note, BCR (IgM, IgD and IgG) surface expression on peripheral B cells was similar between cGVHD phenotypes suggesting that our results were not due to altered receptor expression.BAFF and BCR signaling operate together to convey proliferation, activation and survival signals. To determine if the increased activation of BLNK and SYK were contributing to the B-cell proliferation and survival in cGVHD B cells, we studied the effects of the SYK-inhibitor R406 (the active metabolite of Fostamatinib, kindly provided by Rigel Pharmaceuticals). B cells from patients with and without cGVHD were stimulated with anti-IgM and treated with R406. As expected, B cells from patients with cGVHD hyper-proliferated in response to anti-IgM and SYK inhibition prevented BCR-driven proliferation in vitro in both patient phenotypes. Strikingly, BCR stimulation ex vivo induced a significant increase in surviving B cell number from patients with cGVHD (n = 4) compared to patients without cGVHD (n = 4) (Figure 1). Importantly, inhibition of SYK with low-dose R406 (0.01 μM) abrogated the survival advantage of B cells from patients with cGVHD (Figure 1). These data suggest that B cell hyper-responsiveness in patients with cGVHD is due to elevated levels and activation of proximal BCR signaling components. Our findings suggest novel therapeutic targets in patients with cGVHD. [Display omitted] Disclosures:Rizzieri:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

BackgroundWe previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL.MethodsPrimary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry.ResultsWe showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.ConclusionsThis study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.

Attacking MALT1 for ABC-DLBCL therapy

Diffuse large B-cell lymphoma (DLBCL) comprises the largest group of non-Hodgkin lymphoma (NHL). DLBCL can be classified according to their cellular origin and patients with the activated B-cell (ABC) subtype have an inferior prognosis relative to those with the germinal center B-cell (GCB) subtype. Constitutive anti-apoptotic NF-κB signaling is a hallmark of ABC-DLBCL. NF-κB activation is caused by chronic active B-cell receptor (BCR) signaling or constitutive MYD88 signaling. Oncogenic mutations that contribute to pathogenesis of ABC-DLBCL tumors have been identified in critical components of both pathways (Figure ​(Figure1)1) [1]. Figure 1 Oncogenic mechanisms and target directed therapeutic approaches in ABC-DLBCL Target directed approaches for ABC-DLBCL therapy have largely focused on the inhibition of upstream protein kinase [2]. Chronic BCR signaling engages the adaptors CD79A and CD79B in a Syk-dependent mechanism. Syk is constitutively active in many B-cell lymphomas and a clinical phase I/II trial using the Syk inhibitor fostamatinib disodium (FosD, AstraZeneca) shows some response also in DLBCL patients. However, most oncogenic mutations in ABC-DLBCL occur further downstream revealing that Syk may not be an optimal target. Downstream of CD79A/B, Btk and PKCβ bridge proximal BCR signaling events to the CARMA1 (CARD11)-BCL10-MALT1 (CBM) complex. Over 20% of ABC-DLBCL tumors carry oncogenic mutations in CD79A/B. Indeed, the irreversible Btk inhibitor ibrutinib (PCI-32765, Pharamcyclics) and the panPKC inhibitor sotrastaurin (STN) are inhibiting the outgrowth of CD79 mutant ABC-DLBCL in preclinical models [3, 4]. Furthermore, positive clinical responses in a phase II trial were reported in relapsed/refractory DLBCL with the selective PKCβ inhibitor enzastarin (LY317615, Eli Lilly) [5]. However, none of these potential drugs is able to target ABC-DLBCL tumors with lesions downstream of PKCβ or in parallel pathways, such as CARMA1 of MYD88, respectively. Downstream of PKCβ the CBM subunit MALT1 has attracted great attention as a potential therapeutic target [6]. MALT1 serves a dual role in NF-κB signaling in response to antigen receptor stimulation. MALT1 is an adaptor that recruits and activates the IκB kinase (IKK) complex, the gatekeeper of canonical NF-κB. In addition, MALT1 is a paracaspase with a caspase-like proteolytic activity that is required for full NF-κB signaling and survival of ABC-DLBCL cells. MALT1 cleaves and thereby inactivates negative regulators of canonical NF-κB, such as the tumor suppressor A20 and the NF-κB subunit RelB which both counteract pro-survival functions of canonical IKK/NF-κB signaling. Two classes of MALT1 inhibitors have now been identified that effectively and selectively inhibit the growth of ABC- but not GCB-DLBCL in vitro and in vivo [7, 8]. Interestingly, the compounds are inhibiting MALT1 by two very different mechanisms. Fontan et al have identified a structurally new small molecule inhibitor (MI-2) that is covalently modifying catalytic center of MALT1 [7]. MI-2 was tolerated in mice at the effective dose without obvious signs of toxicity. Usually, irreversible inhibitors require optimal pharmacokinetic properties for clinical development, but recent advances for instance on the irreversible Btk inhibitor ibrutinib reveal that a clinical use may be possible. In a parallel study, we have identified the phenothiazines-derivatives (PD) thioridazine, mepazine and promazine as reversible MALT1 inhibitors [8]. PD are not targeting the active site of MALT1, but exhibit a non-competitive, allosteric mode of action. Mepazine, thioridazine and promazine have a long medical history as antipsychotics and sedatives drugs used for the treatment of psychiatric disorders. Well-established toxicokinetics and pharmacokinetics suggest that targeting MALT1 for cancer therapy by this class of compounds may be safe and feasible. Further, medicinal chemistry could be used to generate novel PD that are more potent MALT1 inhibitors, while reducing their neurological effects. Taken together, both studies demonstrate that MALT1 inhibition is a promising strategy for the treatment of ABC-DLBCL. In fact, targeting MALT1 may possess some advantages over the inhibition of upstream protein kinases. MALT1 inhibition also affects survival of CARMA1 mutant ABC-DLBCL. Further, with an occurrence of 29% the MYD88 mutation L265P is the most frequent oncogenic mutation in ABC-DLBCL. 65% of the MYD88-mutant ABC-DLBCL tumors carry additional mutations in CARMA1 or CD79A/B and MALT1 inhibitors are toxic to ABC-DLBCL with aberrant activation of both pathways [7, 8]. Thus, MALT1 inhibition indeed holds great promises for the treatment of the majority of ABC-DLBCL. Research on ABC-DLBCL provides a paradigm for the power of using advanced diagnostic tools (e.g. gene expression profiling and genome sequencing) to classify lymphoma entities and to identify specific oncogenic lesions. In parallel the generation of target directed therapeutics will promote the development of more personalized treatment protocols. Because of the various oncogenic lesions and the possibilities of drug resistances, there cannot be enough ‘smart’ drugs for ABC-DLBCL therapy. In the future, combinatorial treatment protocols may certainly enhance anti-tumor efficacy of the single agents that are currently in preclinical and/or clinical development.

Chronic Active B-Cell Receptor Signaling in Lymphoma

Abstract SCI-26We have developed loss-of-function, RNA interference-based screens to reveal genes essential for cancer cell proliferation and survival. In parallel, we are using high-throughput RNA resequencing (RNA-seq) to identify somatic mutations and other structural abnormalities in cancer. The intersection of these two data sets has helped us to discover novel pathogenetic pathways in lymphoma that are amenable to therapeutic attack. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) has constitutive activation of the NF-κB pathway, which we traced to the signaling adapter CARD11. In some ABC DLBCL biopsies (∼10%), somatic mutations produce CARD11 isoforms that spontaneously activate NF-κB signaling. In ABC DLBCL tumors with wild-type CARD11, we defined a “chronic active” form of B-cell receptor (BCR) signaling that activates NF-κB. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as Bruton’s tyrosine kinase (BTK), or components of the BCR itself. Over one-fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR. In 18 percent of cases, mutations occur in a single tyrosine residue in the critical “ITAM” signaling motif, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. Based on these findings, we are conducting clinical trials of ibrutinib in relapsed/refractory DLBCL. Ibrutinib is an irreversible and highly selective small-molecule inhibitor of BTK. Thus far, ibrutinib monotherapy has induced many complete and partial responses in patients with ABC DLBCL, including those with “primary refractory” tumors that had never responded to any prior therapy. One patient has been in a sustained complete response for over 19 months, taking ibrutinib daily with no discernible side effects. Of note, ABC DLBCL tumors with and without CD79B mutations have responded, suggesting that BCR pathway addiction may be a prevalent feature in this lymphoma subtype. More recently, we have uncovered a “tonic” form of BCR signaling in Burkitt lymphoma that engages the prosurvival PI(3) kinase pathway. Two-thirds of Burkitt lymphoma cell lines die upon knockdown of BCR subunits or the proximal kinase SYK, due to loss of PI(3) kinase signaling. Moreover, a gene expression signature of PI(3) kinase activity is more highly expressed in Burkitt lymphoma biopsies than in biopsies of other aggressive lymphomas. Tonic BCR signaling in Burkitt lymphoma is mechanistically distinct from chronic active BCR signaling in ABC DLBCL, since it does not engage BTK, CARD11, or NF-κB. RNA-seq revealed that 70 percent of sporadic Burkitt lymphoma cases harbor somatic mutations that potentiate the action of the transcription factor TCF3 by preventing its inhibitory heterodimerization with the DNA-binding inhibitor ID3. TCF3 promotes tonic BCR signaling and PI(3) kinase activity in Burkitt lymphoma by transactivating the immunoglobulin heavy- and light-chain genes, thereby increasing surface BCR expression, and by repressing the phosphatase SHP-1, a potent negative regulator of BCR signaling. Hence, inhibitors of proximal BCR signaling and the PI(3) kinase pathway should be evaluated in Burkitt lymphoma, especially in patients for whom high-dose chemotherapy is infeasible, such as older individuals and those with the endemic form of this lymphoma. Disclosures:Off Label Use: I will be discussing clinical trials of ibrutinib (PCI-32765) in lymphoma. Ibrutinib is an investigational drug that has not yet received FDA approval for any indication.

Quantitative Immunofluorescence Reveals the Signature of Active B-cell Receptor Signaling in Diffuse Large B-cell Lymphoma

B-cell receptor (BCR)-mediated signaling is important in the pathogenesis of a subset of diffuse large B-cell lymphomas (DLBCL) and the BCR-associated kinases SYK and BTK have recently emerged as potential therapeutic targets. We sought to identify a signature of activated BCR signaling in DLBCL to aid the identification of tumors that may be most likely to respond to BCR-pathway inhibition. We applied quantitative immunofluorescence (qIF) using antibodies to phosphorylated forms of proximal BCR signaling kinases LYN, SYK, and BTK and antibody to BCR-associated transcription factor FOXO1 on BCR-cross-linked formalin-fixed paraffin-embedded (FFPE) DLBCL cell lines as a model system and on two clinical cohorts of FFPE DLBCL specimens (n = 154). A robust signature of active BCR signaling was identified and validated in BCR-cross-linked DLBCL cell lines and in 71/154 (46%) of the primary DLBCL patient specimens. Further analysis of the primary biopsy samples revealed increased nuclear exclusion of FOXO1 among DLBCL with qIF evidence of active BCR signaling compared with those without (P = 0.004). Nuclear exclusion of FOXO1 was also detected in a subset of DLBCL without evidence of proximal BCR signaling suggesting that alternative mechanisms for PI3K/AKT activation may mediate FOXO1 subcellular localization in these cases. This study establishes the feasibility of detecting BCR activation in primary FFPE biopsy specimens of DLBCL. It lays a foundation for future dissection of signal transduction networks in DLBCL and provides a potential platform for evaluating individual tumors in patients receiving novel therapies targeting the BCR pathway.

Signal transduction inhibitors in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) therapy has evolved over the past few decades as modern chemo-immunotherapy significantly improved the response and survival of CLL patients. However, treatment toxicity of the intensive chemo-immunotherapy often limits its use in the mostly elderly population of patients. Further, the disease eventually relapses and additional therapy options are required. Of particular interest are molecular targeted therapies that interfere with critical signal transduction pathways controlling cell growth and survival. This review will provide an update on the most recent preclinical and clinical development of signal transduction targeted therapy in CLL. Small molecular kinase inhibitors have been developed to target the proximal B-cell receptor signaling pathway (e.g. spleen tyrosine kinase inhibitors and Bruton's tyrosine kinase inhibitors) or the downstream phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin pathway. These agents showed unique in-vitro activities by inducing apoptosis and blocking interaction of CLL cells and the protective microenvironment. They have also shown promising clinical activity in early-phase clinical studies and appear to alter lymphocyte trafficking. Inhibitors of the B-cell CLL/lymphoma 2 (BCL-2) family of antiapoptotic proteins and Cdk inhibitors are in active clinical development. Strategies modulating the CLL interaction with the microenvironment niche are emerging. Further understanding of novel signaling pathways helps to identify additional potential therapeutic targets. Signal transduction inhibitors are promising new strategy for targeted CLL treatment. Identification of novel molecular targets and therapeutic agents will further expand our therapeutic options.

Transcription factor Zfx controls BCR-induced proliferation and survival of B lymphocytes

AbstractThe development, homeostasis, and function of B lymphocytes involve multiple rounds of B-cell receptor (BCR)–controlled proliferation and prolonged maintenance. We analyzed the role of transcription factor Zfx, a recently identified regulator of hematopoietic stem cell maintenance, in B-cell development and homeostasis. Panhematopoietic or B cell–specific deletion of Zfx in the bone marrow blocked B-cell development at the pre-BCR selection checkpoint. Zfx deficiency in peripheral B cells caused accelerated B-cell turnover, depletion of mature recirculating B cells, and delayed T-dependent antibody responses. In addition, the numbers and function of B-1 cell lineage were reduced. Zfx-deficient B cells showed normal proximal BCR signaling, but impaired BCR-induced proliferation and survival in vitro. This was accompanied by aberrantly enhanced and prolonged integrated stress response and by delayed induction of cyclin D2 and Bcl-xL proteins. Thus, Zfx restrains the stress response and couples antigen receptor signaling to cell expansion and maintenance during B-cell development and peripheral homeostasis. These results identify a novel transcriptional regulator of the B-cell lineage and highlight the common genetic control of stem cell maintenance and lymphocyte homeostasis.