B-cell Receptor Cross-linking Research Articles

Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

A molecular circuit linking the BCR to the NAD biosynthetic enzyme NAMPT is an actionable target in Richter syndrome

AbstractThis works defines, to the best of our knowledge, for the first time a molecular circuit connecting nicotinamide mononucleoside phosphoribosyl transferase (NAMPT) activity to the B-cell receptor (BCR) pathway. Using 4 distinct xenograft models derived from patients with Richter syndrome (RS-PDX), we show that BCR cross-linking results in transcriptional activation of the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme NAMPT, with increased protein expression, in turn, positively affecting global cellular NAD levels and sirtuins activity. NAMPT blockade, by using the novel OT-82 inhibitor in combination with either BTK or PI3K inhibitors (BTKi or PI3Ki), induces rapid and potent apoptotic responses in all 4 models, independently of their mutational profile and the expression of the other NAD biosynthetic enzymes, including nicotinate phosphoribosyltransferase. The connecting link in the circuit is represented by AKT that is both tyrosine- and serine-phosphorylated by PI3K and deacetylated by sirtuin 1 and 2 to obtain full kinase activation. Acetylation (ie, inhibition) of AKT after OT-82 administration was shown by 2-dimensional gel electrophoresis and immunoprecipitation. Consistently, pharmacological inhibition or silencing of sirtuin 1 and 2 impairs AKT activation and induces apoptosis of RS cells in combination with PI3Ki or BTKi. Lastly, treatment of RS-PDX mice with the combination of PI3Ki and OT-82 results in significant inhibition of tumor growth, with evidence of in vivo activation of apoptosis. Collectively, these data highlight a novel application for NAMPT inhibitors in combination with BTKi or PI3Ki in aggressive lymphomas.

B-Cell Receptor Function and Gene Expression Profiling at Single-Cell Level across the Spectrum from Normal to Neoplastic CD5-Positive B-Cell Compartments in Humans

Antigen-independent, autonomous signalling of the B-cell receptor (BCR) is an oncogenic driver common to all cases and subtypes of chronic lymphocytic leukemia (CLL). We have recently shown that the dominant clonal population of low-count (<500 cells/µL) monoclonal B lymphocytosis (MBL) with CD5 +CD20 lowCD79B low CLL phenotype identified prospectively in healthy siblings of CLL patients also exhibit autonomous BCR signalling, albeit with lower signalling strength than CLL BCR (Quinten et al., Haematologica 2023). To gain further insight into the mechanisms of clonal expansion within the CD5 + B-cell compartment as the possible origin of CLL, we performed single-cell gene expression profiling and exemplary analyses of BCR function in normal CD5 + B cells (CD20 hiCD79B hi) and in CLL-phenotype B cells (CD5 +CD20 lowCD79B low) of healthy siblings of CLL patients in comparison to CLL and high-count MBL cells. Screening of blood samples of 16 sibling stem cell donors of CLL patients by flow cytometry revealed the presence of CD5 +CD20 lowCD79B low B cells at low counts in 5 donors. These cells and normal CD5 +CD20 hiCD79B hi B cells were sorted in parallel, pooled at equal ratios, and analyzed together with CLL B cells from their sibling stem cell recipients and 2 unrelated high-count MBL B cells on two 10X Genomics chips for single-cell 5‘ gene expression and BCR sequencing. Data deconvolution per individual was performed based on expressed SNPs and on the individuals’ HLA types. In a combined 2-dimensional t-SNE plot based on the 2000 most variably expressed genes, the normal CD5 +CD20 hiCD79B hi B cells from all 5 donors clustered distinctly from CLL-phenotype cells from all sources (dotted lines, Figure) and indeed showed significantly higher expression of CD20 and CD79B. Gene set enrichment analysis revealed significant upregulation of the hallmark oxidative phosphorylation gene set in all CLL samples compared to non-CLL B-cell populations. BCR analysis demonstrated clonal dominance of >99% for both Ig heavy and light chains in the 5 CLL samples. In accordance with the indication for allogeneic stem cell transplantation, these CLL BCR were unmutated. In marked contrast, normal CD5 +CD20 hiCD79B hi B cells from the healthy SC donors were also unmutated but completely polyclonal, i.e. no identical BCR was detected in any two cells of this compartment. In 4/5 donors, the CLL-phenotype CD5 +CD20 lowCD79B low compartment contained multiple clonal expansions of various degrees. In one high-count MBL, 17 other B-cell clones with various degrees of low-level expansion coexisted with the >90% dominant MBL clone. BCR from the most dominant and one or more of the less expanded CD5 +CD20 lowCD79B low clones of the healthy donors were tested for autonomous signaling as quantified by BCR-induced calcium flux without experimental cross-linking in murine TKO pre-B cells and compared to BCR from nonexpanded CD5 +CD20 hiCD79B hi cells and from their CLL siblings. Autonomous BCR signaling strength was calculated from the fraction of cells with spontaneous calcium flux above test background and calibrated against calcium flux after BCR crosslinking. CLL and high-count MBL BCR had robust median autonomous BCR signaling of 0.326. Expanded CD5 +CD20 lowCD79B low clones of the healthy donors showed significantly weaker BCR signalling strength of 0.163 (p<0.01; Scatter Plot). BCR signalling in nonexpanded CD5 + B cells appeared weaker than in expanded clones but was clearly above the background of BCR from mature B cells isolated from lymph node biopsies expressing a mutated IgG. In conclusion, the low-count CD5 +CD20 lowCD79B low CLL-phenotype B-cell compartment in healthy adults is composed of multiple, predominantly mutated B-cell clones. Therefore, the term “monoclonal B lymphocytosis” is highly misleading for these B-cell populations and should be avoided. Normal CD5 +CD20 hiCD79B hi B cells are exclusively unexpanded and express unmutated BCR, indicating a naïve status. Unexpectedly, these nonexpanded CD5 + B cells express BCR with relatively weak but quantifiable autonomous signalling. Globally, autonomous BCR signalling strength across the CD5 + B-cell compartment appears to be correlated to the degree of clonal expansion. Fully malignant CLL cells overexpress genes of the oxidative phosphorylation pathway, presumably to meet increased energy demand from alternative sources.

Modeling the B-cell receptor signaling on single cell level reveals a stable network circuit topology between nonmalignant B cells and chronic lymphocytic leukemia cells and between untreated cells and cells treated with kinase inhibitors.

B-cell receptor (BCR) signaling is central for the pathomechanism of chronic lymphocytic leukemia (CLL), and inhibitors of BCR signaling have substantially improved treatment options. To model malignant and nonmalignant BCR signaling, we quantified five components of BCR signaling (ZAP70/SYK, BTK, PLCγ2, AKT, ERK1/2) in single cells from primary human leukemic cells and from nonmalignant tissue. We measured signaling activity in a time-resolved manner after stimulation with BCR crosslinking by anti-IgM and/or anti-CD19 and with or without inhibition of phosphatases with H2 O2 . The phosphorylation of BCR signaling components was increased in malignant cells compared to nonmalignant cells and in IGHV unmutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2 O2 led to higher phosphorylation levels of BCR components in CLL cells with mutated IGHV compared to unmutated IGHV. We modeled the connectivity of the cascade components by correlating signal intensities across single cells. The network topology remained stable between malignant and nonmalignant cells. To additionally test for the impact of therapeutic compounds on the network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib), LYN (dasatinib) and SYK (entospletinib). Idelalisib treatment resulted in similar effects in malignant and nonmalignant cells, whereas ibrutinib was mostly active on CLL cells. Idelalisib and ibrutinib had complementary effects on the BCR signaling cascade whose activity was further reduced upon dasatinib and entospletinib treatment. The characterization of the molecular circuitry of leukemic BCR signaling will allow a more refined targeting of this Achilles heel.

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.

B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition.

ABSTRACTAntigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated in vitro by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood. Here, we profiled the small RNAs in reactivated Burkitt lymphoma cells and identified several miRNAs, such as miR-141, that are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. To better understand the functions of these two miRNAs, we examined their molecular targets and experimentally validated multiple candidates commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle. Our data support a model whereby EBV exploits BCR-responsive miR-141 and further mimics activity of this miRNA family via a viral miRNA to promote productive lytic replication.IMPORTANCE EBV is a human pathogen associated with several malignancies. A key aspect of lifelong virus persistence is the ability to switch between latent and lytic replication modes. The mechanisms governing latency, reactivation, and progression of the lytic cycle are only partly understood. This study reveals that specific miRNAs can act to support the EBV lytic phase following BCR-mediated reactivation triggers. Furthermore, this study identifies a role for FOXO3, commonly suppressed by both host and viral miRNAs, in modulating progression of the EBV lytic cycle.

Follicular Lymphoma-associated BTK Mutations are Inactivating Resulting in Augmented AKT Activation.

On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor. Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.See related commentary by Afaghani and Taylor, p. 2123.

Autoantigen Tetramer Silences Autoreactive B Cell Populations.

Many autoimmune therapies focus on immune suppression to reduce symptom severity and halt disease progression; however, currently approved treatments lack specificity for the autoantigen and rely on more global immune suppression. Multivalent antigen arrays can disarm pathogenic autoimmune B cell populations that specifically recognize the antigen of interest via their B cell receptor (BCR). Disarmament may be achieved by BCR engagement, cross-linking, and sustained receptor occupancy as a result of multivalent, high avidity BCR binding. To engage and explore this mechanism, a tetramer display of the encephalogenic proteolipid peptide (PLP139-151), referred to as 4-arm PLP139-151, was synthesized by copper-catalyzed azide-alkyne cycloaddition chemistry. Subcutaneous administration of 4-arm PLP139-151 completely ameliorated symptoms of paralysis in a mouse model of multiple sclerosis known as experimental autoimmune encephalomyelitis. Competitive binding of 4-arm PLP139-151 to PLP139-151-specific IgG in the mouse serum demonstrated the enhanced avidity associated with the multivalent array compared to the free peptide. Furthermore, key PLP139-151-reactive B cells were depleted following 4-arm PLP139-151 treatment, resulting in significant reduction of proinflammatory cytokines. Together, these data demonstrate the potential of 4-arm PLP139-151 to silence autoreactive B cell populations and limit the downstream activation of effector cells.

TLR-mediated activation of Waldenström macroglobulinemia B cells reveals an uncoupling from plasma cell differentiation

AbstractWaldenström macroglobulinemia (WM) is a rare malignancy in which clonal B cells infiltrate the bone marrow and give rise to a smaller compartment of neoplastic plasma cells that secrete monoclonal immunoglobulin M paraprotein. Recent studies into underlying mutations in WM have enabled a much greater insight into the pathogenesis of this lymphoma. However, there is considerably less characterization of the way in which WM B cells differentiate and how they respond to immune stimuli. In this study, we assess WM B-cell differentiation using an established in vitro model system. Using T-cell–dependent conditions, we obtained CD138+ plasma cells from WM samples with a frequency similar to experiments performed with B cells from normal donors. Unexpectedly, a proportion of the WM B cells failed to upregulate CD38, a surface marker that is normally associated with plasmablast transition and maintained as the cells proceed with differentiation. In normal B cells, concomitant Toll-like receptor 7 (TLR7) activation and B-cell receptor cross-linking drives proliferation, followed by differentiation at similar efficiency to CD40-mediated stimulation. In contrast, we found that, upon stimulation with TLR7 agonist R848, WM B cells failed to execute the appropriate changes in transcriptional regulators, identifying an uncoupling of TLR signaling from the plasma cell differentiation program. Provision of CD40L was sufficient to overcome this defect. Thus, the limited clonotypic WM plasma cell differentiation observed in vivo may result from a strict requirement for integrated activation.

Bidirectional linkage between the B-cell receptor and NOTCH1 in chronic lymphocytic leukemia and in Richter’s syndrome: therapeutic implications

NOTCH1 mutations in chronic lymphocytic leukemia (CLL) lead to accumulation of NOTCH1 intracellular domain (NICD) and prolong signaling. These mutations associate with a more aggressive disease compared to wild-type (WT) CLL. In this work we demonstrate a bidirectional functional relationship between NOTCH1 and the B cell receptor (BCR) pathways. By using highly homogeneous cohorts of primary CLL cells, activation of NOTCH1 is shown to increase expression of surface IgM, as well as LYN, BTK, and BLNK, ultimately enhancing BCR signaling responses, including global mRNA translation. Upon BCR cross-linking, NOTCH1 itself is actively translated and increased on cell surface. Furthermore, BCR ligation induces calcium mobilization that can facilitate ligand-independent NOTCH1 activation. These data suggest that the two pathways are functionally linked, providing a rationale for dual inhibition strategies. Consistently, addition of the γ-secretase inhibitor DAPT to ibrutinib significantly potentiates its effects, both in vitro and in a short-term patient-derived xenograft model. While this observation may find limited applications in the CLL field, it is more relevant for Richter's Syndrome (RS) management, where very few successful therapeutic options exist. Treatment of RS-patient-derived xenografts (RS-PDX) with the combination of ibrutinib and DAPT decreases disease burden and increases overall survival.

Distinct roles of BCNP1 in B-cell development and activation.

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

TAK-243, a Small Molecule Inhibitor of Ubiquitin-Activating Enzyme (UAE), Induces ER Stress and Apoptosis in CLL Cells

Introduction: Despite the promise of B-cell receptor-associated kinase inhibitors (BCRi) in CLL, resistance to these agents is inevitable. Ubiquitin-proteasome systems are altered in cancer, leading to destabilization of tumor suppressors, overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. Neoplastic B cells exhibit a state of heightened cellular stress and are thereby susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The UPR is activated in CLL cells upon sIgM signaling and inhibited by ibrutinib. Proteasome inhibitors demonstrate clinical activity in certain types of B-cell neoplasia but are inactive in CLL. Here, we investigated an alternative approach to harness the pro-apoptotic UPR in CLL by using TAK-243, a first-in-class small molecule UAE inhibitor.Methods: Peripheral blood cells were obtained from patients with CLL (N=20) and isolated using Ficoll-Hypaque techniques. TAK-243 was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). TAK-243 efficacy was assessed in CLL cells and 10 DLBCL (diffuse large B-cell lymphoma) cell lines.Results: TAK-243 induced ER stress and the UPR in CLL cells, followed by rapid apoptosis within 2 hours. Following 24-hour incubation, we established an IC50 of ~50 nM (Annexin V+ cells) in CLL cells. By contrast, primary B cells and T cells were less sensitive to TAK-243. Given the importance of tumor microenvironment in CLL cell survival, we evaluated the effect of TAK-243 in a CD40L-expressing stromal co-culture model. Whereas CD40L-stimulated CLL cells were resistant to BCRi, they were fully sensitive to UAE inhibition. TAK-243 had a similar IC50 (~50 nM) across DLBCL cell lines, independent of cell of origin.Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and degradation of proteins controlled by the UPS in CLL cells and DLBCL cell lines. UPR induction occurred within 2 hours, as shown by activation of eIF2α (in both CLL and DLBCL cells) and oligomerization and autophosphorylation of PERK (in DLBCL cells). After 4 hours, neoplastic B cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34, and NOXA. These events were accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of Mcl-1 and, ultimately, cleavage of caspase-3 and PARP. TAK-243 inhibited NFκB pathway, as shown by accumulation of IκBα, a negative pathway regulator. The extent of the UPR in CLL cells varies depending on the initiating signal. For example, B-cell receptor crosslinking induced expression of CHOP and GRP78 in CLL cells, but only weak activation of PERK and no IRE1-dependent processing of XBP1 (Krysov S, et al. Blood. 2014). Targeting UAE in CLL cells induced robust activation of eIF2α, upregulation of CHOP, GADD34 as well as NOXA mRNA, indicating high sensitivity to this pathway.TAK-243 induced a more rapid UPR and exhibited lower IC50 compared with the proteasome inhibitor bortezomib in CLL and DLBCL cells. While both drugs induced autophagy as shown by LC3 processing, only bortezomib treatment led to p62 degradation, suggesting that autophagy was inefficient in response to TAK-243 due to lack of ubiquitin conjugation. Our findings were confirmed in a mouse lymphoma xenograft model. OCI-LY3 cells were inoculated subcutaneously in the right flank of NSG mice and treatment with TAK-243 (10 or 20 mg/kg IV twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression, induction of ER stress, and decreased cell proliferation and survival.Conclusions: The UAE inhibitor TAK-243 induces ER stress and promotes apoptosis in CLL cells in vitro and restricts lymphoma growth in vivo. TAK-243 exhibited greater in-vitro cytotoxicity in lymphoma cells compared to bortezomib. Targeting UAE is a novel approach to disrupt the UPS which may hold promise in therapy of CLL and other B-cell malignancies. DisclosuresSpurgeon:Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:TG Therapeutics: Consultancy; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Takeda Oncology: Research Funding.

The Sjögren's syndrome-associated autoantigen Ro52/TRIM21 modulates follicular B cell homeostasis and immunoglobulin production.

Systemic rheumatic diseases are characterized by abnormal B cell activation with autoantibody production and hypergammaglobulinaemia. Ro52/SSA, also denoted tripartite motif (TRIM)21, is a major autoantigen in Sjögren's syndrome and systemic lupus erythematosus. Interestingly, TRIM21-deficient mice develop systemic autoimmunity with B cell-driven manifestations such as autoantibodies, hypergammaglobulinaemia and glomerulonephritis following tissue injury. The mechanisms by which TRIM21-deficiency leads to enhanced B cell activation and antibody production are, however, not well understood, and to further elucidate the role of TRIM21 in systemic autoimmunity, we investigated the B cell phenotype and antibody responses of Trim21-/- mice following immunization with thymus-dependent (TD) and thymus-independent (TI) antigens. We found that TRIM21-deficient mice developed significantly higher specific antibody titres than their wild-type counterparts upon B cell receptor (BCR) engagement by TD and TI type II antigens, and this was accompanied by an altered B cell phenotype. Furthermore, BCR cross-linking, but not anti-CD40 stimulation, in vitro resulted in a significantly higher proliferation of Trim21-/- cells. We also observed that splenic follicular B cells were expanded not only in immunized mice but also already in young, unmanipulated Trim21-/- mice, and transcriptomic analysis of these cells revealed an up-regulation of genes associated with B cell differentiation, indicating a role for TRIM21 in their regulation. In conclusion, in this study we describe a link between the rheumatic autoantigen Ro52/TRIM21 and increased antibody production associated with follicular B cell expansion, implicating a potential role for Ro52/TRIM21 in the pathogenesis of systemic autoimmune diseases.

Dissecting the contribution of antigen-dependent B cell receptor engagement and T cell help to Germinal Center B cell selection and differentiation.

Abstract Proper selection of somatically hyper mutated B cells in the Germinal Centers (GCs) and their differentiation into long-lived plasma cells is key for the development of high-affinity long-term humoral immunity. However, relative contribution towards GC B cell selection by the two key factors: antigen-driven B cell receptor (BCR) signaling and T cell help, remains an open question. We have addressed this long-standing question in the field using an experimental model that enables independent manipulation of both antigen-driven BCR engagement and antigenic peptide presentation in GC B cells. Our studies suggest that in the absence of antigen-driven BCR engagement, T cell help is sufficient for robust selection of GC B cells and their differentiation into plasmablasts. In contrast, neither is induced by antigen-dependent BCR crosslinking in the absence of T cell help. Interestingly, when the levels of MHCII/peptide presentation by GC B cells are suboptimal, antigen-dependent BCR engagement can synergize with T cell help to promote selection of GC B cells and their differentiation into plasmablasts. Our findings reconcile somewhat contradictory data reported in previous studies and point towards the importance of the two signal model in the GC response.

Generation of T follicular helper cells invitro: requirement for B-cell receptor cross-linking and cognate B- and T-cell interaction.

SummaryThe minimum requirements for in vitro modelling of natural CD4+ T‐cell differentiation into T follicular helper (Tfh) cells are still under investigation. We co‐cultured wild‐type and T‐cell receptor (TCR) transgenic CD4+ T cells from naive mice with dendritic cells and B‐cell receptor (BCR) transgenic B cells in the presence of HIV‐derived virus‐like particles containing matched B‐cell and T‐cell epitopes. This co‐culturing induced co‐expression of Tfh‐master regulator transcription factor BCL‐6 and CXCR5 in up to 10% of the wild‐type and up to 40% of the TCR‐transgenic CD4+ T cells. Phenotypic markers, production of interleukin‐21 and isotype switching of the B cells to IgG1 further indicated a helper function of the induced Tfh cells in vitro. Dendritic cells supported the generation of functional Tfh cells, but were unable to induce them without cognate B cells. Hence, our study presents a robust experimental system for efficient generation of functionally active Tfh cells in vitro and confirms the importance of cognate B‐ and T‐cell cross‐talk for the Tfh differentiation process.

Malaria-induced interferon-γ drives the expansion of Tbethi atypical memory B cells

Many chronic infections, including malaria and HIV, are associated with a large expansion of CD21−CD27− ‘atypical’ memory B cells (MBCs) that exhibit reduced B cell receptor (BCR) signaling and effector functions. Little is known about the conditions or transcriptional regulators driving atypical MBC differentiation. Here we show that atypical MBCs in malaria-exposed individuals highly express the transcription factor T-bet, and that T-bet expression correlates inversely with BCR signaling and skews toward IgG3 class switching. Moreover, a longitudinal analysis of a subset of children suggested a correlation between the incidence of febrile malaria and the expansion of T-bethi B cells. The Th1-cytokine containing supernatants of malaria-stimulated PBMCs plus BCR cross linking induced T-bet expression in naïve B cells that was abrogated by neutralizing IFN-γ or blocking the IFN-γ receptor on B cells. Accordingly, recombinant IFN-γ plus BCR cross-linking drove T-bet expression in peripheral and tonsillar B cells. Consistent with this, Th1-polarized Tfh (Tfh-1) cells more efficiently induced T-bet expression in naïve B cells. These data provide new insight into the mechanisms underlying atypical MBC differentiation.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma

AbstractWe used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO–induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

Abstract 666: CD37 tetraspanin as a novel biomarker for PD-1 blockade in diffuse large B-cell lymphoma

Abstract PD-1 immune checkpoint blockade reconstituting antitumor immunity has changed the cancer treatment paradigm. PD-1 blockade has been successful in many types of solid tumors and Hodgkin lymphoma, but not yet for diffuse large B cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. We found several biomarkers including loss of CD37 tetraspanin expression in DLBCL correlated with increased PD-1 expression suggesting potential sensitivity of these DLBCL subsets to PD-1 blockade. CD37 (TSPAN26) is a member of the tetraspanin superfamily, widely expressed on normal and malignant mature B-cells and downregulated in plasma cells. It has been documented that CD37 plays important roles in T-cell-B-cell interactions, B-cell humoral response triggered by B-cell receptor cross-linking, and a balance between immune responses and tolerance. Interestingly, in a large cohort of DLBCL patients, we found that loss of CD37 expression in DLBCL predicts strikingly decreased overall and progression-free survival rates, and that PDCD1 gene expression was upregulated in CD37-negative activated B-cell-like (ABC) DLBCL by gene expression profiling, whereas the costimulatory molecule ICOSLG was upregulated in CD37+ germinal center B-cell-like (GCB) DLBCL. Using the new fluorescent multiplex technology, we further measured PD-1 and PD-L1 expression at the protein level on lymphoma cells and immune cells in the tumor microenvironment, and found that PD-1 protein levels were increased on both cytotoxic and helper T-cells infiltrating in CD37-negative DLBCL either of GCB or ABC subtype. These novel discoveries suggest that CD37 is important for sustained antitumor adaptive immunity, that immune dysregulation plays an important role for poor clinical outcomes in DLBCL, and that CD37-negative DLBCL may be sensitive to PD-1 blockade. In summary, loss of a CD37 tetraspanin was found to correlate with PD-1 overexpression in DLBCL clinical samples, and CD37 may serve as a novel biomarker for anti-PD-1 immunotherapy clinical trials in DLBCL. Note: This abstract was not presented at the meeting. Citation Format: Zijun Y. Xu-Monette, Ken H. Young. CD37 tetraspanin as a novel biomarker for PD-1 blockade in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2017-666

Quantitative in vitro and in vivo models to assess human IgE B cell receptor crosslinking by IgE and EMPD IgE targeting antibodies

Targeting plasma IgE by therapeutic mABs like Omalizumab (Xolair®) is current clinical practice for severe allergic conditions or other IgE related diseases like chronic urticaria. As an alternative to soluble IgE targeting, IgE supply can be lowered by targeting the Extracellular Membrane Proximal Domain (EMPD) of the IgE B cell receptor (BCR) present on IgE switched B cells. This ultimately leads to apoptosis of these cells upon IgE BCR crosslinking. Since tools to selectively assess the efficacy of IgE BCR crosslinking by IgE targeting antibodies are limited, a readily quantifiable cell model was developed that allows to specifically address IgE BCR crosslinking activity in vitro. The new cell model allowed for a direct quantitative comparison of anti-EMPD IgE therapeutic prototype antibody 47H4 with anti-IgE(Ce3) directed therapeutic antibody Omalizumab and with a newly selected anti-human EMPD IgE monoclonal antibody, designated mAB 15cl12. Furthermore, a complementing mouse model was developed that allows for in vivo validation of antibodies addressing human EMPD IgE. It carries a targetable humanized EMPD IgE sequence that has been introduced by seamless genomic replacement of the endogenous EMPD encoding sequence. The model allowed to directly compare IgE lowering activity of two anti-human EMPD IgE therapeutic antibodies in vivo. Our tools provide the means for quantitative assessment of IgE BCR crosslinking activity which is increasingly gaining attention with respect to forthcoming second generation anti-IgE clinical candidates such as Ligelizumab or other clinical candidates featuring additional effector functions such as IgE BCR crosslinking activity.