B Cell Antigen Receptor Signaling Research Articles

A Review of Resistance Mechanisms to Bruton's Kinase Inhibitors in Chronic Lymphocytic Leukemia.

Bruton's Tyrosine Kinase (BTK) inhibitors have become one of the most vital drugs in the therapy of chronic lymphocytic leukemia (CLL). Inactivation of BTK disrupts the B-cell antigen receptor (BCR) signaling pathway, which leads to the inhibition of the proliferation and survival of CLL cells. BTK inhibitors (BTKi) are established as leading drugs in the treatment of both treatment-naïve (TN) and relapsed or refractory (R/R) CLL. Furthermore, BTKi demonstrate outstanding efficacy in high-risk CLL, including patients with chromosome 17p deletion, TP53 mutations, and unmutated status of the immunoglobulin heavy-chain variable region (IGHV) gene. Ibrutinib is the first-in-class BTKi which has changed the treatment landscape of CLL. Over the last few years, novel, covalent (acalabrutinib, zanubrutinib), and non-covalent (pirtobrutinib) BTKi have been approved for the treatment of CLL. Unfortunately, continuous therapy with BTKi contributes to the acquisition of secondary resistance leading to clinical relapse. In recent years, it has been demonstrated that the predominant mechanisms of resistance to BTKi are mutations in BTK or phospholipase Cγ2 (PLCG2). Some differences in the mechanisms of resistance to covalent BTKi have been identified despite their similar mechanism of action. Moreover, novel mutations resulting in resistance to non-covalent BTKi have been recently suggested. This article summarizes the clinical efficacy and the latest data regarding resistance to all of the registered BTKi.

B cell receptor signaling and associated pathways in the pathogenesis of chronic lymphocytic leukemia.

B cell antigen receptor (BCR) signaling is a key driver of growth and survival in both normal and malignant B cells. Several lines of evidence support an important pathogenic role of the BCR in chronic lymphocytic leukemia (CLL). The significant improvement of CLL patients' survival with the use of various BCR pathway targeting inhibitors, supports a crucial involvement of BCR signaling in the pathogenesis of CLL. Although the treatment landscape of CLL has significantly evolved in recent years, no agent has clearly demonstrated efficacy in patients with treatment-refractory CLL in the long run. To identify new drug targets and mechanisms of drug action in neoplastic B cells, a detailed understanding of the molecular mechanisms of leukemic transformation as well as CLL cell survival is required. In the last decades, studies of genetically modified CLL mouse models in line with CLL patient studies provided a variety of exciting data about BCR and BCR-associated kinases in their role in CLL pathogenesis as well as disease progression. BCR surface expression was identified as a particularly important factor regulating CLL cell survival. Also, BCR-associated kinases were shown to provide a crosstalk of the CLL cells with their tumor microenvironment, which highlights the significance of the cells' milieu in the assessment of disease progression and treatment. In this review, we summarize the major findings of recent CLL mouse as well as patient studies in regard to the BCR signalosome and discuss its relevance in the clinics.

Quantitative description of the phase-separation behavior of the multivalent SLP65-CIN85 complex.

Biomolecular condensates play a major role in cell compartmentalization, besides membrane-enclosed organelles. The multivalent SLP65 and CIN85 proteins are proximal B-cell antigen receptor (BCR) signal effectors and critical for proper immune responses. In association with intracellular vesicles, the two effector proteins form phase separated condensates prior to antigen stimulation, thereby preparing B lymphocytes for rapid and effective activation upon BCR ligation. Within this tripartite system, 6 proline-rich motifs (PRMs) of SLP65 interact promiscuously with 3 SH3 domains of the CIN85 monomer, establishing 18 individual SH3-PRM interactions whose individual dissociation constants we determined. Based on these 18 dissociation constants, we measured the phase-separation properties of the natural SLP65/CIN85 system as well as designer constructs that emphasize the strongest SH3/PRM interactions. By modeling these various SLP65/CIN85 constructs with the program LASSI (LAttice simulation engine for Sticker and Spacer Interactions), we reproduced the observed phase-separation properties. In addition, LASSI revealed a deviation in the experimental measurement, which was independently identified as a previously unknown intramolecular interaction. Thus, thermodynamic properties of the individual PRM/SH3 interactions allow us to model the phase-separation behavior of the SLP65/CIN85 system faithfully.

Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

Autoinhibition in the Signal Transducer CIN85 Modulates B Cell Activation.

Signal transduction by the ligated B cell antigen receptor (BCR) depends on the preorganization of its intracellular components, such as the effector proteins SLP65 and CIN85 within phase-separated condensates. These liquid-like condensates are based on the interaction between three Src homology 3 (SH3) domains and the corresponding proline-rich recognition motifs (PRM) in CIN85 and SLP65, respectively. However, detailed information on the protein conformation and how it impacts the capability of SLP65/CIN85 condensates to orchestrate BCR signal transduction is still lacking. This study identifies a hitherto unknown intramolecular SH3:PRM interaction between the C-terminal SH3 domain (SH3C) of CIN85 and an adjacent PRM. We used high-resolution nuclear magnetic resonance (NMR) experiments to study the flexible linker region containing the PRM and determined the extent of the interaction in multidomain constructs of the protein. Moreover, we observed that the phosphorylation of a serine residue located in the immediate vicinity of the PRM regulates this intramolecular interaction. This allows for a dynamic modulation of CIN85's valency toward SLP65. B cell culture experiments further revealed that the PRM/SH3C interaction is crucial for maintaining the physiological level of SLP65/CIN85 condensate formation, activation-induced membrane recruitment of CIN85, and subsequent mobilization of Ca2+. Our findings therefore suggest that the intramolecular interaction with the adjacent disordered linker is effective in modulating CIN85's valency both in vitro and in vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell.

Reduced Proliferation and Cell Death of Acute Myeloid Leukemia Cell Lines after Acalabrutinib Treatment

Background Acute myeloid leukemia (AML) still remains a significant challenge, with intensive chemotherapy treatments showing limited efficacy and lack of druggable targets in contrast to other hematologic malignancies. Exosomes, nanomolecules derived from cells reflect the parent cell's state, present opportunities for disease biomarker discovery and targeted therapy. Proteomic analysis of AML-derived exosomes may identify druggable targets despite limited prior research. In this study we investigated the potential of exosomes as a tool for target discovery in AML. Methods We conducted a proteomic analysis of AML-derived exosomes from HL-60, KG-1, and THP-1 cell lines, using fibroblast and mesenchymal stem cells as controls. Exosomes of each cell line were isolated, and digested via filter-aided sample preparation (FASP), followed by isobaric tagging using tandem mass tag (TMT) reagents. The samples were then fractionated using high-pH reversed-phase liquid chromatography and analyzed using Q-Exactive Orbitrap Hybrid Mass Spectrometry. Proteomic data were evaluated using SEQUEST in Proteome Discoverer 2.1 with a false discovery rate set to 1%. Differentially expressed proteins (DEPs) were identified and visualized in an interaction network and was analyzed for the gene ontology biological process (GOBP) using DAVID software. Protein-protein interactions were analyzed using the STRING software. Based on proteomic analysis, we identified the BTK-related pathway as potential druggable target for AML cells. AML cells were incubated with ACT, a BTK inhibitor, in various exposure time and density. Cell viability and proliferation rate was assessed using CCK-8 assay. BTK activity and signaling were assessed via Western blot analysis and immunocytochemistry of AML-derived exosomes. Cell death was evaluated using Annexin-V & PI apoptosis kit and FACS analysis. Statistical analysis was performed to ascertain significance. Results Proteomics of exosomes from three AML cell types revealed distinct protein profiles via LC-MS, with 2450 unique proteins identified (Figure 1A). For KG1, HL60, and THP1, the number of up-regulated DEPs were 438, 434, and 408, and the number of down-regulated DEPs was 126, 118, and 89 (fold change>1.5 or <0.67, P<0.05). Gene ontology analysis identified four main categories of biological processes, including cell migration, T-cell pathways, cell damage, and vesicle formation pathways. Investigation into protein-protein interactions using the STRING software allowed selection of tyrosine kinases SYK and LYN as biomarker candidates. Both kinases regulate BTK, a critical molecule in B-cell antigen receptor signaling. SYK and LYN were found to be enriched in exosomes, promoting phosphorylation of BTK in recipient cells. (Figure 1B) Cell viability experiments with AML cell lines revealed reduced viability upon treatment with ACT. Viability decreased with increased exposure time and density, with the highest effects observed at 1,000nM ACT concentration for 48 hours (Figure 2A). This was accompanied by decreased cell numbers and proliferation rate which was significant in the HL-60 and KG-1 cell lines (Figure 2B). Western blot analysis demonstrated significant decrease of the BTK downstream signaling (P-BTK, PKC and p-ERK) by acalabrutinib treatment (1,000nM) at 48 hours (Figure 2C). Proliferative markers, such as PCNA, and Cyclin D1 were also significantly decreased. LYN and SYK was also decreased in the exosomes of HL-60 and THP-1 cell line. The inhibition of P-BTK and Cyclin D1 by ACT was further confirmed through Immunofluorescence assay. Finally, ACT was shown to promote cell death in AML cell lines, as evidenced by an apoptosis assay, with the most significant increases observed in cells treated exposed to 1,000nM ACT for 72 h (Figure 2D). Conclusion Overall, the findings of this study revealed that acalabrutinib reduced proliferation and induced cell death in AML cells suggesting the potential of BTK inhibitors in AML treatment. This was explained through proteomic analysis of AML-derived exosomes. However, further studies are required to validate the clinical use of exosomes as biomarkers and druggable target.

WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins.

B cell antigen receptor (BCR) signaling induces actin cytoskeleton remodeling by stimulating actin severing, actin polymerization, and the nucleation of branched actin networks via the Arp2/3 complex. This enables B cells to spread on antigen-bearing surfaces in order to increase antigen encounters and to form an immune synapse (IS) when interacting with antigen-presenting cells (APCs). Although the WASp, N-WASp, and WAVE nucleation-promoting factors activate the Arp2/3 complex, the role of WAVE2 in B cells has not been directly assessed. We now show that both WAVE2 and the Arp2/3 complex localize to the peripheral ring of branched F-actin when B cells spread on immobilized anti-Ig antibodies. The siRNA-mediated depletion of WAVE2 reduced and delayed B cell spreading on immobilized anti-Ig, and this was associated with a thinner peripheral F-actin ring and reduced actin retrograde flow compared to control cells. Depleting WAVE2 also impaired integrin-mediated B cell spreading on fibronectin and the LFA-1-induced formation of actomyosin arcs. Actin retrograde flow amplifies BCR signaling at the IS, and we found that depleting WAVE2 reduced microcluster-based BCR signaling and signal amplification at the IS, as well as B cell activation in response to antigen-bearing cells. Hence, WAVE2 contributes to multiple actin-dependent processes in B lymphocytes.

SH2 domain-containing inositol 5-phosphatases support the survival of Burkitt lymphoma cells by promoting energy metabolism.

Burkitt lymphoma cells (BL) exploit antigen-independent tonic signals transduced by the B-cell antigen receptor (BCR) for their survival, but the molecular details of the rewired BL-specific BCR signal network remain unclear. A loss of function screen revealed the SH2 domain-containing 5`-inositol phosphatase 2 (SHIP2) as a potential modulator of BL fitness. We characterized the role of SHIP2 in BL survival in several BL cell models and show that perturbing SHIP2 function renders cells more susceptible to apoptosis, while attenuating proliferation in a BCR-dependent manner. Unexpectedly, SHIP2 deficiency did neither affect PI3K survival signals nor MAPK activity, but attenuated ATP production. We found that an efficient energy metabolism in BL cells requires phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2), which is the enzymatic product of SHIP proteins. Consistently, interference with the function of SHIP1 and SHIP2 augments BL cell susceptibility to PI3K inhibition. Notably, we provide here a molecular basis of how tonic BCR signals are connected to energy supply, which is particularly important for such an aggressively growing neoplasia. These findings may help to improve therapies for the treatment of BL by limiting energy metabolism through the inhibition of SHIP proteins, which renders BL cells more susceptible to the targeting of survival signals.

B cell receptor-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics.

Successful B cell activation, critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15 min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodelling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through Akt and MAPK axes.

Combined immunodeficiency and impaired PI3K signaling in a patient with biallelic LCP2 variants

Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T-and B-cell defects. We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.

Siglec cis-ligands and their roles in the immune system.

Sialic acid-binding immunoglobulin-like lectins are a family of membrane molecules primarily expressed in immune cells. Most of them are inhibitory receptors containing immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. On the cell surface, sialic acid-binding immunoglobulin-like lectins are mostly bound by sialylated glycans on membrane molecules expressed in the same cell (cis-ligands). Although ligands of sialic acid-binding immunoglobulin-like lectins are not efficiently identified by conventional methods such as immunoprecipitation, in situ labeling including proximity labeling is useful in identifying both cis-ligands and the sialylated ligands expressed by other cells (trans-ligands) of sialic acid-binding immunoglobulin-like lectins. Interaction of the inhibitory sialic acid-binding immunoglobulin-like lectins with cis-ligands including both those with and without signaling function modulates the inhibitory activity of sialic acid-binding immunoglobulin-like lectins by multiple different ways. This interaction also modulates signaling function of the cis-ligands. So far, little is known about the role of the interaction between sialic acid-binding immunoglobulin-like lectins and the cis-ligands. Nonetheless, recent studies showed that the inhibitory activity of CD22 (also known as Siglec-2) is regulated by endogenous ligands, most likely cis-ligands, differentially in resting B cells and those in which B-cell antigen receptor is ligated. This differential regulation plays a role in quality control of signaling-competent B cells and also partial restoration of B-cell antigen receptor signaling in immunodeficient B cells.

Therapeutic effect of ibrutinib, a selective Bruton's tyrosine kinase inhibitor, in orbital fibroblasts from patients with Graves' orbitopathy.

Bruton's tyrosine kinase (BTK) is an essential protein in B-cell antigen receptor (BCR) signaling pathway and is known to be related to pathogenetic effect on B-cell related malignancies and various autoimmune diseases. In this study, we investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK inhibitor in the pathogenesis of Graves' orbitopathy (GO) in in vitro model. Expression of BTK in orbital tissues from GO and normal control subjects were evaluated by real-time polymerase chain reaction (PCR). Primary cultured orbital fibroblasts from each subject were exposed to ibrutinib and stimulated with interleukin (IL)-1β or insulin like growth factor (IGF)-1. Production of inflammatory cytokines was evaluated by real time PCR and enzyme-linked immunosorbent assays (ELISA). The downstream transcription factors were also determined by western blot assays. The expression of BTK in GO tissues were significantly higher than in healthy controls. After stimulation of GO orbital fibroblasts with IL-1β or IGF-1, BTK mRNA and phosphorylated (p)- BTK protein expression was also enhanced. Ibrutinib reduced the expression of BTK mRNA and proteins of p-BTK, and inhibited the IL-1β- and IGF-1-induced production of proinflammatory cytokines including IL-6, IL-8 and COX-2 in both GO and normal cells. Ibrutinib also significantly attenuated phosphorylation of Akt, p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) in IL-1β stimulated GO cells and Akt, JNK, and NF-κB in IL-1ß stimulated normal cells. BTK expression is enhanced in GO tissue and orbital fibroblasts. Ibrutinib, a BTK inhibitor suppresses proinflammatory cytokine production as well as phosphorylation of Akt and NF-κB protein. Our results suggest the potential role of BTK in GO inflammatory pathogenesis and possibility of a novel therapeutic target of GO.

FcγRIIB inhibits inflammation in a murine model of psoriasis

BackgroundPsoriasis is a chronic, inflammatory cutaneous disease. FcγRIIB is a low-affinity receptor for the IgG Fc fragment that provides a negative feedback pathway to down-regulate B-cell antigen receptor signaling. ObjectiveThe aim of this study was to investigate the role of FcγRIIB in the development of murine imiquimod (IMQ)-induced, psoriasis-like skin inflammation. MethodsThe experimental psoriasis-like skin inflammation was induced by the topical application of IMQ to the ears of FcγRIIB deficient (FcγRIIB-/-) and wild-type (WT) mice. After 6 days, epidermal thickness and inflammatory cell infiltration of the skin were histopathologically assessed and cytokine and chemokine expression levels were measured with RT-PCR. ResultsSkin inflammation was significantly worse in FcγRIIB-/- mice than WT mice. In the skin, the numbers of Gr-1+ neutrophils, CD11c+ dendritic cells, and Foxp3+ T cells were significantly higher in FcγRIIB-/- mice than WT mice. In the spleen, the numbers of CD25+Foxp3+ T cells and CD19+IL-10+ B cells were also significantly higher in FcγRIIB-/-mice than WT mice. The mRNA expression of Il-6, Il-17a, and Il-23a was significantly enhanced in FcγRIIB-/- mice. An adoptive transfer of splenic leukocytes from FcγRIIB-/- mice into WT mice also exacerbated skin inflammation compared to WT mice that received splenic leukocytes from WT mice. Intravenous immunoglobulin significantly reduced skin inflammation in WT mice, but this improvement was not observed in FcγRIIB-/- mice. ConclusionThese results indicate that FcγRIIB likely plays a suppressive role in IMQ-induced, psoriasis-like skin inflammation. Furthermore, signal modulation via FcγRIIB is a potential therapeutic target for psoriasis.

Abelson tyrosine kinase controls BCR signalling and B-cell differentiation by promoting B-cell metabolism.

As a nonreceptor tyrosine kinase, Abelson tyrosine kinase (c-Abl) was first studied in chronic myelogenous leukaemia, and its role in lymphocytes has been well characterised. c-Abl is involved in B-cell development and CD19-associated B-cell antigen receptor (BCR) signalling. Although c-Abl regulates different metabolic pathways, the role of c-Abl is still unknown in B-cell metabolism. In this study, B-cell-specific c-Abl knockout (KO) mice (Mb1Cre+/- c-Ablfl/fl ) were used to investigate how c-Abl regulates B-cell metabolism and BCR signalling. We found that the levels of activation positive BCR signalling proximal molecules, phosphorylated spleen tyrosine kinase (pSYK) and phosphorylated Bruton tyrosine kinase (pBTK), were decreased, while the level of key negative regulator, phosphorylated SH2-containing inositol phosphatase 1 (pSHIP1), was increased in Mb1Cre+/- c-Ablfl/fl mice. Furthermore, we found c-Abl deficiency weakened the B-cell spreading, formation of BCR signalosomes, and the polymerisation of actin during BCR activation, and also impaired the differentiation of germinal center (GC) B-cells both in quiescent condition and after immunisation. Moreover, B-cell mitochondrial respiration and the expression of B-cell metabolism-regulating molecules were downregulated in c-Abl deficiency mice. Overall, c-Abl, which involved in actin remodelling and B-cell metabolism, positively regulates BCR signalling and promotes GC differentiation.

Next-generation Bruton's Tyrosine Kinase (BTK) Inhibitors Potentially Targeting BTK C481S Mutation- Recent Developments and Perspectives.

Bruton's tyrosine kinase (BTK) plays a vital role in B-cell antigen receptor (BCR) signalling transduction pathway. Controlling BCR signalling by BTK inhibitors is a promising therapeutic approach for the treatment of inflammatory and autoimmune diseases. Since the approval of ibrutinib for the treatment of different haematological cancers in 2013, great efforts have been made to explore new BTK inhibitors. Despite the remarkable potency and efficacy of first and second generation irreversible BTK inhibitors against various lymphomas and leukaemia, there are also some clinical limitations, such as off-target toxicity and primary/acquired drug resistance. Acquired drug resistance due to the C481S mutation in BTK is the major challenging problem of irreversible inhibitors. After, the BTK C481S mutation, the irreversible covalent inhibitors cannot form covalent bond with BTK and drop activities. Hence, there is an urgent need to develop novel BTK inhibitors to overcome the mutation problem. In recent years, a few reversible BTK inhibitors have been developed and are under clinical evaluation stages. In addition, a few reversible BTK-PROTACs have been explored and under developments. A number of reversible non-covalent BTK inhibitors, including MK1026/ ARQ531, LOXO305, fenebrutinib are at different stages of clinical trials for autoimmune diseases. In this review, we summarized the discovery and development of nextgeneration BTK inhibitors, especially targeting BTK C481S mutation and their applications for the treatment of lymphomas and autoimmune diseases.

P686: A PHASE 1 FIRST IN-HUMAN STUDY OF BGB-16673, A BRUTON TYROSINE KINASE PROTEIN DEGRADER, IN PATIENTS (PTS) WITH B-CELL MALIGNANCIES (TRIAL IN PROGRESS)

Background: Bruton tyrosine kinase (BTK) functions downstream of the B-cell antigen receptor (BCR) and plays a critical role within the BCR signaling pathway and the pathogenesis of several B-cell malignancies. BTK inhibitors (BTKi) have revolutionized management of B-cell malignancies. However, resistance caused by BTK mutations, which can abrogate BTKi binding capacity or BTK scaffold function, or cause kinase hyper activation, may limit therapeutic options in subsequent lines of therapy. Re-challenging patients with agents that can overcome the resistance due to BTK mutations may provide a novel treatment option. BGB-16673 is an investigational, orally available agent with preclinically demonstrated BTK degradation activity against both wild type and mutant forms commonly identified in pts who have progressed on BTKi. Aims: In the dose-escalation part of the study, we aim to assess the safety and tolerability of BGB-16673 in selected relapsed or refractory (R/R) B-cell malignancies, to characterize its pharmacokinetic (PK) and pharmacodynamic (PD) profiles, to determine a recommended phase 2 dose (RP2D) and to evaluate anti-tumor activity. In the dose-expansion part of the study, we aim to evaluate the safety, tolerability, PK, PD, and anti-tumor activity under the RP2D in pts with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL). Methods: BGB-16673-101 (NCT05006716) is a phase 1 open-label, dose-escalation and -expansion study evaluating BGB-16673 in adult pts with R/R B-cell malignancies. Key inclusion criteria include confirmed diagnosis of R/R B-cell malignancy (including marginal zone lymphoma, follicular lymphoma, MCL, CLL/SLL, and Waldenström macroglobulinemia [WM]), Eastern Cooperative Oncology Group performance status score of 0 to 2, and adequate organ function. Key exclusion criteria include current or history of central nervous system involvement, autologous stem cell transplant (ASCT) within 3 months or chimeric antigen receptor T cell therapy or allogeneic SCT within 6 months of the first dose of BGB-16673 or requiring treatment with strong inhibitors or inducers of CYP3A. All pts will be followed for safety and tolerability, including treatment-emergent adverse events that occur during treatment and up to 30 days after treatment discontinuation, or until the initiation of another anti-cancer therapy, whichever occurs first. The totality of the available safety, efficacy, PK, and PD data from the dose-escalation part will be used by the Safety Monitoring Committee to determine the RP2D. The dose-expansion part will commence subsequent to RP2D determination. Responses will be evaluated per the 2014 Lugano Classification, the 2018 International Workshop on CLL guidelines response assessment with modification for treatment-related lymphocytosis, or the 6th International Workshop on WM consensus criteria. Additional efficacy analyses will include progression-free survival and overall survival. All pts will give informed consent. Results: This is a trial in progress; safety and tolerability results of BGB-16673 are expected. Summary/Conclusion: BGB-16673-101 is the first in-human study of the BTK degrader BGB-16673.

Conformational, Toxic, Physicochemical and Molecular Docking Analysis of the Anticancer Acalabrutinib Molecule

Acalabrutinib is an inhibitor of Bruton's tyrosine kinase (BTK) activity and prevents the activation of the B-cell antigen receptor (BCR) signaling pathway. For having these properties acalabrutinib recently was approved for medical use as an anticancer drug. Determining the conformational properties of a bioactive molecule is necessary to reveal its bioactivity. For this reason, the conformational states of the acalabrutinib were examined first. The AM1, a semi-experimental method, was used to examine the stable conformations of the acalabrutinib molecule. Nine lowest energy conformers of the acalabrutinib molecule were determined and their relative energies were calculated. Afterwards, the interactions of the most stable conformer of acalabrutinib with DNA and integrin were examined by docking simulations, and the most active interaction sites and binding affinities were determined.

Fumarate suppresses B-cell activation and function through direct inactivation of LYN.

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

Bruton's Kinase Inhibitors for the Treatment of Immunological Diseases: Current Status and Perspectives.

The use of Bruton’s tyrosine kinase (BTK) inhibitors has changed the management of patients with B-cell lymphoid malignancies. BTK is an important molecule that interconnects B-cell antigen receptor (BCR) signaling. BTK inhibitors (BTKis) are classified into three categories, namely covalent irreversible inhibitors, covalent reversible inhibitors, and non-covalent reversible inhibitors. Ibrutinib is the first covalent, irreversible BTK inhibitor approved in 2013 as a breakthrough therapy for chronic lymphocytic leukemia patients. Subsequently, two other covalent, irreversible, second-generation BTKis, acalabrutinib and zanubrutinib, have been developed for lymphoid malignancies to reduce the ibrutinib-mediated adverse effects. More recently, irreversible and reversible BTKis have been under development for immune-mediated diseases, including autoimmune hemolytic anemia, immune thrombocytopenia, multiple sclerosis, pemphigus vulgaris, atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus, Sjögren’s disease, and chronic spontaneous urticaria, among others. This review article summarizes the preclinical and clinical evidence supporting the role of BTKis in various autoimmune, allergic, and inflammatory conditions.

The Small GTPase RHOA Links SLP65 Activation to PTEN Function in Pre B Cells and Is Essential for the Generation and Survival of Normal and Malignant B Cells.

The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA-deficient mice. The RhoA-deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA-deficient cells with RhoA or Foxo1, a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function.