Btk Expression Research Articles

In vivo Bruton's tyrosine kinase inhibition attenuates alcohol-associated liver disease by regulating CD84-mediated granulopoiesis.

Severe alcohol-associated hepatitis (AH) is a life-threatening form of alcohol-associated liver disease. Liver neutrophil infiltration is a hallmark of AH, yet the effects of alcohol on neutrophil functions remain elusive. Identifying therapeutic targets to reduce neutrophil-mediated liver damage is essential. Bruton's tyrosine kinase (BTK) plays an important role in neutrophil development and function; however, the role of BTK in AH is unknown. Using RNA sequencing of circulating neutrophils, we found an increase in Btk expression (P = 0.05) and phosphorylated BTK (pBTK) in patients with AH compared with healthy controls. In vitro, physiologically relevant doses of alcohol resulted in a rapid, TLR4-mediated induction of pBTK in neutrophils. In a preclinical model of AH, administration of a small-molecule BTK inhibitor (evobrutinib) or myeloid-specific Btk knockout decreased proinflammatory cytokines and attenuated neutrophil-mediated liver damage. We found that pBTK was essential for alcohol-induced bone marrow granulopoiesis and liver neutrophil infiltration. In vivo, BTK inhibition or myeloid-specific Btk knockout reduced granulopoiesis, circulating neutrophils, liver neutrophil infiltration, and liver damage in a mouse model of AH. Mechanistically, using liquid chromatography-tandem mass spectrometry, we identified CD84 as a kinase target of BTK, which is involved in granulopoiesis. In vitro, CD84 promoted alcohol-induced interleukin-1β and tumor necrosis factor-α in primary human neutrophils, which was inhibited by CD84-blocking antibody treatment. Our findings define the role of BTK and CD84 in regulating neutrophil inflammation and granulopoiesis, with potential therapeutic implications in AH.

Bruton’s tyrosine kinase inhibition ameliorated neuroinflammation during chronic white matter ischemia

Chronic cerebral hypoperfusion (CCH), a disease afflicting numerous individuals worldwide, is a primary cause of cognitive deficits, the pathogenesis of which remains poorly understood. Bruton’s tyrosine kinase inhibition (BTKi) is considered a promising strategy to regulate inflammatory responses within the brain, a crucial process that is assumed to drive ischemic demyelination progression. However, the potential role of BTKi in CCH has not been investigated so far. In the present study, we elucidated potential therapeutic roles of BTK in both in vitro hypoxia and in vivo ischemic demyelination model. We found that cerebral hypoperfusion induced white matter injury, cognitive impairments, microglial BTK activation, along with a series of microglia responses associated with inflammation, oxidative stress, mitochondrial dysfunction, and ferroptosis. Tolebrutinib treatment suppressed both the activation of microglia and microglial BTK expression. Meanwhile, microglia-related inflammation and ferroptosis processes were attenuated evidently, contributing to lower levels of disease severity. Taken together, BTKi ameliorated white matter injury and cognitive impairments induced by CCH, possibly via skewing microglia polarization towards anti-inflammatory and homeostatic phenotypes, as well as decreasing microglial oxidative stress damage and ferroptosis, which exhibits promising therapeutic potential in chronic cerebral hypoperfusion-induced demyelination.

AnGong NiuHuang (AGNH) pill attenuated traumatic brain injury through regulating NF-κB/Nlrp3 axis and glycerophospholipid metabolism

BackgroundTraumatic brain injury (TBI), especially neuroinflammation after TBI persists for a long time and causes significant neurodegenerative pathologies and neuropsychiatric problems. PurposeIn this study, the neuroprotective effect of AnGong NiuHuang (AGNH) on TBI was investigated and the mechanism was revealed by integrating multiple omics. MethodsThe rats with TBI were administrated with AGNH for 5 consecutive days and the effect was evaluated by using modified neurologic severity score (mNSS), brain edema, H&E staining, Nissl staining and TUNEL staining. The mechanism was revealed by using RNA sequencing (RNA-seq) and metabolomic analysis. The inflammatory factors, apoptosis-related proteins and identified vital targets were validated by enzyme-linked immunosorbent assay, western blotting and immunofluorescence staining. ResultsAdministration of AGNH decreased mNSS, brain edema, brain structure damage, but increased Nissl body density in the rats with TBI. Additionally, AGNH reduced IL-1β, IL-17A, TNF-α, MMP9, MCP-1, IL-6, Bax and TUNEL staining,but elevated Bcl2 level. Integrating transcriptomic analysis and metabolomic analysis identified vital targets and critical metabolic pathways. Importantly, AGNH treatment reduced the expression of TLR4, MYD88, NLRP3, BTK, IL-18 and Caspase 1 as well as glycerophospholipid metabolism-related protein AGPAT2 and PLA2G2D, and decreased the nuclear translocation of NF-κB p65 in the brain of TBI rats. Additionally, AGNH increased phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), but decreased 1-acyl-sn-glycero-3-phosphocholine (LysoPC) in the metabolic pathway of glycerophospholipid metabolism. ConclusionTaken together, AGNH inhibited NF-κB/NLRP3 axis to suppress neuroinflammation, cell apoptosis and pyroptosis, and improved metabolic pathways of glycerophospholipid metabolism after TBI.

Mmu-miR-185 regulates osteoclasts differentiation and migration by targeting Btk.

Bones undergo a constant remodeling, a process involving osteoclast-mediated bone resorption and osteoblast-mediated bone formation, crucial for maintaining healthy bone mass. We previously observed that miR-185 depletion may promote bone formation by regulating Bgn expression and the BMP/Smad signaling pathway. However, the effects of miR-185-5p on the osteoclasts and bone remodeling have not been elucidated, warranting further exploration. Tartrate-resistant acid phosphatase staining was utilized to assess the differentiation ability of bone marrow mononuclear macrophages (BMMs) from mmu-miR-185 gene knockout (KO) mice and wild-type (WT) mice. A reverse transcriptase-quantitative PCR was conducted to compare differences in miR-185-5p and osteoclast marker molecules, including Trap, Dcstamp, Ctsk and Nfatc1, between the KO group and WT group BMMs. Western blot analysis was employed to observe the expression of osteoclast marker molecules. A cell-counting kit-8 was used to analyze cell proliferation ability. Transwell experiments were conducted to detect cell migration. Dual-luciferase reporter assays were employed to confirm whether Btk is a downstream target gene of miR-185-5p. miR-185 depletion promoted osteoclast differentiation in bone marrow-derived monocytes/macrophages. Overexpression of miR-185-5p in RAW264.7 cells inhibited differentiation and migration of osteoclasts. Furthermore, Btk was identified as a downstream target gene of miR-185-5p, suggesting that miR-185-5p may inhibit osteoclast differentiation and migration by targeting Btk. miR-185 regulates osteoclasts differentiation, with overexpression of miR-185-5p inhibiting osteoclast differentiation and migration in vitro. Additionally, miR-185-5p may modulate osteoclastic differentiation and migration by regulating Btk expression.

Bruton’s tyrosine kinase inhibitors in brain diseases

Bruton’s tyrosine kinase (BTK) is a non-receptor-bound intracellular signaling protein. It is well known for its importance in the growth and malignancy of B cells, but recent studies suggested that the BTK is also associated with many other innate immune cells. As reported, a comparatively high level of BTK expression can be observed in monocytes, neutrophils, macrophages, and even central nervous system-resident immune cells like microglia. This suggests that BTK activation occurs in various acute and chronic inflammatory conditions. Here, we discuss how BTK inhibitors might be used to treat certain conditions, concentrating on ischemic stroke, multiple sclerosis, neuromyelitis optica spectrum disorders, and Alzheimer’s disease. We specifically show the significance of targeting B cells in controlling the inflammatory component of the disease in multiple sclerosis treatment. In addition, we draw attention to the role of BTK in the NLRP3 inflammasome activation, which is essential for brain damage and neuroinflammation. In summary, we conclude that therapeutic targeting of BTK in brain diseases is a potential strategy that can complement the existing therapies.

Formononetin inhibits IgE by huPlasma/PBMCs and mast cells/basophil activation via JAK/STAT/PI3-Akt pathways.

Food allergy is a prevalent disease in the U.S., affecting nearly 30 million people. The primary management strategy for this condition is food avoidance, as limited treatment options are available. The elevation of pathologic IgE and over-reactive mast cells/basophils is a central factor in food allergy anaphylaxis. This study aims to comprehensively evaluate the potential therapeutic mechanisms of a small molecule compound called formononetin in regulating IgE and mast cell activation. In this study, we determined the inhibitory effect of formononetin on the production of human IgE from peripheral blood mononuclear cells of food-allergic patients using ELISA. We also measured formononetin's effect on preventing mast cell degranulation in RBL-2H3 and KU812 cells using beta-hexosaminidase assay. To identify potential targets of formononetin in IgE-mediated diseases, mast cell disorders, and food allergies, we utilized computational modeling to analyze mechanistic targets of formononetin from various databases, including SEA, Swiss Target Prediction, PubChem, Gene Cards, and Mala Cards. We generated a KEGG pathway, Gene Ontology, and Compound Target Pathway Disease Network using these targets. Finally, we used qRT-PCR to measure the gene expression of selected targets in KU812 and U266 cell lines. Formononetin significantly decreased IgE production in IgE-producing human myeloma cells and PBMCs from food-allergic patients in a dose-dependent manner without cytotoxicity. Formononetin decreased beta-hexosaminidase release in RBL-2H3 cells and KU812 cells. Formononetin regulates 25 targets in food allergy, 51 in IgE diseases, and 19 in mast cell diseases. KEGG pathway and gene ontology analysis of targets showed that formononetin regulated disease pathways, primary immunodeficiency, Epstein-Barr Virus, and pathways in cancer. The biological processes regulated by formononetin include B cell proliferation, differentiation, immune response, and activation processes. Compound target pathway disease network identified NFKB1, NFKBIA, STAT1, STAT3, CCND1, TP53, TYK2, and CASP8 as the top targets regulated at a high degree by formononetin. TP53, STAT3, PTPRC, IL2, and CD19 were identified as the proteins mostly targeted by formononetin. qPCR validated genes of Formononetin molecular targets of IgE regulation in U266 cells and KU812 cells. In U266 cells, formononetin was found to significantly increase the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. In basophils KU812 cells, formononetin significantly increased the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK, TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. These findings comprehensively present formononetin's mechanisms in regulating IgE production in plasma cells and degranulation in mast cells.

Inhibition of Lysine Acetyltransferases p300/CBP Overcomes BTK Inhibitor Resistance in Mantle Cell Lymphoma Cells

Background: Although Bruton's Tyrosine Kinase inhibitors (BTKis) are effective against relapsed refractory mantle cell lymphoma (MCL), the limited duration of therapeutic response and the emergence of resistance remain clinical issues. We conducted a CRISPR/Cas9 screen to identify the genes responsible for BTKi sensitivity and resistance. Materials and Methods: To identify the genes responsible for BTKi resistance, we used a CRISPR library (Kosuke Yusa, V3), targeting 18,740 human genes. We compared three BTKis, ibrutinib, acalabrutinib and tirabrutinib, using DMSO as a control. We transduced Granta519, an ibrutinib-resistant MCL cell line expressing Cas9 with the CRISPR library lentiviruses. Next, we counted sgRNA copy numbers using next generation sequencing after a two-week drug treatment. We identified 76 genes that sensitized cells to the BTKis upon knockout. After excluding common essential genes based on the Cancer Dependency Map portal (depmap.org/portal/), we ranked these genes according to the extent to which the combination of their knockout and BTKis inhibited cell proliferation. Results: Lysine acetyltransferases (KATs), p300 and CBP were ranked first and ninth, respectively. Knockout of p300 or CBP alone moderately decreased cell numbers, where as their combination with BTKi treatment significantly decreased cell numbers. To further validate this synergistic effect, we performed an MTS assay in combination with p300/CBP inhibitors (C-646 and A-485) and ibrutinib in MCL cell lines (Granta519 and Mino). The p300/CBP inhibitor increased ibrutinib sensitivity, leading to a up to four-fold greater suppression of cell viability. This effect was even greater than that of existing drugs, including venetoclax (a BCL-2 inhibitor), and idelalisib (a PI3K inhibitor), which are thought to have synergistic effects. The synergistic effect of a p300/CBP inhibitor and ibrutinib was also observed in long-term cultures for up to two weeks in both ibrutinib-resistant and ibrutinib-sensitive MCL cell lines. Furthermore, combination treatment resulted in an enhancement of G0/G1 cell cycle arrest and an increase in apoptotic cells in the MCL cell lines. To elucidate the mechanism underlying this synergistic effect, we measured the expression of IκBα mRNA as an indicator of NFκB activity by real-time PCR. In Granta519, ibrutinib alone caused an initial decrease in IκBα mRNA expression followed by a rebound increase. However, the addition of A-485 decreased the rebound and suppressed IκBα mRNA expression. These results indicate that ibrutinib and A-485 cooperatively suppress NFκB activity. The combination of ibrutinib and A-485 synergistically suppressed phosphorylation of PLCγ2 and of AKT as confirmed by Western blotting. This suggests that downstream PLCγ2 and AKT activation might be involved in ibrutinib resistance. Lentiviral overexpression of p300 in Granta519 cells enhanced phosphorylation of AKT. On the other hand, ibrutinib suppressed the expression of p300, and conversely, p300/CBP inhibitors suppressed the expression of BTK. These data suggest that the expression of p300 and BTK are dependent on each other. We hypothesize that direct or indirect interactions may occur through phosphorylation by BTK and acetylation by p300. Conclusion: Our CRISPR screen identified lysine acetyltransferases p300/CBP as BTK inhibitor resistance genes in human MCL cells. The combination of a p300/CBP inhibitor and a BTK inhibitor synergistically suppresses both BTK and AKT, leading to NFκB suppression. Taken together, we first identified that the combination of p300/CBP inhibition and BTK inhibition could be a promising strategy for treating patients with MCL.

Abstract 18390: Ibrutinib Increases Ventricular Arrhythmia Burden Independently of Atrial Arrhythmia

Introduction: Use of Bruton Tyrosine Kinase inhibitors (BTKi) such as ibrutinib is frequently limited by atrial arrhythmia, possibly through off-target inhibition of C-Terminal-Src-Kinase (CSK). Whether BTKi can directly trigger ventricular arrhythmia is less clear. Case reports and retrospective analysis have suggested a link, but causal effect is not established. The association is further put into doubt by absent expression of CSK or BTK in ventricular cardiomyocytes, and absence of ventricular arrhythmia signals in meta-analyses of BTKi RCTs. Further study is critically needed. Hypothesis: Ibrutinib induces ventricular arrhythmia through a unique mechanism. Methods: We performed a retrospective crossover study of ibrutinib-treated patients with serial cardiac monitoring both on and off ibrutinib (pacemakers, loop recorders, or multiple holters) across a 10-year span in a large academic center to compare burden of atrial/ventricular ectopy on and off ibrutinib. A separate group of Chronic Lymphocytic Leukemia (CLL) patients with serial cardiac monitoring not on BTKi was used as additional controls. To determine biological correlation, we tested the ability of ibrutinib to induce arrhythmia in cultured human Induced Pluripotent Stem Cell derived ventricular cardiomyocytes (iPSC-vCMs). Results: 88% (15/17) of patients on ibrutinib with serial cardiac monitoring had increased burden of Premature Ventricular Contractions (PVCs), with an average increase of 155 PVCs/hour (p<0.0001). An increase was not observed in the control cohort. Ventricular arrhythmia burden was not associated with atrial arrhythmia burden. Removing patient-related confounders, in vitro analysis of iPSC-vCMs demonstrated that ibrutinib induced arrhythmia following three days of treatment in a dose-dependent manner. Conclusions: In patients with continuous/serial cardiac monitoring, ibrutinib is associated with a significant increase in ventricular ectopy (average of 155 PVCs/hr). Lack of ventricular and atrial arrhythmia concordance suggests an alternative mechanism of arrhythmia induction. Increased arrhythmogenicity of human ventricular cardiomyocytes treated with ibrutinib indicates a direct myocardial mechanism.

Modeling Gilteritinib Resistance By Enforced cxcl8a Expression in Zebrafish HSPCs

In Acute Myeloid Leukemia (AML), the FMS-like tyrosine kinase III Internal Tandem Duplication (FLT3-ITD) is common and increases the risk of relapse which is only partially mitigated by tyrosine kinase inhibitor (TKI) therapy. Gilteritinib is the current mainstay for treating relapsed FLT3-ITD AML, but therapy is non-curative, and the duration of remission is transient. Mutations in the RAS pathway are known to promote gilteritinib resistance, however non-mutational mechanisms of resistance remain under-explored and could provide new avenues for combination therapy. Our previous work has associated AML cell-autonomous expression of chemokines such as CXCL8 (IL-8) as a mechanism of resistance to gilteritinib therapy. Here we enforce expression of cxcl8a ( cxcl8, ENSDARG00000104795) by hematopoietic stem and progenitor cells (HSPCs) in transgenic zebrafish embryos using a defined Runx1 enhancer element. In the absence of gilteritinib, Runx1:cxcl8(+) animals had greater numbers of HPSCs compared to Runx1:cxcl8(-) animals (4.0 vs 2.1, p = 0.04). When treated with gilteritinib at 100 nM from 48-72 hours post-fertilization (hpf), HSPC numbers were decreased in both groups; however, Runx1:cxcl8(+) animals still had more HSPCs compared to Runx1:cxcl8(-) animals (1.6 vs 0.4, p = 0.02). This suggests that cxcl8 may act as a resistance factor for gilteritinib in vivo and that understanding regulators of cxcl8 expression may provide insight into gilteritinib resistance. Reanalysis of published single cell RNA-seq data in AML patients revealed an association between expression of CXCL8 and BTK and BMX but not other Tec-family kinases such as ITK and TEC itself. Zebrafish do not have an annotated BMX gene; BTK is expressed in HSPCs and neutrophils but not the erythroid lineage. CHMFL is a specific and potent inhibitor of human BMX with an unknown spectrum of activity in zebrafish. In Runx1:cxcl8(-) Runx1:GFP zebrafish, CHMFL treatment reduced HSPC numbers, but this was not rescued in Runx1:cxcl8(+) animals (untreated vs treated, cxcl8(-): 2.9 vs 1.5, p = 0.02, untreated vs treated, cxcl8(+): 4.0 vs 1.4, p = 0.0006). This contrasts with our prior work with the non-specific Tec family kinase inhibitor, ibrutinib, where HSPC numbers were rescued in Runx1:cxcl8(+) animals. Ongoing studies are focused on understanding the targets of ibrutinib and CHMFL in the zebrafish and how these pathways may affect gilteritinib resistance.

Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK.

Cytotoxic CD8+ T cell (CTL) exhaustion is a dysfunctional state of T cells triggered by persistent antigen stimulation, with the characteristics of increased inhibitory receptors, impaired cytokine production and a distinct transcriptional profile. Evidence from immune checkpoint blockade therapy supports that reversing T cell exhaustion is a promising strategy in cancer treatment. Ibrutinib, is a potent inhibitor of BTK, which has been approved for the treatment of chronic lymphocytic leukemia. Previous studies have reported improved function of T cells in ibrutinib long-term treated patients but the mechanism remains unclear. We investigated whether ibrutinib directly acts on CD8+ T cells and reinvigorates exhausted CTLs. We used an established in vitro CTL exhaustion system to examine whether ibrutinib can directly ameliorate T cell exhaustion. Changes in inhibitory receptors, transcription factors, cytokine production and killing capacity of ibrutinib-treated exhausted CTLs were detected by flow cytometry. RNA-seq was performed to study transcriptional changes in these cells. Btk deficient mice were used to confirm that the effect of ibrutinib was independent of BTK expression. We found that ibrutinib reduced exhaustion-related features of CTLs in an in vitro CTL exhaustion system. These changes included decreased inhibitory receptor expression, enhanced cytokine production, and downregulation of the transcription factor TOX with upregulation of TCF1. RNA-seq further confirmed that ibrutinib directly reduced the exhaustion-related transcriptional profile of these cells. Importantly, using btk deficient mice we showed the effect of ibrutinib was independent of BTK expression, and therefore mediated by one of its other targets. Our study demonstrates that ibrutinib directly ameliorates CTL exhaustion, and provides evidence for its synergistic use with cancer immunotherapy.

Co-Targeting of BTK and TrxR as a Therapeutic Approach to the Treatment of Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a haematological malignancy representing the most diagnosed non-Hodgkin's lymphoma (NHL) subtype. Despite the approved chemotherapies available in clinics, some patients still suffer from side effects and relapsed disease. Recently, studies have reported the role of the Trx system and the BCR signalling pathway in cancer development and drug resistance. In this regard, we assessed a potential link between the two systems and evaluated the effects of [Au(d2pype)2]Cl (TrxR inhibitor) and ibrutinib (BTK inhibitor) alone and in combination on the cell growth of two DLBCL lymphoma cell lines, SUDHL2 and SUDHL4. In this study, we show higher expression levels of the Trx system and BCR signalling pathway in the DLBCL patient samples compared to the healthy samples. The knockdown of TrxR using siRNA reduced BTK mRNA and protein expression. A combination treatment with [Au(d2pype)2]Cl and ibrutinib had a synergistic effect on the inhibition of lymphoma cell proliferation, the activation of apoptosis, and, depending on lymphoma cell subtype, ferroptosis. Decreased BTK expression and the cytoplasmic accumulation of p65 were observed after the combination treatment in the DLBCL cells, indicating the inhibition of the NF-κB pathway. Thus, the co-targeting of BTK and TrxR may be an effective therapeutic strategy to consider for DLBCL treatment.

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.

Orelabrutinib and venetoclax synergistically induce cell death in double-hit lymphoma by interfering with the crosstalk between the PI3K/AKT and p38/MAPK signaling.

Double-hit lymphoma (DHL) is a rare and aggressive mature B-cell malignancy with concurrentMYCandBCL2rearrangements. When DHL becomes relapsed or refractory, it becomes resistant to the majority of therapeutic approaches and has subpar clinical results. Therefore, innovative therapeutics for this particular patient population are urgently needed. Orelabrutinib, a new oral BTK inhibitor, combined with the Bcl-2 inhibitor venetoclax, was used to confirm the antitumor effect of DHL. Cell counting kit-8 and Annexin V-FITC/PI assays were used to examine the interaction of this combined regimen on DHL cell lines and primary lymphoma cells. RNA sequencing, EdU incorporation assay, mitochondrial membrane potential assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of orelabrutinib with or without venetoclax against DHL cell lines. In this study, orelabrutinib combined with venetoclax synergistically induced DHL cell death, as evidenced by the inhibition of cell proliferation, the induct of cell cycle arrest, and the promotion of cell apoptosis via the mitochondrial pathway. Orelabrutinib treatment alters genome-wide gene expression in DHL cells. The combined regimen decreases the expression of BTK and Mcl-1, potentially interfering with the activity and crosstalk of PI3K/AKT signaling and p38/MAPK signaling. In addition, the combination of orelabrutinib and venetoclax shows cytotoxic activity in primary B-lymphoma cells. In summary, these findings reveal a novel therapy targeting BCR signaling and the Bcl-2 family for DHL patients with a poor prognosis.

Bcl-2 Family (Bcl-2, Mcl-1, Bim and Bax) and Btk Expression in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Lymphocytes in Different Lymphoid Compartments and Changes during Btk Inhibitor Therapies (ibrutinib or acalabrutinib)

B-cell chronic lymphocytic leukemia (B-CLL) is characterized with variable infiltration by B -CLL lymphocytes of various lymphoid compartments, i.e peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) and lymphoid organs. Interactions with various microenvironments may result in different disease activity and resistance to apoptosis. Novel agents targeting Btk and Bcl-2 show remarkable activity in B-CLL. However, they may have different activity in different lymphoid compartments while Btk inhibitors also may cause significant B-CLL lymphocyte redistribution between compartments. Aim of this study was to evaluate: 1) is there a different expression of Bcl-2 family of anti- and pro-apoptotic proteins (Bcl-2, Bax, Bim, mcl-1) and Btk in B-CLL lymphocytes from different lymphoid compartments; 2) relationship of observed expressions to disease parameters (TTM, TD, stage, B2MG, LDH); 3) changes during treatment with Btk inhibitors. We have included 28 B-CLL patients (18/10 M/F, median age 71 yr, range 48-85) treated with Btk inhibitors (ibrutinib 24, acalabrutinib 4). There were 15 previously treated patients and 13 treatment naïve patients. All evaluated patients had samples from PB and BM before treatment while 19 patients had also LN samples. At least one follow-up sample from PB had 20 patients. Median TTM score was 10.2 (range 3.4-23.9) and TD score was 0.68. Rai stage III/IV had 8 patients. Expression of Bcl-2, Mcl-1, Bim, Bax and Btk was determined by flow cytometry in CD19+CD5+ lymphocytes from fresh samples taken simultaneously by venipuncture (PB) or fine needle aspiration (BM and LN). There is higher expression of Bcl-2, Mcl-1 in LN compared to PB and BM (p<0.05), while there is no change in follow-up PB during ibrutinib/acalbrutinib treatment. There is no significant difference in Bim expression between compartments, while there is trend for higher expression in PB follow-up sample (p=0.054). There is no significant difference in Bax expression between PB, BM and LN compartments, while there is significant drop in Bax expression in PB follow-up sample during treatment (p<0.05). p-Btk has higher expression in LN compared to PB and BM (p<0.05). Follow-up PB p-Btk expression is comparable to pretreatment expression in PB (despite redistribution). We haven't found significant correlation of Bcl-2 family expression and p-Btk expression to the parameters of tumor mass (TTM) and tumor distribution (TD). In conclusion, there is a different pattern of expression of anti-apoptotic and pro-apoptotic Bcl-2 family members and Btk between lymphoid compartments. Our results may indicate higher BCR signaling and higher antiapoptotic activity in B-CLL lymphocytes in lymph node microenvironment. With redistribution during Btk inhibitor treatment B-CLL lymphocytes assume PB phenotype with downregulation of Btk and anti-apoptotic Bcl-2 and Mcl-1 proteins. Downregulation of pro-apoptotic Bax and possible upregulation of Bim protein indicate that there may be more complex interplay between various proteins resulting in different disease course. These results again emphasize the role of LN microenvironment in pathogenesis of B-CLL and may provide insights for treatment strategies combining targeted agents (Btk inhibitors and Bcl-2 antagonists).

Stilbene B10 induces apoptosis and tumor suppression in lymphoid Raji cells by BTK-mediated regulation of the KRAS/HDAC1/EP300/PEBP1 axis

Lymphoma is a cancer of the lymphoid cells that originated in matured B or T cells. The bioactive natural compounds can efficiently treat this disease with lesser side effects. Thus, in this study, a natural stilbene B10 (3-methoxy 5-hydroxy stilbene) isolated from Cajanus cajan (Pigeon Pea) was screened for its anti-proliferative efficacy against 13 cancer cell lines. B10 showed a potential effect on the human lymphoma (Raji) cells. Cytotoxicity analysis of B10 has revealed IC50 concentrations in Raji cells at low doses (18 µM) than other cancer cell lines. The B10 could significantly cause dose and time-dependent inhibition in the proliferation of Raji cells triggering intrinsic apoptosis and S/G1 phase cellular arrest. There was an increased expression of phospho-γ-H2A.X and decreased expression of cyclin D1, causing DNA damage and cell cycle arrest, post- B10 treatments. The mitochondrial membrane potential (MMP) variations observed after B10 treatment led to changes in Bax/Bcl-2 ratio, cytochrome C release, and enhanced expression of cleaved caspase3, 9, PARP-1, and APAF-1. The B10 inhibited the proliferation of Raji cells by significantly downregulating the expression of KRAS, BTK, MDM2, P-JAK2, P-STAT3, PI3K, HDAC1/2, SIRT7, and EP300. The treatment upregulated the tumor suppressor genes PEBP1 and SAP18. Thus, the study could reveal the selective inhibitory effects of B10 on lymphoma, suggesting it as a probable innovative chemotherapeutic agent.

The Inhibition of Bruton Tyrosine Kinase Alleviates Acute Liver Failure via Downregulation of NLRP3 Inflammasome.

There is no effective treatment for acute liver failure (ALF) except for an artificial liver support system (ALSS) and liver transplant. Bruton tyrosine kinase (Btk) plays important immunoregulatory roles in the inflammatory diseases, but its possible function in ALF remains to be characterized. In this study, we detected the phosphorylation level of Btk in ALF mouse liver and analyzed the protective effects of Btk inhibitor on survival rate and liver damage in ALF mouse models. We measured the expression levels of various inflammatory cytokines in the ALF mouse liver and primary human monocytes. In addition, we examined the expression of the NLRP3 inflammasome in mouse models with or without Btk inhibition. Clinically, we observed the dynamic changes of Btk expression in PBMCs of ALSS-treated patients. Our results showed that Btk was upregulated significantly in the experimental ALF mouse models and that Btk inhibition alleviated liver injury and reduced the mortality in these models. The protective effect of Btk inhibitors on ALF mice partially depended on the suppression of NLRP3 inflammasome signaling. Clinical investigations revealed that the dynamic changes of Btk expression in PBMCs could predict the effect of ALSS treatment. Our work shows that Btk inhibition is an effective therapeutic strategy for ALF. Moreover, Btk is a useful indicator to predict the therapeutic effect of ALSS on liver failure, which might have great value in clinical practice.

A Prognostic Nomogram Based on Response to Bortezomib and BTK Expression for Treatment-Experienced Multiple Myeloma Patients

To establish a prognostic nomogram based on response to bortezomib and BTK expression for treatment-experienced multiple myeloma patients. The Oncomine database was utilized to determine BTK expression, sex, age, albumin, Mayo index, response to bortezomib treatment, follow-up time and survival status in multiple myeloma(MM) patients. Cut-off point for BTK expression was calculated using R software. Univariate and multivariate analyses by Cox proportional hazards regression were then performed. Significant prognostic factors were combined to build a nomogram. The discrimination ability and predictive accuracy of the nomogram were evaluated using the index of concordance (C-index) and calibration curves. Multivariate analysis showed that response to bortezomib, BTK expression and sex were independent risk factors for prognosis. The C-index value of the nomogram made according to the independent risk factors was 0.729 (95%CI, 0.642-0.8164). The calibration curves showed good consistency between predicted and actual survivals for 1-year and 2-year overall survival. The proposed nomogram is accurate in predicting the prognosis of patients with MM.

Bruton’s Tyrosine Kinase in Neutrophils Is Crucial for Host Defense against Klebsiella pneumoniae

Humans with dysfunctional Bruton’s tyrosine kinase (Btk) are highly susceptible to bacterial infections. Compelling evidence indicates that Btk is essential for B cell-mediated immunity, whereas its role in myeloid cell-mediated immunity against infections is controversial. In this study, we determined the contribution of Btk in B cells and neutrophils to host defense against the extracellular bacterial pathogen Klebsiella pneumoniae, a common cause of pulmonary infections and sepsis. Btk−/− mice were highly susceptible to Klebsiella infection, which was not reversed by Btk re-expression in B cells and restoration of natural antibody levels. Neutrophil-specific Btk deficiency impaired host defense against Klebsiella to a similar extent as complete Btk deficiency. Neutrophil-specific Btk deficiency abolished extracellular reactive oxygen species production in response to Klebsiella. These data indicate that expression of Btk in neutrophils is crucial, while in B cells, it is dispensable for in vivo host defense against K. pneumoniae.

Computational and Mechanistic Study of the Therapeutic Potential of Compound Formononetin in Preventing Mast Cell Activation and IgE Production in Food Anaphylaxis

The prevalence of allergic diseases such as Asthma, Rhinitis, Anaphylaxis, Eczema, Urticaria, and Angioedema have been rising dramatically in low‐ and middle‐income countries around the world. According to American College of Allergy, Asthma &amp; Immunology, allergies are the 6th leading cause of chronic illness in the U.S. and more than 50 million Americans suffer from allergies each year. Extensive data suggest that food allergies affect up to 10% of the world population and have been increasing in the last two to three decades. Globally 300 million people have asthma and about 200 to 250 million people suffer from food allergies. Previously we have shown that a small molecule compound, formononetin have shown decreases IgE production from B cells. We hypothesize that formononetin will be a potential candidate for preventing food anaphylaxis in food allergy by regulating molecular pathways that involved in IgE production in B cells, mast cell activation and degranulation. The objective of this study is to reveal potential therapeutic mechanisms of formononetin in prevention and treatment of food anaphylaxis from food allergy, by using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein‐protein interaction analyses and in silico molecular docking to identify gene and protein targets that are regulated in food allergy and mast cells diseases. The targets identified were further validated using qRT‐PCR in B cell multiple myeloma cell line. U266 cells were cultured to 1.0 × 106 cells/mL and then incubated with formononetin at different concentrations of 20 μg/mL for 72 hours. Supernatants were collected for measuring IgE levels by ELISA and cell viability was determined using Trypan blue dye. mRNA expression was determined for the top targets identified and compared with GAPDH. Mining formononetin targets into obtained disease targets uncovers 25 targets for food allergy, 51 targets for IgE diseases and 19 targets for mast cell diseases. This targets where further used for gene ontology, KEGG pathway analysis, CTPD network and protein‐protein interaction. Top regulated KEGG pathways include the primary immunodeficiency pathway, Epstein Barr virus infection pathway, Th17 cell differentiation, PI3K‐Akt pathway. Regulated gene ontology biological processes include B cell proliferation, B cell differentiation, B cell activation and apoptotic process. Formononetin decreased IgE at after 72 hours, P &lt; 0.05 without cytotoxicity. Formononetin decreased mRNA expression of P53, TYK2, CASP8 significantly (P &lt; 0.05) and IgEH very significantly (P &lt; 0.01) and increased the mRNA expression of BCL2, NFKBIA and BTK significantly (P &lt; 0.05) after 72 hours in the culture group compared to the untreated group. These findings suggest that formononetin may be a novel therapeutic candidate for treatment and prevention of IgE/mast cell mediated food allergy and other allergic diseases.

Clinical, immunological and genomic characteristics of children with X-linked agammaglobulinemia from Kerala, South India

X-linked agammaglobulinemia (XLA) is an X-linked recessive primary immunodeficiency disorder caused due to a pathogenic variant in the Bruton tyrosine (BTK) gene with an incidence of 1:379,000 live births and 1:190,000 male births. Patients affected with XLA present with recurrent infections of the gastrointestinal and respiratory tracts. Here we report the first case series of 17 XLA patients of 10 South Indian families with a wide spectrum of clinical and genetic features. In our cohort, patients presented mainly with recurrent pneumonia, gastrointestinal infection, otitis media, pyoderma, abscesses, empyema, arthritis, and osteomyelitis. Using next-generation and Sanger sequencing we have identified 10 unique pathogenic and likely pathogenic variants in 17 patients. This encompasses three nonsynonymous, two stop-gain, two frameshifts, two structural, and one splicing variant, out of which two of them are novel. Based on the type of variant, patients had variable clinical features and treatment responses. We have also evaluated Btk protein expression for six patients in comparison to the healthy individuals and determined mosaic Btk expression patterns in four mothers. We have also performed family screening in 6 families using Sanger sequencing and identified 19 carriers for the variant. The diagnosis for the patients led to the proper treatment i.e. 15 patients were on intravenous immunoglobulin (IVIG) and the other two had successful hematopoietic stem cell transplantation (HSCT). Unfortunately, two of our patients died due to sepsis, while on IVIG. We envision the present study could help in better understanding of patients with XLA and help in family screening and prenatal diagnosis. To the best of our knowledge, this is the largest case series of patients affected with XLA from South India.