Phosphorylation Of BTK Research Articles

Tuning the B-CLL microenvironment: evidence for BAG3 protein- mediated regulation of stromal fibroblasts activity

The Bcl2-associated athanogene-3 (BAG3) protein, a critical regulator of cellular survival, has been identified as a potential therapeutic target in various malignancies. This study investigates the role of BAG3 within stromal fibroblasts and its interaction with B-cell chronic lymphocytic leukemia (B-CLL) cells. Previous research demonstrated that BAG3 maintains the active state of pancreatic stellate cells (PSCs) and aids pancreatic ductal adenocarcinoma (PDAC) spread via cytokine release. To explore BAG3’s role in bone marrow-derived stromal fibroblasts, BAG3 was silenced in HS-5 cells using siRNA. In co-culture experiments with PBMCs from B-CLL patients, BAG3 silencing in HS-5 cells increased apoptosis and decreased phosphorylation of BTK, AKT, and ERK in B-CLL cells, thus disrupting their pro-survival key signaling pathways. The observation of fibroblast-activated protein (FAP) positive cells in infiltrated bone marrow specimens co-expressing BAG3 further support the involvement of the protein in fibroblast-mediated tumor survival. Additionally, BAG3 appears to support B-CLL survival by modulating cytokine networks, including IL-10 and CXCL12, which are essential for leukemic cell survival and proliferation. A robust correlation between BAG3 expression and the levels of CXCL12 and IL-10 was observed in both co-cultures and patient specimens. These findings point out the need for a more in-depth comprehension of the intricate network of interactions within the tumor microenvironment and provide valuable insights for the selection of new potential therapeutic targets in the medical treatment of CLL.

#1261 A small-molecule inhibitor of Bruton's tyrosine kinase reduces production of galactose-deficient IgA1 in IgA nephropathy

Abstract Background and Aims IgA nephropathy (IgAN) is a chronic kidney disease characterized by glomerular immunodeposits that contain aberrantly glycosylated IgA1, i.e., IgA1 with some hinge-region O-glycans deficient in galactose (galactose-deficient IgA1; Gd-IgA1). These immunodeposits originate from circulating immune complexes that contain Gd-IgA1 bound by IgG autoantibodies. Consistent with this hypothesis, serum levels of Gd-IgA1 and IgG autoantibodies independently predict disease progression in IgAN patients. Biochemical studies using IgA1-secreting cell lines derived from peripheral blood of IgAN patients and healthy controls have revealed that Gd-IgA1 production is driven by altered biosynthetic pathways of IgA1 O-glycans. Specifically, cell lines from IgAN patients have altered expression and activity of several glycosyltransferases, C1GalT1 and ST6GalNAc2, and a C1GalT1-specific chaperone, C1GalT1C1. Genetic studies have shown that serum levels of Gd-IgA1 are genetically co-determined and genome-wide association studies (GWAS) identified genetic variations that impact the expression C1GALT1 and C1GALT1C1 genes. Moreover, macroscopic hematuria during an upper-respiratory-tract infection is common at the clinical onset of IgAN and during episodes of accentuated disease activity, indicating a connection between IgAN and mucosal inflammation. Notably, a pro-inflammatory cytokine interleukin-6 (IL-6), enhances production of Gd-IgA1 exclusively in the cell lines derived from IgAN patients. Leukemia inhibitory factor (LIF) has similar effects. LIF-induced effects are mediated by abnormal STAT1 signaling and pathways that include Lyn, a Src-family kinase. The corresponding gene, LYN, was recently identified in one of the 16 new IgAN-associated GWAS loci. Lyn, together with spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk), is involved in B-cell-receptor (BCR)-signaling pathways that include activation and inhibitory signals. The aim of this study was to assess if Btk may be involved in Gd-IgA1 production in IgAN. Method IgA1-producing cell lines were cloned by limiting dilution of Epstein-Barr-virus-immortalized peripheral-blood B cells from patients with IgAN (n = 5) and healthy controls (n = 5). Cell signaling, induced by LIF or by BCR crosslinking with anti-IgA antibody, was assessed by flow cytometry or SDS-PAGE/immunoblotting using antibodies for specific phosphorylated sites of selected proteins. Data were normalized using antibodies for specific proteins. Total IgA and Gd-IgA1 were quantified in cell-culture medium using standard ELISA and lectin-ELISA, respectively. Gd-IgA1, normalized to IgA, was expressed in Units (U) of standard Gd-IgA1 protein. Results IgA1-producing cell lines from patients with IgAN and healthy controls secreted similar amounts of total IgA (17.2 ± 9.3 ug/ml vs. 14.6 ± 9.5 ug/ml), but the cells from IgAN patients secreted more Gd-IgA1 (24.2 ± 7.5 U vs. 15.5 ± 2.7 U; p < 0.05). Baseline phosphorylation of Btk (Y223), but not Syk (Y551), was higher in the cells from patients with IgAN than in those from healthy controls. We used an IgA1-secreting cell line from an IgAN patient to confirm that BCR crosslinking with F(ab’)2 anti-IgA antibody in IgA + CD19+ cells lines induced phosphorylation of Btk (Y223). Conversely, anti-IgM antibody, used as a negative control, did not activate Btk. LIF stimulation did not affect phosphorylation of Btk or Syk. A Btk inhibitor, PCI-32765 (ibrutinib), reduced Gd-IgA1 production in a dose-dependent fashion exclusively in the IgAN-derived cells. Notably, 1 uM PCI-32765 reduced Gd-IgA1 production to that for healthy controls (25.7 ± 1.2 U to 17.3 ± 1.8 U; p < 0.01). Conclusion Inhibition of Btk with a small-molecule inhibitor normalized Gd-IgA1 production in IgA1-secreting cells from patients with IgAN, revealing a previously unknown role of Btk in aberrant O-glycosylation of IgA1. Btk may thus represent a potential new target for disease-specific therapy of IgAN.

Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbβ3 Integrin Activation and Degranulation.

Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase Cγ 2 (PLCγ2) and protein kinase C (PKC). Here, the activation sequence, crosstalk, and downstream effects of this Syk-Btk-PKC signalosome in human platelets were analyzed. Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk, or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT, and PLCγ2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk, and downstream kinases MEK1/2, Erk1/2, p38, and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLCγ2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182, and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib, but not GF109203X, suppressed convulxin-induced intracellular Ca2+ mobilization, whereas all three protein kinase inhibitors abolished degranulation and αIIbβ3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation. Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk, and PKC controls platelet granule exocytosis and αIIbβ3 integrin activation.

498 Bruton’s tyrosine kinase (BTK) inhibitors impede platelet aggregation but not adhesion to collagen.

OBJECTIVES/GOALS: The research objectives of this project are to elucidate the effects of Bruton’s tyrosine kinase inhibitors (BTKi) of varying target specificity on platelet function with regard to platelet aggregation, adhesion, spreading, and intracellular signaling as measured by kinase phosphorylation. METHODS/STUDY POPULATION: Blood from healthy volunteers was obtained and processed to obtain both washed platelets and platelet-rich plasma. The samples were then treated with one of the BTKi drugs or with vehicle (DMSO) at concentrations matching patient blood concentrations derived from clinical trials and pharmacokinetic studies. The incubated samples were then analyzed in an aggregometer using one of several agonists. Aggregation was stopped after five minutes with a perchloric acid-based lysis buffer. The samples were then analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation. Adhesion was assessed by incubating washed platelets treated with BTKi on microtiter wells coated with fibrinogen or collagen and quantifying adherent platelets by their endogenous acid phosphatase activity. RESULTS/ANTICIPATED RESULTS: We found that ibrutinib, zanubrutinib, and pirtobrutinib all completely inhibited collagen-induced platelet aggregation, whereas they did not inhibit aggregation induced by thrombin, ristocetin, arachidonic acid, or the PAR1 activator peptide SFLLRN (T6). Acalabrutinib inhibited collagen-induced platelet aggregation only at high concentrations (1-2 micromolar). At the lower concentration of 200 nanomolar, comparable to the concentration required for the other BTK inhibitors to completely inhibit platelet aggregation, acalabrutinib failed to inhibit aggregation but did inhibit auto-phosphorylation, indicating an impact on signaling. None of the BTKi drugs inhibited adhesion of platelets to collagen-coated surfaces. DISCUSSION/SIGNIFICANCE: Our data show the inhibitory effect of BTKi on collagen-induced platelet aggregation and signaling. However, it remains unclear whether the inhibition is due to an effect on BTK itself or other related kinases. Better insight into the mechanisms of platelet inhibition by BTKi may help guide the development of BTKi with a lower risk of hemorrhage.

Abstract 7586: TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL)

Abstract DLBCL originate from germinal center B cells (GCB). The ABC-DLBCL subtype, characterized by activation of B-cell receptor (BCR) signaling and downstream NF-kB pathway, carries a poor outcome. Up to 30% of ABC-DLBCL present TOXLOF mutations, suggesting that TOX function as a tumor suppressor. Our analysis of TOX expression along the germinal center B-cell differentiation program (naïve → GCB → plasma cells) showed that TOX expression paralleled NF-kB activity with highest levels in plasma cells (P<0.001, vs. either naïve and GCB). We thus propose that TOX acts as a transcriptional gatekeeper in GCB cells to prevent early activation of NF-kB thus enabling differentiation into plasma cells while restraining lymphomagenesis. We generated a conditional GCB TOX KO mouse model, induced the GC reaction with a T-cell antigen and found that TOX KO (vs. TOX WT, 10 mice per condition) had impaired differentiation of plasma cells (p<0.001) and hyperproliferative Ki67+ GCB cells (p<0.05). We then generated (by CRISPR) an isogenic TOXLOF ABC-DLBCL cell line (TMD8) and found upregulation of genes in the NF-kB pathway (q-value <0.05, vs. TMD8). Furthermore, TMD8-TOXLOF cells showed increased secretion of autocrine cytokines IL6 and IL10, and higher STAT3 phosphorylation (p<0.05 for all comparisons, vs. TMD8-TOXWT cells) all compatible with NF-kB activation. Tumor microenvironmental cues can hyperactivate BCR and downstream NF-kB in ABC-DLBCL. We thus implanted isogenic TMD8 cells in mice (n = 10) and determined pathway activation by phospho-flow. We found massive activation of SYK, BTK, PLCG2 (from the BCR) and p65 (from NF-kB) in TMD8-TOXLOF vs. TMD8-TOXWT tumors (all p<0.01), and vs. these cell lines cultured in vitro (all p<0.01). To determine whether these features can be therapeutically exploited, we conducted a viability screening using a panel of 20 clinical-phase targeted therapies relevant to ABC-DLBCL. We found that BTK inhibitor acalabrutinib (BCR pathway) and MALT1 inhibitor JNJ67690246 (NF-kB pathway) were 2-3 fold more active in TMD8-TOXLOF vs. TMD8-TOXWT cells (all p<0.05). We then xenografted isogenic TMD8 cells (n = 10 mice per condition) and, when tumors reached 150 mm3, we randomized mice to vehicle vs. acalabrutinib 30 mg/kg/day by oral gavage (human dose equivalent). Acalabrutinib was significantly more active in TMD8-TOXLOF tumors (vs. TMD8-TOXWT, p=0.0008), which translated into significantly prolonged survival (p<0.05). This acalabrutinib effect was accompanied by significant reduction of BTK, SYK and PLCG2 phosphorylation in TMD8-TOXLOF tumors (p<0.0001, all comparisons). Thus, TOXLOF leads to hyperactivation of the BCR and NF-kB signaling pathways in ABC-DLBCL cells, rendering them hypersensitive to BCR-targeted therapies, delineating a potential mechanism-based therapeutic approach for ABC-DLBCL TOXLOF patients. Citation Format: Eloisi Caldas Lopes, Maria Revuelta, Rossella Marullo, Leandro Cerchietti. TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7586.

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.

Bruton’s tyrosine kinase is a possible therapeutic target in microscopic polyangiitis

BackgroundBruton’s tyrosine kinase (Btk) is an enzyme expressed in leukocytes other than T lymphocytes and plasma cells and involved in B-cell receptor- and Fcγ receptor (FcγR)-mediated signal transduction. Btk inhibitors potentially suppress autoantibody production due to the expected inhibitory ability of B lymphocyte differentiation into antibody-producing plasma cells and reduce FcγR-mediated neutrophil activation, including the release of neutrophil extracellular traps (NETs). Microscopic polyangiitis (MPA) is a systemic small-vessel vasculitis characterized by the pathogenic autoantibody, antineutrophil cytoplasmic antibody (ANCA) that reacts with myeloperoxidase (MPO). MPO and MPO-ANCA immune complex (IC)-induced FcγR-mediated NETs are critically involved in MPA pathogenesis. This study aimed to demonstrate the therapeutic efficacy of the Btk inhibitor tirabrutinib on MPA.MethodsVarious doses of tirabrutinib or vehicle were orally administered to Sprague–Dawley rats daily. Four weeks later, the number of peripheral B lymphocytes was counted, and Btk phosphorylation in B lymphocytes was evaluated by flow cytometry. Human peripheral blood neutrophils were stimulated by MPO and anti-MPO antibody ICs (MPO and anti-MPO-ICs), and Btk and its downstream Vav phosphorylation were assessed by western blotting. The effects of tirabrutinib on MPO and anti-MPO-IC-induced NET formation were examined in vitro. Wistar Kyoto rats were immunized with human MPO to induce experimental MPA and given drug-free or tirabrutinib-containing feed (0.0037% or 0.012%) from day 0 or 28. All rats were euthanized on day 42 for serological and histological evaluation.ResultsTirabrutinib inhibited Btk phosphorylation without decreasing B lymphocytes in vivo. Neutrophil Btk and Vav were phosphorylated when stimulated with MPO and anti-MPO-ICs. Tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA in a dose-dependent manner in vivo. Although MPO-ANCA production was not affected, NET-forming neutrophils in the blood were significantly reduced by tirabrutinib.ConclusionsThe Btk inhibitor tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA by reducing NET-forming neutrophils but not decreasing MPO-ANCA titer in vivo. This study suggests that Btk is a possible therapeutic target in MPA.

Targeting Covalent and Non-Covalent Btki-Resistant CLL Using the Dual Irreversible/Reversible 4 th Generation BTK Inhibitor LP-168

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p<0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p<0.001, n=10 and 51%, p<0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p<0.001, n=8; CCL4: 93%, p<0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p<0.0001, n=3; T474I: 99%, p<0.0001, n=3) and CCL4 (C481S: 97%, p<0.0001, n=3; T474I: 98%, p<0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p<0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).

Anlotinib Inhibits Proliferation and Induces Apoptosis in B-cell Acute Lymphoblastic Leukemia by Targeting the BTK and AKT/mTOR Pathway.

Acute lymphoblastic leukemia (ALL) is the second most common acute leukemia in adults, whose known drug treatments are limited and expensive. This investigation aimed to investigate the therapeutic potential of anlotinib in B-cell acute lymphoblastic leukemia (B-ALL). The B-ALL cell lines Nalm-6 and BALL-1 were used to verify the therapeutic potential of anlotinib in BALL. The cell activity was measured by Cell Counting Kit-8. Apoptosis was detected by Annexin V-FITC/PI double staining combined with flow cytometry. Afterward, the binding capacity of anlotinib to the critical protein was predicted by molecular docking, and the protein changes in the related pathways downstream of the target proteins were verified by western blot. Finally, the effect of anlotinib on the survival rate was verified in B-ALL nude mice. Anlotinib inhibited the proliferation of the B-ALL cell lines, Nalm-6, and BALL-1, and promoted apoptosis. Molecular docking results showed that it had the potential binding ability to BTK. Western blot revealed that anlotinib was able to inhibit the phosphorylation of BTK, AKT, and mTOR, thereby inhibiting the proliferation of B-ALL cells. In addition, anlotinib suppressed weight loss and prolonged the survival time of mice. To summarize, anlotinib can inhibit the proliferation of B-ALL and promotes apoptosis by inhibiting the phosphorylation of BTK and AKT, and mTOR.

Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets.

Bruton's tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.

Abstract 2780: Unique pharmacodynamic properties conferred by differential binding to BTK, pirtobrutinib vs covalent inhibitors

Abstract Covalent Bruton tyrosine kinase inhibitors (cBTKi) have transformed the treatment of B cell malignancies. Despite the efficacy of cBTKi, treatment failure often occurs through development of resistance or intolerance. Pirtobrutinib, a highly selective, non-covalent BTKi, potently inhibits both WT and C481 mutant BTK. Pirtobrutinib has favorable oral pharmacology, is well tolerated, and has shown promising efficacy in patients with poor prognosis B cell malignancies following prior therapy, including prior cBTKi. Here we report in vitro BTK binding, conformation, and activation differences between cBTKi and pirtobrutinib. Cellular studies showed pirtobrutinib inhibited BTK phosphorylation at both Y223 and Y551 in REC-1 and Ramos A1 human lymphoma cell lines. In contrast, despite inhibition of Y223 phosphorylation, both cell lines treated with cBTKi, ibrutinib, acalabrutinib or zanubrutinib, retained Y551 phosphorylation. The differential effects on Y551 were also observed in PBMC isolated from treatment naïve human CLL donors treated in vitro with pirtobrutinib or ibrutinib. To investigate the binding effects of cBTKi and pirtobrutinib on BTK stability and conformation, a series of biophysical and structural studies were performed. In a hydrogen/deuterium exchange mass spectrometry study using full length BTK, all compounds significantly inhibited exchange in regions surrounding the ATP binding site in the kinase domain. While cBTKi induced significantly increased exchange for peptides in the SH3 and SH2 domains, pirtobrutinib significantly reduced exchange in a portion of the SH3 domain, nearly the entire SH2 domain, and SH2 linker, suggesting that pirtobrutinib uniquely stabilizes BTK in a closed/inactive conformation. Consistent with this proposed model, crosslinking mass spectrometry results of inhibitor bound BTK showed that pirtobrutinib promoted different interactions between the SH2 domain and kinase domain than cBTKi. To confirm that pirtobrutinib promotes greater stabilization of BTK, a mass spectrometry based cellular thermal shift assay was performed in both Ramos and BTK overexpressing HEK293 cells. In both cell lines, pirtobrutinib showed significantly greater stabilization of BTK compared to cBTKi. In addition, crystallographic studies of BTK in complex with pirtobrutinib revealed domain organization consistent with stabilized SH2-SH3 interactions with the kinase domain. These data suggest pirtobrutinib may differentially impact BTK stability and conformation compared to cBTKi. The cellular and biophysical data are consistent with a model in which cBTKi shift BTK toward an open conformation, whereas pirtobrutinib allosterically stabilizes a closed BTK conformation, preventing activation by upstream kinases. Additional studies are underway to further characterize the differential effects of pirtobrutinib on BTK conformation. Citation Format: Joshua A. Ballard, Kevin Ebata, Hetal S. Randeria, Garrett Tinline, Thomas Lee, Lauren M. Hanson, John A. Latham, E. Peder Cedervall, Jenny Chong, Kyle B. Del Valle, Bernard C. Collins, Tony H. Morales, Thomas C. Benedict, Marc A. Schureck, Ethan T. Bender, Christopher Mendoza, David Molina, Meagan Nakamoto, Hsiao-Chiao Shiah, Hao Xu, Alfonso Espada, Leticia Cano, Charles K. Allerston, Paul Schnier, Barbara J. Brandhuber. Unique pharmacodynamic properties conferred by differential binding to BTK, pirtobrutinib vs covalent inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2780.

Luxeptinib interferes with LYN-mediated activation of SYK and modulates BCR signaling in lymphoma.

Luxeptinib (LUX) is a novel oral kinase inhibitor that inhibits FLT3 and also interferes with signaling from the BCR and cell surface TLRs, as well as activation of the NLRP3 inflammasome. Ongoing clinical trials are testing its activity in patients with lymphoma and AML. This study sought to refine understanding of how LUX modulates the earliest steps downstream of the BCR following its activation by anti-IgM in lymphoma cells in comparison to ibrutinib (IB). LUX decreased anti-IgM-induced phosphorylation of BTK at Y551 and Y223 but its ability to reduce phosphorylation of kinases further upstream suggests that BTK is not the primary target. LUX was more effective than IB at reducing both steady state and anti-IgM-induced phosphorylation of LYN and SYK. LUX decreased phosphorylation of SYK (Y525/Y526) and BLNK (Y96) which are necessary regulators of BTK activation. Further upstream, LUX blunted the anti-IgM-induced phosphorylation of LYN (Y397) whose activation is required for phosphorylation of SYK and BLNK. These results indicate that LUX is targeting autophosphorylation of LYN or a step further upstream of LYN in the cascade of signal generated by BCR and that it does so more effectively than IB. The fact that LUX has activity at or upstream of LYN is important because LYN is an essential signaling intermediate in multiple cellular signaling processes that regulate growth, differentiation, apoptosis, immunoregulation, migration and EMT in normal and cancer cells.

The immunomodulatory molecule TIGIT is expressed by chronic lymphocytic leukemia cells and contributes to anergy.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory checkpoint receptor that negatively regulates Tcell responses. CD226 competes with TIGIT for binding to the CD155 ligand, delivering a positive signal to the T cell. Here we studied the expression of TIGIT and CD226 in a cohort of 115 patients with chronic lymphocytic leukemia (CLL) and report expression of TIGIT and CD226 by leukemic cells. By devising a TIGIT/CD226 ratio, we showed that CLL cells favoring TIGIT over CD226 are typical of a more indolent disease, while those favoring CD226 are characterized by a shorter time to first treatment and shorter progression-free survival after first treatment. TIGIT expression was inversely correlated to the B-cell receptor (BCR) signaling capacity, as determined by studying BTK phosphorylation, cell proliferation and interleukin- 10 production. In CLL cells treated with ibrutinib, in which surface IgM and BCR signaling capacity are temporarily increased, TIGIT expression was downmodulated, in line with data indicating transient recovery from anergy. Lastly, cells from patients with Richter syndrome were characterized by high levels of CD226, with low to undetectable TIGIT, in keeping with their high proliferative drive. Together, these data suggest that TIGIT contributes to CLL anergy by downregulating BCR signaling, identifying novel and actionable molecular circuits regulating anergy and modulating CLL cell functions.

Resistance to the Non-Covalent BTK Inhibitor Pirtobrutinib

Pirtobrutinib is a non-covalent BTK inhibitor (BTKi) designed to maintain activity despite the most common resistance mutation to covalent inhibitors, at BTK C481. To investigate mechanisms of disease progression on pirtobrutinib, we evaluated the effect of pirtobrutinib in vitro on the BCR pathway in pre- and post-treatment patient samples (n=5), and on 2 of these patients, we performed whole exome sequencing longitudinally including prior to and at relapse on pirtobrutinib. Phylogenetics and subclonal dynamics associated with resistance were evaluated using the PhylogicNDT and Concerti tools. To investigate the impact of identified BTK mutations on BCR activation, we generated 6 single and 3 double mutants using site directed mutagenesis and expressed them in the BTK null DT40 B cell line. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, reduced CCL3/CCL4 chemokine secretion, as well as effective induction of apoptosis and inhibition of proliferation, in response to pirtobrutinib. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition and cytokine production, with reduced inhibition of proliferation and induction of apoptosis. In WES analysis, patient #1 had 16 samples evaluated prior to, during, and at relapse on acalabrutinib, vecabrutinib and pirtobrutinib. Clonal analysis of samples collected during acalabrutinib shows steady selection of a clone harboring BTK p.C481S mutation with CCF 92% at relapse but then steadily decreases during pirtobrutinib treatment. Concerti's time-scaled phylogenetic tree shows the birth of a new clone containing the BTK gatekeeper mutation, p.T474I, during acalabrutinib treatment, which then grows rapidly under pirtobrutinib treatment, taking over nearly the entire cancer cell population and replacing the prior p.C481S clone. This complete clonal shift during pirtobrutinib treatment suggests that pirtobrutinib effectively inhibits the p.C481S clone, while the p.T474I gatekeeper clone is likely driving resistance in this patient. We also observed an additional gatekeeper clone BTK p.T474L develop at low levels, as well as another previously undescribed BTK mutation at p.M477I. Manual inspection showed that BTK mutations p.M477I and p.T474I are in cis and therefore in the same clone. Patient #2 had 10 samples evaluated prior to, during and at relapse on ibrutinib and pirtobrutinib. During ibrutinib therapy, we observed a steady increase in a clone with TP53 p.S240G and SF3B1 p.K666N mutations, reaching CCFs >40% at relapse on ibrutinib. We also noted a significant increase in BTK p.C481R (CCF 33%), BTK p.C481S (CCF 11%) and TP53 p.R196* (CCF 5%) at progression on ibrutinib. Under pirtobrutinib treatment the clone carrying BTK p.C481R decreased to CCF 20%, while BTK p.C481S (28%) and TP53 p.R197* (35%) both increased. Concerti's phylogenetic tree captures the birth of a resistant clone, harboring BTK p.L528W which significantly increases to CCF 30% at progression on pirtobrutinib. Functional characterization of the identified BTK mutations demonstrated that only T474I, T474L and C481S mutants showed adequate kinase activity, while all the other mutants including M477I, C48IR and L528W essentially lacked kinase activity as judged by phosphorylation at BTK Y223 and at PLCG2 Y753. As expected, the C481S variant was resistant to ibrutinib, but not to pirtobrutinib, while the T474I/L variants were sensitive to ibrutinib but resistant to pirtobrutinib. Interestingly, phosphorylation of AKT and ERK were retained downstream, even with mutations that failed to activate proximal BCR signaling. Furthermore, phosphorylation of AKT and ERK was also observed in the B7.10 cell line which lacks endogenous BTK, demonstrating significant activation of these pathways independent of BTK. In this study, we demonstrate that ex vivo efficacy of pirtobrutinib declines as patients’ CLL starts to progress, in concert with the development of gatekeeper and alternative site BTK mutations that lead to resistance to pirtobrutinib. Interestingly, many of the second-site BTK mutations fail to activate BTK phosphorylation but are still associated with downstream activation of phospho-AKT and phospho-ERK; the mechanism of this activation remains to be elucidated.

Bruton's Tyrosine Kinase Inhibitors Impair FcγRIIA-Driven Platelet Responses to Bacteria in Chronic Lymphocytic Leukemia.

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton’s tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbβ3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro. While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbβ3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions.

The Tigit/CD226/CD155 Immunomodulatory Axis Is Deregulated in CLL and Contributes to B-Cell Anergy

The Tigit/CD226/CD155 Immunomodulatory Axis Is Deregulated in CLL and Contributes to B-Cell Anergy

SHP1 Loss Augments DLBCL Cellular Response to Ibrutinib: A Candidate Predictive Biomarker

BackgroundB-cell receptor (BCR) signaling plays a key role in B-cell non-Hodgkin lymphoma (NHL). Protein tyrosine kinases such as LYN, SYK and BTK are essential in the activation of BCR leading to B-cell survival, proliferation and differentiation. SHP1 is a cytosolic protein tyrosine phosphatase functioning as a negative regulator of the BCR signaling cascade. It dephosphorylates and counteracts the activities of the tyrosine kinases, including CD79A/CD79B, SYK and BLNK (Curr. Opin. Immunol.12, 307).Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive NHL. DLBCL demonstrates heterogeneity in clinical presentation, genomic alterations and cell signaling. BCR signature has been identified in a subset of DLBCL (Monti Blood 105, 1851). Chronic active BCR plays a particular role in the subtype of activated B-cell like (ABC) DLBCL by cell-of-origin subclassification. Based on the success of ibrutinib in other B-cell NHL, clinical trials of ibrutinib in DLBCL was conducted and the initial results showed a 37% response rate in ABC-lymphomas vs 5% in GCB-DLBCL (Wilson Nat Med 21, 922). However, a large phase 3 clinical trial of ibrutinib-containing regimen in >800 non-GCB patients did not find any substantial difference in response between those received ibrutinib vs placebo. Thus, biomarker based on cell-of-origin did not seem to help stratify patients as expected (Younes JCO 27, 1285). New predictive biomarkers are needed to identify patients who likely benefit from ibrutinib or other BCR-directed therapeutic agents.We hypothesized that SHP1 may play a role in defining the level of the activity of the BCR pathway. Suppressed SHP1 expression in various types of NHL has been reported. We hypothesized that lack of SHP1 may contribute to the hyperactivity of BCR in DLBCL; and tumors with low SHP1 may respond better to ibrutinib. In this work, we set out to explore the relationship between SHP1 & ibrutinib sensitivity in DLBCL.Methods and ResultsWe first reviewed the published literature on SHP1 in NHL using a meta-analysis to provide a quantitative and thus unbiased analysis. Seventeen articles were selected based on criteria including inclusion of primary tumors with normal benign controls. The analysis of the SHP1 protein expression by immunohistochemistry (IHC) revealed that lack of SHP1 expression is found in 178 of 242 (73.6%) NHL tissues vs. 32 of 264 (12%) normal/benign controls. The odds ratio derived by the meta-analysis was 119.58 with a 95% confidence interval of 37.60-380.15, suggesting loss of SHP1 is strongly associated with NHL including DLBCL. Likewise, the analysis of the methylation studies revealed a strong association between SHP1 promoter hypermethylation and NHL (OR 69.46, 95% CI 12.32-391.74).To further understand the range of variability of SHP1 expression in subtypes of DLBCL tumors, we constructed a tissue microarray with 48 DLBCL tumors and 12 cell lines. By immunohistochemistry, we found a wide range of variability with regards to the expression levels and no substantial difference between the GCB and non-GCB subtypes.Using 12 cell lines together with resting B-cells from normal donors, we identified a strong reverse linear correlation between SHP1 protein expression and promoter methylation (r = 0.85, P=0.0002) suggesting that promoter hypermethylation may be responsible for reduced SHP1 expression in DLBCL cells.Using CRISPR, we knocked out SHP1 in both GCB and ABC cell lines. The phosphorylation of early BCR components, BTK, SYK and LYN, were increased in the SHP1 knockout clones compared to the wild type ones. The results demonstrate that SHP1 depletion results in increased proximal BCR signaling activity. Moreover, the knock out clones demonstrated increased sensitivity of ibrutinib. We further showed that re-introduction of SHP1 vector to the knockout clones via stable lentiviral transduction restored normal sensitivity of cells to ibrutinib.ConclusionsTogether, our results show that SHP1 expression is suppressed in a substantial fraction of DLBCL tumors regardless of cell-of-origin subclassifications. Using genetic approaches, we demonstrated that loss of SHP1 increases BCR signaling activity and sensitizes tumor cells to the inhibition by ibrutinib. Our results suggest that loss of SHP1 may be used as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment. DisclosuresWang: Incyte Research Institute: Ended employment in the past 24 months.

The BTK/PI3K/BRD4 axis inhibitor SRX3262 overcomes Ibrutinib resistance in mantle cell lymphoma

SummaryMantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma and one of the most challenging blood cancers to combat due to frequent relapse after treatment. Here, we developed the first-in-class BTK/PI3K/BRD4 axis inhibitor SRX3262, which simultaneously blocks three interrelated MCL driver pathways – BTK, PI3K-AKT-mTOR and MYC. SRX3262 concomitantly binds to BTK, PI3K, and BRD4, exhibits potent in vitro and in vivo activity against MCL, and overcomes the Ibrutinib resistance resulting from the BTK-C481S mutation. Our results reveal that SRX3262 inhibits IgM-induced BTK and AKT phosphorylation and abrogates binding of BRD4 to MYC loci. SRX3262 promotes c-MYC destabilization, induces cell cycle arrest and apoptosis, and shows antitumor activity in in vivo xenograft models. Together, our study provides mechanistic insights and rationale for the use of the triple BTK/PI3K/BRD4 activity inhibitors as a new approach to treat MCL.

Mutated MYD88 Activates the BCR Component SYK and Provides a Rationale Therapeutic Target in Waldenstrom's Macroglobulinemia

Background: Mutations in the Toll receptor (TLR) pathway are highly prevalent in Waldenstrom's Macroglobulinemia (WM) and ABC DLBCL, wherein mutated MYD88 triggers NF-kB pro-survival signaling through BTK/IRAK. Activation of B-cell receptor (BCR) signaling can also be triggered by activating mutations in CD79A/B in ABC DLBCL, though are rare in WM (5-8%). Despite these findings, there is evidence for BCR activation in WM (Argyropoulos et al, Leukemia 2016). We hypothesized that crosstalk between TLR and BCR might account for aberrant BCR signaling in WM. We therefore performed PhosFLow studies on MYD88 and BCR signaling components and observed the hyperactivation of SYK (pY525-pY526) in MYD88 mutated WM (BCWM.1, MWCL-1) cells compared to wild-type MYD88 expressing control cells. We next treated MYD88 mutated WM cells with a peptide inhibitor of MYD88 homodimerization, and observed that the phosphorylation of SYK at tyrosine 525 and 526 (pY525-pY526) together with phosphorylation of BTK at tyrosine 223 (pY223) was markedly reduced. In contrast, the MYD88 inhibitor had no impact on either SYK or BTK activation in MYD88 wild-type Ramos cells. Knockdown of MYD88 by lentiviral transduction in MYD88 mutated BCWM.1 cells confirmed the reduction of SYK (pY525-pY526) by two shRNAs. Cell viability analysis by CellTiter-Glo® assay showed that the SYK inhibitor R406 was cytotoxic to MYD88 mutated WM cells at pharmacologically attainable drug levels (EC50 0.247 uM for BCWM.1; 0.315 uM for MWCL-1), versus >1 uM for MYD88 wild-type control cells. Furthermore, the combination of R406 with ibrutinib produced synergistic cytotoxicity in MYD88 mutated WM cells. Conclusion: Our findings demonstrate that in addition to TLR path activation, mutated MYD88 activates the BCR component SYK tyrosine kinase. These findings provide the rationale for combined therapeutic targeting of BCR and TLR pathways in MYD88 mutated WM. DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Ibrutinib As a Possible Therapeutic Strategy for Epstein-Barr Virus-Positive Lymphoma/Leukemia

Epstein-Barr virus (EBV) is associated with many types of lymphoproliferative disorders. EBV-coded latent membrane protein 2A (LMP2A) functions in promoting B cell survival partly through Bruton's tyrosine kinase (Btk) -dependent signaling pathway. In the present study, we investigated whether a Btk inhibitor, Ibrutinib, shows therapeutic efficacy for EBV-related lymphoma. Ibrutinib has been already approved as a therapeutic drug for several B cell malignancies, such as chronic lymphocytic leukemia. Hence, once it shows efficacy, Ibrutinib could be quickly moved to clinical usage.First, we examined the effects of Ibrutinib on B cells transformed by EBV infection in vitro, called lymphoblastic cell lines (LCLs). We cultured LCLs in the presence or absence of Ibrutinib and analyzed cell survival using flow cytometry. As a result, Ibrutinib treatment decreased survival rate of the LCLs compared with a vehicle (Fig. A). Indeed, Btk phosphorylation was detected in the LCLs by western blot analysis. Collectively, Btk signal inhibition by Ibrutinib is probably lethal for EBV-LCLs. LCLs are classified as latency type III and have a polyclonal nature like lymphoproliferative disorders in post-transplant and HIV-associated immune suppressed individuals. Hence, Ibrutinib possibly be applicable to treatment for such disorders. To examine whether Ibrutinib is effective in vivo, we are now planning to administer Ibrutinib to EBV-infected humanized mice, which can be used as a model for EBV-related lymphoproliferative disorders (Yajima et al., J. Infect. Dis. 2008).Further, we obtained peripheral blood mononuclear cells (PBMCs) from EBV+ aggressive NK cell leukemia (ANKL) patient. Approximately 90% of the PBMCs were positive for NK cell marker CD56 and hence probably be leukemia cells. First, we examined in vitro effects of Ibrutinib on the PBMCs. The PBMCs were cultured in the presence or absence of Ibrutinib, and survival rate was analyzed using flow cytometry. Results showed that Ibrutinib treatment decreased survival of CD56High cells (Fig. B). Next, we examined Btk phosphorylation in the PBMCs using western blot. Unexpectedly, even total Btk was not detected. This suggests Ibrutinib-induced lethality of the ANKL cells is dependent on other pathways. Ibrutinib could target other kinases than Btk including those reported to be expressed in NK cells, such as ITK, TXK, and JAK3. Hence, it is possible that Ibrutinib decreases the ANKL cells through inhibiting these kinases.Next, we tried to establish a mouse model of ANKL xenograft for in vivo assays. We transplanted the PBMCs from ANKL patient into immunodeficient NOG mice after X-ray irradiation. Around 2 weeks after the transplantation, a large number of the transplanted PBMCs were detected at least in spleen and bone marrow. These ANKL cells in spleen and bone marrow could be successively transplanted into other NOG mice. After transplantation, the recipient mice gradually decreased their body weight and died in several weeks. Now, we are analyzing these mice in detail and are investigating the effects of Ibrutinib administration on the survival of these mice.Collectively, we found that Ibrutinib inhibits survival of EBV+ LCLs and ANKL cells in vitro . This raises a possibility that Ibrutinib can be used for EBV-related lymphoproliferative disorders and EBV+ ANKL, although we should confirm the effectiveness of Ibrutinib in vivo . [Display omitted] DisclosuresKotani:Janssen Pharma: Research Funding.