Bruton's Tyrosine Kinase Phosphorylation Research Articles

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL

AbstractThe t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre–B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=<0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=<0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

CXCR4-BTK axis mediate pyroptosis and lipid peroxidation in early brain injury after subarachnoid hemorrhage via NLRP3 inflammasome and NF-κB pathway

C-X-C chemokine receptor type 4 (CXCR4) is critical for homeostasis of the adaptive and innate immune system in some CNS diseases. Bruton's tyrosine kinase (BTK) is an essential kinase that regulates inflammation in immune cells through multiple signaling pathways. This study aims to explore the effect of CXCR4 and BTK on neuroinflammation in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Our results showed that the expression of CXCR4 and p-BTK increased significantly at 24 h after SAH in vivo and in vitro. Ibrutinib improved neurological impairment, BBB disruption, cerebral edema, lipid peroxidation, neuroinflammation and neuronal death at 24 h after SAH. Inhibition of BTK phosphorylation promoted the in vitro transition of hemin-treated proinflammatory microglia to the anti-inflammatory state, inhibited the p-P65 expression and microglial pyroptosis. NLRP3 deficiency can significantly reduce pyroptosis in SAH mice. Moreover, CXCR4 inhibition can suppress NLRP3-mediated pyroptosis, NF-κB activation and NOX2 expression in vitro, and ibrutinib can abolish CXCR4-aggravated BBB damage and pyroptosis in EBI after SAH. The levels of CXCR4 in CSF of SAH patients is significantly increased, and it is positively correlated with GSDMD and IL-1β levels, and have a moderate diagnostic value for outcome at 6-month follow-up. Our findings revealed the effect of CXCR4 and P-BTK on NLRP3-mediated pyroptosis and lipid peroxidation after SAH in vivo and in vitro, and the potential diagnostic role of CXCR4 in CSF of SAH patients. Inhibition of CXCR4-BTK axis can significantly attenuate NLRP3-mediated pyroptosis and lipid peroxidation by regulating NF-κB activation in EBI after SAH.

Platelet caspase‐1 and Bruton tyrosine kinase activation in patients with COVID‐19 is associated with disease severity and reversed in vitro by ibrutinib

BackgroundSeverity of coronavirus disease 2019 (COVID‐19) is often associated with thrombotic complications and cytokine storm leading to intensive are unit (ICU) admission. Platelets are known to be responsible for abnormal hemostasis parameters (thrombocytopenia, raised D‐dimers, and prolonged prothrombin time) in other viral infections through the activation of the nucleotide‐binding domain leucine repeat rich containing protein 3 inflammasome induced by signaling pathways driven by Bruton tyrosine kinase (BTK) and leading to caspase‐1 activation. ObjectivesWe hypothesized that caspase‐1 activation and the phosphorylation of BTK could be associated with the severity of the disease and that ibrutinib, a BTK inhibitor, could inhibit platelet activation. Methods and ResultsWe studied caspase‐1 activation by flow cytometry and the phosphorylation of BTK by Western blot in a cohort of 51 Afro‐Carribean patients with COVID‐19 disease (19 not treated in ICU and 32 treated in ICU). Patients with a platelet count of 286.7 × 109/L (69–642 × 109/L) were treated by steroids and heparin preventive anticoagulation. Caspase‐1 and BTK activation were associated with the severity of the disease and with the procoagulant state of the patients. Furthermore, we showed in vitro that the plasma of ICU patients with COVID‐19 was able to increase CD62P expression and caspase‐1 activity of healthy platelets and that ibrutinib could prevent it. ConclusionsOur results show that caspase‐1 and BTK activation are related to disease severity and suggest the therapeutic hope raised by ibrutinib in the treatment of COVID‐19 by reducing the procoagulant state of the patients.

Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib.

Recent clinical trials have shown promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti–VLA-4 antibody) treatment. Under in vitro T follicular helper–like conditions, BTK phosphorylation was enhanced by T-bet–inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet–associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

Inhibition of Bruton Tyrosine Kinase Reduces Neuroimmune Cascade and Promotes Recovery after Spinal Cord Injury.

Microglia/astrocyte and B cell neuroimmune responses are major contributors to the neurological deficits after traumatic spinal cord injury (SCI). Bruton tyrosine kinase (BTK) activation mechanistically links these neuroimmune mechanisms. Our objective is to use Ibrutinib, an FDA-approved BTK inhibitor, to inhibit the neuroimmune cascade thereby improving locomotor recovery after SCI. Rat models of contusive SCI, Western blot, immunofluorescence staining imaging, flow cytometry analysis, histological staining, and behavioral assessment were used to evaluate BTK activity, neuroimmune cascades, and functional outcomes. Both BTK expression and phosphorylation were increased at the lesion site at 2, 7, 14, and 28 days after SCI. Ibrutinib treatment (6 mg/kg/day, IP, starting 3 h post-injury for 7 or 14 days) reduced BTK activation and total BTK levels, attenuated the injury-induced elevations in Iba1, GFAP, CD138, and IgG at 7 or 14 days post-injury without reduction in CD45RA B cells, improved locomotor function (BBB scores), and resulted in a significant reduction in lesion volume and significant improvement in tissue-sparing 11 weeks post-injury. These results indicate that Ibrutinib exhibits neuroprotective effects by blocking excessive neuroimmune responses through BTK-mediated microglia/astroglial activation and B cell/antibody response in rat models of SCI. These data identify BTK as a potential therapeutic target for SCI.

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

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

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.

Target modulation and pharmacokinetics/pharmacodynamics translation of the BTK inhibitor poseltinib for model-informed phase II dose selection

The selective Bruton tyrosine kinase (BTK) inhibitor poseltinib has been shown to inhibit the BCR signal transduction pathway and cytokine production in B cells (Park et al.Arthritis Res. Ther.18, 91, https://doi.org/10.1186/s13075-016-0988-z, 2016). This study describes the translation of nonclinical research studies to a phase I clinical trial in healthy volunteers in which pharmacokinetics (PKs) and pharmacodynamics (PDs) were evaluated for dose determination. The BTK protein kinase inhibitory effects of poseltinib in human peripheral blood mononuclear cells (PBMCs) and in rats with collagen-induced arthritis (CIA) were evaluated. High-dimensional phosphorylation analysis was conducted on human immune cells such as B cells, CD8 + memory cells, CD4 + memory cells, NK cells, neutrophils, and monocytes, to map the impact of poseltinib on BTK/PLC and AKT signaling pathways. PK and PD profiles were evaluated in a first-in-human study in healthy donors, and a PK/PD model was established based on BTK occupancy. Poseltinib bound to the BTK protein and modulated BTK phosphorylation in human PBMCs. High-dimensional phosphorylation analysis of 94 nodes showed that poseltinib had the highest impact on anti-IgM + CD40L stimulated B cells, however, lower impacts on anti-CD3/CD-28 stimulated T cells, IL-2 stimulated CD4 + T cells and NK cells, M-CSF stimulated monocytes, or LPS-induced granulocytes. In anti-IgM + CD40L stimulated B cells, poseltinib inhibited the phosphorylation of BTK, AKT, and PLCγ2. Moreover, poseltinib dose dependently improved arthritis disease severity in CIA rat model. In a clinical phase I trial for healthy volunteers, poseltinib exhibited dose-dependent and persistent BTK occupancy in PBMCs of all poseltinib-administrated patients in the study. More than 80% of BTK occupancy at 40 mg dosing was maintained for up to 48 h after the first dose. A first-in-human healthy volunteer study of poseltinib established target engagement with circulating BTK protein. Desirable PK and PD properties were observed, and a modeling approach was used for rational dose selection for subsequent trials. Poseltinib was confirmed as a potential BTK inhibitor for the treatment of autoimmune diseases.Trial registration: This article includes the results of a clinical intervention on human participants [NCT01765478].

Bortezomib enhances the anti-cancer effect of the novel Bruton's tyrosine kinase inhibitor (BGB-3111) in mantle cell lymphoma expressing BTK.

BGB-3111, a novel Bruton’s tyrosine kinase (BTK) inhibitor, shows promising anti-cancer effects in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia (WM). This study aimed to investigate the anti-cancer effects of BGB-3111 combined with bortezomib (BTZ) against the BTK-expressing MCL. We found that BTK, which was overexpressed in 59.4% of patients with MCL, was mainly characterized by high Ki67 and elevated MIPI scores. BGB-3111 strongly inhibited cell proliferation, induced cell cycle arrest in the G1/G0-phase, and promoted cell apoptosis in the MCL cells expressing BTK. BGB-3111 provides better safety than another BTK inhibitor, ibrutinib as ibrutinib inhibits the inducible T-cell kinase (ITK) as an off-target effect but BGB-3111 does not inhibit ITK. Low doses of BTZ enhanced the anti-cancer effect induced by the low dose of BGB-3111 by downregulating the expression levels of PARP and Bcl-2 and increasing the expression levels of cleaved PARP and cleaved caspase-9. In addition, low doses of BGB-3111, but not of BTZ, inhibited BTK phosphorylation. However, low-doses of BTZ strengthened the anti-cancer effect induced by the low-doses of BGB-3111 via synergistically suppressing the IκBα and P65 phosphorylation. Taken together, our findings validate that BGB-3111 is a novel and effective BTK inhibitor for MCL-expressing BTK. Hence, it can be harnessed as a potential therapeutic strategy through a combinatorial treatment comprising low-dose BGB-3111 and low-dose BTZ to gain strong anti-cancer effects and better safety for MCL patients.

SY-1530, a highly selective BTK inhibitor, effectively treats B-cell malignancies by blocking B-cell activation.

Objective:B-cell antigen receptor (BCR) signaling is required to maintain the physiological functions of normal B cells and plays an important pathogenic role in B-cell malignancies. Bruton tyrosine kinase (BTK), a critical mediator of BCR signaling, is an attractive target for the treatment of B-cell malignancies. This study aimed to identify a highly potent and selective BTK inhibitor.Methods:Homogeneous time-resolved fluorescence assays were used to screen BTK inhibitors. Typhoon fluorescence imaging and Western blot analysis were used to confirm the effects of SY-1530 on the BCR signaling pathway. Additionally, the anti-tumor activities of SY-1530 were evaluated in TMD8 xenografts and spontaneous canine B-cell lymphoma.Results:We found a novel irreversible and non-competitive inhibitor of BTK, SY-1530, which provided dose-dependent and time-dependent inhibition. SY-1530 selectively bound to BTK rather than inducible T-cell kinase; consequently, it did not significantly affect T-cell receptor signaling and caused limited off-target effects. SY-1530 blocked the BCR signaling pathway through down-regulation of BTK activity, thus leading to impaired phosphorylation of BTK and its downstream kinases. Moreover, SY-1530 induced apoptosis in a caspase-dependent manner and efficaciously inhibited tumor growth in mouse xenograft models of B-cell malignancy (P < 0.001). SY-1530 also induced positive clinical responses in spontaneous canine B-cell lymphoma.Conclusions:SY-1530 is an irreversible and selective BTK inhibitor that shows inhibitory effects on B-cell malignancies by blocking the BCR signaling pathway. Therefore, it may be a promising therapeutic approach for the treatment of B-cell malignancies.

Role of Bruton's tyrosine kinase in endotoxin/lipopolysaccharide-induced pyroptosis of intestinal cells in scalded mice

Objective: To investigate the role of Bruton's tyrosine kinase (BTK) in pyroptosis of intestinal cells caused by endotoxin/lipopolysaccharide (LPS) in scalded mice. Methods: The experimental research method was applied. One hundred and twenty-eight male C57BL/6 mice aged 6-8 weeks were divided into sham injury group, scald alone group, scald+LPS group, scald+LPS+3 mg/kg LFM-A13 group, scald+LPS+10 mg/kg LFM-A13 group, and scald+LPS+30 mg/kg LFM-A13 group. There were 8 mice in sham injury group, and there were 24 mice in the other 5 groups, respectively. Mice in 5 scald groups were inflicted with 10% total body surface area full-thickness scald on the back, and mice in sham injury group were sham injured on the back. At post injury hour (PIH) 0 (immediately), mice in sham injury group and scald alone group were intraperitoneally injected with normal saline, mice in scald+LPS group were intraperitoneally injected with LPS, and mice in scald+LPS+3 mg/kg LFM-A13 group, scald+LPS+10 mg/kg LFM-A13 group, and scald+LPS+30 mg/kg LFM-A13 group were intraperitoneally injected with LPS and LFM-A13 in corresponding doses. Mice in sham injury group were sacrificed at PIH 0 to collect serum and intestinal tissue, and 8 mice in each group of 5 scald groups were sacrificed at PIH 0, 12, and 24 to collect intestinal tissue and serum at PIH 12. Immunohistochemistry was used to detect phosphorylation of BTK in intestinal tissue of mice. Western blotting was used to detect the protein expressions of phosphorylated BTK (p-BTK), cleaved cysteine aspartic acid specific protease 1 (caspase-1), and cleaved caspase-11 in intestinal tissue of mice. Enzyme-linked immunosorbent assay method was used to detect interleukin-1β (IL-1β) in serum and intestinal tissue of mice. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: There was no obvious phosphorylation of BTK in intestinal tissue of mice in 6 groups at PIH 0 and scald alone group at PIH 12 and 24. Phosphorylation of BTK in intestinal tissue of mice in scald+LPS group at PIH 12 and 24 were obviously increased compared with those in scald alone group. Phosphorylation of BTK in intestinal tissue of mice in scald+LPS+3 mg/kg LFM-A13 group, scald+LPS+10 mg/kg LFM-A13 group, and scald+LPS+30 mg/kg LFM-A13 group were obviously decreased compared with those in scald+LPS group, and the degrees of decline gradually increased with increase of dose in LFM-A13. Compared with (0.130±0.010) of sham injury group and (0.120±0.040 and 0.110±0.040) of scald alone group, protein expressions of p-BTK in intestinal tissue of mice in scald+LPS group at PIH 12 and 24 were obviously increased (0.470±0.090 and 0.430±0.080, P<0.01). Compared with those in scald+LPS group, protein expressions of p-BTK in intestinal tissue of mice in scald+LPS+3 mg/kg LFM-A13 group at PIH 24, and scald+LPS+10 mg/kg LFM-A13 group and scald+LPS+30 mg/kg LFM-A13 group at PIH 12 and 24 were obviously decreased (0.280±0.060, 0.300±0.120, 0.150±0.050, 0.280±0.090, 0.140±0.040, P<0.05 or P<0.01). Compared with those in scald+LPS+3 mg/kg LFM-A13 group, protein expressions of p-BTK in intestinal tissue of mice in scald+LPS+10 mg/kg LFM-A13 group and scald+LPS+30 mg/kg LFM-A13 group at PIH 24 were obviously decreased (P<0.01). Compared with those in sham injury group and scald alone group, protein expressions of cleaved caspase-1 and caspase-11 in intestinal tissue of mice in scald+LPS group were obviously increased at PIH 12 and 24 (P<0.01). Compared with those in scald+LPS group, protein expressions of cleaved caspase-1 at PIH 12 and cleaved caspase-11 at PIH 12 and 24 in intestinal tissue of mice in scald+LPS+3 mg/kg LFM-A13 group and protein expressions of cleaved caspase-1 and caspase-11 in intestinal tissue of mice in scald+LPS+10 mg/kg LFM-A13 group and scald+LPS+30 mg/kg LFM-A13 group at PIH 12 and 24 were obviously decreased (P<0.01). Compared with those in scald+LPS+3 mg/kg LFM-A13 group, protein expressions of cleaved caspase-1 and caspase-11 in intestinal tissue of mice in scald+LPS+10 mg/kg LFM-A13 group and scald+LPS+30 mg/kg LFM-A13 group at PIH 12 and 24 were obviously decreased (P<0.05 or P<0.01). At PIH 12, content of IL-1β in intestinal tissue and serum of mice in scald+LPS group were obviously higher than those in sham injury group and scald alone group (P<0.01), and content of IL-1β in intestinal tissue and serum of mice in scald+LPS+30 mg/kg LFM-A13 group were obviously lower than those in scald+LPS group (P<0.01). Conclusions: Phosphorylation of BTK is related to increases of cleaved caspase-1 and caspase-11 in intestinal tissue, and IL-1β content in intestinal tissue and serum of scalded septic mice caused by LPS. Phosphorylation of BTK mediates intestinal cell pyroptosis of scalded mice caused by LPS. Inhibiting phosphorylation of BTK can alleviate intestinal cell pyroptosis of scalded mice, with protective effect on intestinal injury intestine.

Abstract PO-59: Anti-membrane IgM monoclonal antibody, mAb4, is a novel, next-generation BTK inhibitor mediating B-cell leukemia and lymphoma cell death

Abstract Introduction: The concept that the B-cell receptor complex (BCRC) initiates a driver pathway in leukemia-lymphoma has been validated by clinical data. However, the BCRC has been overlooked as a therapeutic target due to homology between the BCRC's membrane IgM (mIgM) subunit and circulating serum IgM (sIgM). Previously, we reported the generation of a first-in-class antibody, mAb4, to a neoepitope specific to the BCRC, which inhibits cell growth (Welt et al. 2016; US Patent Nos. 9,926,381 and 10,227,419). Bruton's tyrosine kinase (BTK) inhibitors have emerged as valuable clinical therapies but can induce severe off-target effects. BTK is a downstream mediator of the BCRC's signaling pathway. We aim to characterize the impact of targeting the BCRC on BTK phosphorylation and its resulting biologic effects. We hypothesize that mAb4 binding to the BCRC initiates antitumor effects in part by inhibition of BTK phosphorylation. Methods: Antibody reagents detecting phosphorylation of BTK at Tyr551 (clones A16064A, 797837, and M4G3LN), Tyr223 (clones A16128B and 720101), and Ser180 (clones 3i5 and 3D3), were used to compare inhibition of phosphorylation by a BTK inhibitor drug, and by mAb4, in Burkitt lymphoma cells (CA46) over 0-96 hours, by ELISA. Flow cytometry will be used to further confirm these biologic effects and examine cell cycle phase inhibition. Results: We find that both mAb4 and a BTK inhibitor drug inhibit phosphorylation at Tyr551 and Tyr223, but not Ser180, across all Ab reagents used in ELISA. mAb4-treated leukemia and lymphoma cells undergo growth inhibition and apoptosis while the BTK inhibitor drug mediates only cell growth inhibition, as reported by others. Preliminary flow cytometry results suggest that mAb4 treatment induces a specific cell cycle arrest. Conclusions: Here we show that phospho-tyrosine BTK inhibition is mediated upon mAb4 binding to the BCRC's mIgM. Though BTK exhibits identical patterns of phosphorylation inhibition following treatment with either mAb4 or a BTK inhibitor drug, each treatment results in different cell survival outcomes. While studies show that BTK inhibitors induce a cell growth inhibitory effect, we find that mAb4 mediates BCRC internalization, and in low-density cultures, cell growth inhibition, anticlonogenic activity, and apoptosis. Further studies will examine spleen tyrosine kinase (SYK) phosphorylation and regulation of CXCR4 (a chemokine receptor), as mobilization of tumor cells into the blood may additionally be an important clinical parameter in BTK inhibitor drug efficacy. Due to the specific targeting of mAb4 to IgM-expressing B-cells, we expect that the toxicities associated with small-molecule BTK inhibitors upon interaction with nonlymphatic tissue, including bleeding, infection, cytopenia, arrhythmia, and secondary malignancies, will be avoided with mAb4 treatment. With increased antitumor activity and expected reduced toxicities, mAb4 represents a compelling candidate for clinical development. Citation Format: Rachel Welt, Jonathan Welt, Virginia Raymond, David Kostyal, Sydney Welt. Anti-membrane IgM monoclonal antibody, mAb4, is a novel, next-generation BTK inhibitor mediating B-cell leukemia and lymphoma cell death [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-59.

Discovery of BIIB068: A Selective, Potent, Reversible Bruton's Tyrosine Kinase Inhibitor as an Orally Efficacious Agent for Autoimmune Diseases.

Autoreactive B cell-derived antibodies form immune complexes that likely play a pathogenic role in autoimmune diseases. In systemic lupus erythematosus (SLE), these antibodies bind Fc receptors on myeloid cells and induce proinflammatory cytokine production by monocytes and NETosis by neutrophils. Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that signals downstream of Fc receptors and plays a transduction role in antibody expression following B cell activation. Given the roles of BTK in both the production and sensing of autoreactive antibodies, inhibitors of BTK kinase activity may provide therapeutic value to patients suffering from autoantibody-driven immune disorders. Starting from an in-house proprietary screening hit followed by structure-based rational design, we have identified a potent, reversible BTK inhibitor, BIIB068 (1), which demonstrated good kinome selectivity with good overall drug-like properties for oral dosing, was well tolerated across preclinical species at pharmacologically relevant doses with good ADME properties, and achieved >90% inhibition of BTK phosphorylation (pBTK) in humans.

Modulation of CD81 By Epigenetic Drug Combination Sensitizes Acute Lymphoblastic Leukemia Via Decreased BTK Signaling

Background We previously discovered that the epigenetic drug combination - azacitidine (aza, DNMTi) and panobinostat (pano, HDACi) can sensitize ALL cells to chemotherapy (Quagliano et al., Leuk Res, 44:101, 2017). This sensitization occurred through a decrease in cellular adhesion and a modulation of the cell surface expression of the tetraspanin protein CD81 (Quagliano et al., Blood, 132:3957, 2018). This study aims to elucidate the molecular mechanism by which CD81 surface modulation by pre-treatment with aza/pano leads to chemosensitization. Methods CD81 knockout Nalm6 (CD81KO) cells were generated by CRISPR/Cas9 mutagenesis. IHC was performed using anti-human mitochondria antibody on femurs harvested from NSG-SGM3 mice at 72 h post transplantation with WT or CD81KO cells. For adhesion assay, ALL cells stained with VPD450 were co-cultured with Saos-2 cells for 24 h. Unbound cells were washed, and bound cells were collected for flow cytometry analysis. CD19 surface expression was determined after 48 h treatment with aza/pano (500 nM/1.5 nM) using flow cytometry. For western blot analysis, aza/pano treated cells were transferred on to Saos-2 monolayers and treated with Ara-C (30 nM) for 16 h. Results CD81KO cells not only had decreased homing and engraftment visualized by reduced ALL cell presence in bone marrow (Fig. 1A), but also had 45% reduction in cellular adhesion to osteoblasts compared to WT Nalm6 cells (Fig. 1B), indicating that CD81 downregulation results in reduced bone marrow interactions. To investigate the signaling effects downstream of aza/pano-mediated CD81 modulation, we first tested if CD19 was affected due to its prominent role in the formation of the B-cell co-receptor and because CD19 requires CD81 for proper membrane trafficking (Cherukuri et al., J Immunol, 172:370, 2004). CD19 surface expression was reduced in aza/pano-treated cells and completely knocked out in CD81KO cells (Fig. 1C). CD19 is known to prolong and amplify the activation of Bruton's tyrosine kinase (BTK), so we also analyzed the phosphorylation of BTK in aza/pano-treated cells and found it to be reduced by 63% (Fig. 1D). This reduction in BTK phosphorylation may be responsible for the decrease in cell adhesion following treatment with aza/pano because prior studies observed reduced adhesion following BTK inhibition (Herman et al., Clin Cancer Res, 21:4642, 2015). Taken together, these data suggest that aza/pano induced reduction in cell adhesion was mediated by downregulation of CD81 and BTK dephosphorylation. We studied how the expression of p53 and its target BCL2 associated X protein (BAX), which are known to be induced by Ara-C, were affected following pre-treatment with aza/pano. Cells with Ara-C treatment alone or the aza/pano pre-treatment both had minor induction of p53 expression compared to control, while aza/pano pre-treated cells that received Ara-C had further increase in p53 protein (1.7-fold increase compared to Ara-C alone, Fig. 1E). Bax mRNA and protein were increased 4.2-fold in aza/pano-treated cells following Ara-C treatment compared to Ara-C alone. Increased Bax expression following Ara-C treatment in aza/pano pre-treated cells was accompanied by a 3.9-fold increase in cleavage of caspase-3 compared to cells treated with Ara-C alone, which in turn causes cleavage of Poly-ADP Ribose Polymerase (PARP) (Fig. 1E). Both p53 and Bax protein levels were higher in CD81KO cells than in WT cells (Fig. 1E), suggesting that aza/pano-mediated reduction of CD81 is involved in inducing the expression of p53. CD81KO cells that were treated with Ara-C also had further increase in p53 and Bax expression compared to Ara-C treated WT cells (Fig. 1E), consistent with an increased sensitivity to chemotherapy. To confirm the role of BTK inhibition in the induction of p53, Nalm6 cells were treated with the BTK inhibitor fenebrutinib for 24 h (10 nM). Following treatment, expression of p53 was induced and Bax expression was 2.6-fold higher in treated cells compared to untreated cells (Fig. 1E). Taken together, these data suggest that modulation of the surface expression of CD81 and the phosphorylation of BTK by aza/pano induces sensitization via upregulation of p53 and overexpression of Bax. Conclusion We identify a novel mechanism by which aza/pano treatment induces chemosensitization by reducing cell adhesion via modulating CD81 surface expression, reducing BTK phosphorylation, and inducing p53. Figure 1 Disclosures No relevant conflicts of interest to declare.

High Surface IgM Levels Associate with Shorter Response Duration and Bypass of the BTK Blockade during Ibrutinib Therapy in CLL Patients

The circulating tumor cells of chronic lymphocytic leukemia (CLL) are characterized by variably low surface IgM (sIgM) levels and signaling capacity, consequent to stimulation by tissue-based antigen. The variability is evident in both CLL subsets with unmutated and mutated immunoglobulin genes and affects tumor cell survival and proliferation. This appears clinically important because CLL with relatively higher sIgM levels/signaling capacity progress more rapidly than those with lower sIgM levels/signaling capacity (D'Avola, Blood 2016), likely due to a larger proliferative component at tissue sites. B-cell receptor (BCR)-signaling inhibitor ibrutinib exerts its therapeutic activity in CLL by irreversibly binding the Bruton's tyrosine kinase (BTK) at Cys481 in the active site. This leads to redistribution of CLL cells from tissue sites into peripheral blood and to a selective recovery of sIgM levels and function, consequent to release from antigen drive at tissue sites (Drennan, Clin Cancer Res 2019). We have now investigated the hypothesis that sIgM levels on the CLL cells affect duration of response to ibrutinib therapy possibly consequent to circumvention of the pharmacological BTK inhibition. Seventy CLL patients participating in the "real-world" observational study at the University of Southampton (NIHR/UKCRN ID: 31076) were investigated for phenotypic, molecular and functional characteristics associated with response to single-agent ibrutinib. Time to progression requiring a new treatment from ibrutinib start (TTNT) was used as primary endpoint to measure duration of response. The levels of sIgM were measured by flow cytometry. Anti-IgM induced signaling was determined by immunoblotting or iCa2+ mobilization. BTK and PLCγ2 mutations were determined using the Illumina NexteraXT kit and a MiSeq at the University of Southampton and validated independently at Karolinska Institutet. Our previous cut-offs of 50 (MFI) or 5 (iCa2+ % mobilization) were used to distinguish patients with high/low sIgM levels or signaling capacity, respectively. Median follow-up from ibrutinib start was 42 months (range 12-70). Pre-ibrutinib sIgM levels (range 7-618, median 65; high sIgM MFI 46/70, 66%) and sIgM signaling capacity (range 1-98; median 45; high sIgM signalers 56/70, 80%) were broadly heterogeneous. However, median values were significantly higher than the general CLL population at diagnosis, as expected in progressive CLL. By univariate analyses of clinical, phenotypic, FISH and immunogenetic characteristics for TTNT, high sIgM was the only parameter predicting shorter duration of response to ibrutinib (p=0.02). Only 2/24 CLL with low sIgM levels (CLLIgM_lo) had progressed (median TTNT not reached) compared to 15/46 CLL with high sIgM (CLLIgM_hi, 14% at 12 months, 18% at 24 months, 34% at 36 months, 44% at 42 months) during therapy. The levels of sIgM correlated significantly with sIgM signaling capacity prior to treatment (r= 0.68; p&lt;0.0001) and were selectively maintained during ibrutinib therapy. From in vitro analysis of 13 therapy-naive CLL samples, the degree of inhibition by ibrutinib of anti-IgM induced signals downstream to BTK correlated negatively with sIgM levels (r=-0.68,p=0.01) and signaling (r=-0.71,p=0.009). Anti-IgM induced signals were then measured in 12 patients (7 CLLIgM_hi, 5 CLLIgM_lo) after 1, 4 and 12 weeks of ibrutinib therapy. These patients had not acquired BTK or PLCγ2 mutations and BTK phosphorylation appeared completely inhibited at all time-points. Mean anti-IgM induced iCa2+ mobilization and ERK1/2 phosphorylation were significantly reduced but not abolished during therapy and degree of inhibition was variable between individuals. Remarkably, degree of inhibition of anti-IgM-induced iCa2+ mobilization and pERK1/2 was significantly lower in CLLIgM_hi than in CLLIgM_lo (p&lt;0.05 at all time-points). These results confirm that sIgM signaling is dependent on sIgM levels and that it can circumvent BTK blockade when sIgM levels are high. They suggest that high sIgM signaling can drive ibrutinib resistance despite ability of ibrutinib to fully occupy the BTK phosphorylation pocket. A possibility is that those CLL cells with high sIgM represent a potentially dangerous subpopulation equipped to migrate to tissue and to receive proliferative stimuli. These cells might be targeted by a combinatorial therapeutic approach with ibrutinib. Disclosures Johnson: Takeda: Honoraria; Kite: Honoraria; Bristol-Myers Squibb: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Epizyme: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria. Duncombe:Abbvie: Other: Advisory Board membership;Educational support; Gilead: Other: Advisory Board membership; Janssen: Other: Advisory Board membership;Educational support; Novartis: Other: Advisory Board membership. Scarfo:Janssen: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria. Sutton:Gilead: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Ghia:Pharmacyclics LLC, an AbbVie Company: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; ArQule: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy, Honoraria; Novartis: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding. Forconi:Roche: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Gilead Sciences: Research Funding.

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

BCR-Induced VLA-4 Activation in the TCL1 Transgenic Mouse Model for Chronic Lymphocytic Leukemia

Introduction. Ibrutinib, a small molecule inhibitor of Bruton's tyrosine kinase (BTK), has proven to be an efficient treatment for chronic lymphocytic leukemia (CLL). A distinct characteristic of ibrutinib therapy is transient lymphocytosis. Recently, we have demonstrated that CLL patients with high levels of CD49d show reduced lymphocytosis and inferior nodal response under ibrutinib due to residual activity of BCR-induced inside-out activation of the CD49d/CD29 integrin VLA-4 (Tissino E et al. J Exp Med. 2018;215(2):681-697). Here, we used Tcl1 transgenic (tg) mice as a model to further validate the observation of VLA-4 activation under ibrutinib and to study involved signaling pathways and the effect of VLA-4 inhibition in vivo. Methods. Surface receptor expression analysis of various receptors was performed by flow cytometry. The phosphorylation of signaling molecules was measured by phosflow and western blotting. VLA-4 affinity state was determined by a real-time kinetic assay described in Chigaev A et al. J Biol Chem. 2001;276(52):48670-8. To analyze the distribution of individual VLA-4 molecules on the cell surface, immunofluorescence approaches and superresolution microscopy (STORM, Abbelight) were employed. Mouse treatment studies were performed upon transplantation of TCL1-tg splenocytes to wild-type C57BL/6J mice using the small molecule VLA-4 inhibitor firategrast in drinking water. Tumor infiltration of different organs was measured by flow cytometry. Results. Analyzing the surface expression of CD49d and other homing receptors, we found that TCL1-tg mice correspond with the CD49d-high CLL cohort. We found that both CLL cells from TCL1-tg mice and human CD49d-high CLL show similar CD49d expression levels as the corresponding healthy B cells (human: N = 116 CD49d-high CLL and 32 healthy donor, P = 0.8717; mouse: N = 12 per group, P = 0.6845). Next, we analyzed the impact of BCR pathway inhibitors on the phosphorylation of signaling molecules involved in the BCR pathway after activation by anti-IgM (aIgM) in TCL1-tg leukemic cells. Ibrutinib and idelalisib showed specific patterns of inhibition of BTK and PI3K, respectively. The combination of ibrutinib and idelalisib proved to be the most efficient in reducing the phosphorylation of BTK, SYK, ERK1/2 and Akt upon IgM activation, compared to the phosphorylation of stimulated cells without inhibition (N = 6, P = 0.0003, 0.0305, 0.0039, 0.0019, respectively). IgM stimulation induced VLA-4 high affinity as well as a reorganization of VLA-4 molecules on the cell surface, forming areas of high VLA-4 density. BCR-induced inside-out activation of VLA-4 remained functional upon treatment with ibrutinib (N = 5, cnt vs aIgM P = 0.0017, cnt vs ibrutinib+aIgM P = 0.0499), while idelalisib reduced VLA-4 activation more effectively (N = 5, cnt vs aIgM P = 0.0014, cnt vs idelalisib+aIgM P = 0.0803), suggesting a pivotal role of PI3K in the transmission of the exogenous antigen signal to the integrin. Finally, to analyze the potential of VLA-4 blockage in a tumor setting similar to VLA-4-high CLL patients, we treated wild-type C57BL/6J mice (N = 6 mice per group), which were transplanted with TCL1-tg splenocytes, with the CD49d inhibitor firategrast. This treatment reduced the tumor load in spleen and bone marrow. Conclusion. We found that the TCL1-tg mouse model is adequate to study the activity of the BCR-VLA-4 axis in CLL. Using this model, we show that a) BCR stimulation induces both, an increase in VLA-4 affinity as well as avidity (clustering), b) that PI3K is an essential transmitter between BCR and VLA-4, and c) that VLA-4 inhibition alters tumor infiltration patterns in vivo. Synergies of VLA-4 blockage with established therapy options as a possible way of reducing microenvironment-induced resistance development are currently been investigated. Disclosures Egle: Celgene: Honoraria, Other: Advisory board and Travel support. Greil:Eisai: Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Sandoz: Honoraria; Genentech: Honoraria, Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Honoraria; Janssen-Cilag: Honoraria; AstraZeneca: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; GSK: Research Funding; Boehringer Ingelheim: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Celgene: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; MSD: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Ratiopharm: Research Funding; Gilead: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding.

Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE‐dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI‐1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC50 values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI‐1 cells. In C2 cells, the combination “ibrutinib + midostaurin” produced synergistic growth‐inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE‐dependent histamine release in primary MCT cells, with IC50 values ranging from 0.05 to 0.1 μM in NI‐1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti‐proliferative effects in canine neoplastic MCs and counteracts IgE‐dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.

Abivertinib, a novel BTK inhibitor: Anti-Leukemia effects and synergistic efficacy with homoharringtonine in acute myeloid leukemia.

Ibrutinib, an inhibitor of Bruton tyrosine kinase (BTK), has shown promising pharmacologic effects in acute myeloid leukemia (AML). In this study, we report that abivertinib or AC0010, a novel BTK inhibitor, inhibits cell proliferation, reduces colony-forming capacity, and induces apoptosis and cell cycle arrest in AML cells, especially those harboring FLT3-ITD mutations. Abivertinib was also found to be more sensitive than ibrutinib in treating AML. We demonstrate that in addition to targeting the phosphorylation of BTK, abivertinib also targeted the crucial PI3K survival pathway. Furthermore, abivertinib suppressed the expression of p-FLT3 and the downstream target p-STAT5 in AML cells harboring FLT3-ITD mutations. Moreover, in vitro and in vivo data revealed synergistic activity between abivertinib and homoharringtonine (HHT), a natural plant alkaloid commonly used in China, in treating AML cells with or without FLT3-ITD mutations. Collectively, these preclinical data suggest that abivertinib may be a promising novel agent for AML, with potential for combination treatment with HHT. Clinical studies on abivertinib-involved therapy are planned.