BTK inhibition limits microglia-perpetuated CNS inflammation and promotes myelin repair.

In multiple sclerosis (MS), persisting disability can occur independent of relapse activity or development of new central nervous system (CNS) inflammatory lesions, termed chronic progression. This process occurs early and it is mostly driven by cells within the CNS. One promising strategy to control progression of MS is the inhibition of the enzyme Bruton's tyrosine kinase (BTK), which is centrally involved in the activation of both B cells and myeloid cells, such as macrophages and microglia. The benefit of BTK inhibition by evobrutinib was shown as we observed reduced pro-inflammatory activation of microglia when treating chronic experimental autoimmune encephalomyelitis (EAE) or following the adoptive transfer of activated T cells. Additionally, in a model of toxic demyelination, evobrutinib-mediated BTK inhibition promoted the clearance of myelin debris by microglia, leading to an accelerated remyelination. These findings highlight that BTK inhibition has the potential to counteract underlying chronic progression of MS.

Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma.

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.

Computational Investigation of the Covalent Inhibition Mechanism of Bruton's Tyrosine Kinase by Ibrutinib.

Covalent inhibitors represent a promising class of therapeutic compounds. Nonetheless, rationally designing covalent inhibitors to achieve a right balance between selectivity and reactivity remains extremely challenging. To better understand the covalent binding mechanism, a computational study is carried out using the irreversible covalent inhibitor of Bruton tyrosine kinase (BTK) ibrutinib as an example. A multi-µs classical molecular dynamics trajectory of the unlinked inhibitor is generated to explore the fluctuations of the compound associated with the kinase binding pocket. Then, the reaction pathway leading to the formation of the covalent bond with the cysteine residue at position 481 via a Michael addition is determined using the string method in collective variables on the basis of hybrid quantum mechanical-molecular mechanical (QM/MM) simulations. The reaction pathway shows a strong correlation between the covalent bond formation and the protonation/deprotonation events taking place sequentially in the covalent inhibition reaction, consistent with a 3-step reaction with transient thiolate and enolates intermediate states. Two possible atomistic mechanisms affecting deprotonation/protonation events from the thiolate to the enolate intermediate were observed: a highly correlated direct pathway involving proton transfer to the Cα of the acrylamide warhead from the cysteine involving one or a few water molecules and a more indirect pathway involving a long-lived enolate intermediate state following the escape of the proton to the bulk solution. The results are compared with experiments by simulating the long-time kinetics of the reaction using kinetic modeling.

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

CD5 Gene Signature Identifies Diffuse Large B-Cell Lymphomas Sensitive to Bruton's Tyrosine Kinase Inhibition.

PURPOSE: A genetic classifier termed LymphGen accurately identifies diffuse large B-cell lymphoma (DLBCL) subtypes vulnerable to Bruton's tyrosine kinase inhibitors (BTKis), but is challenging to implement in the clinic and fails to capture all DLBCLs that benefit from BTKi-based therapy. Here, we developed a novel CD5 gene expression signature as a biomarker of response to BTKi-based therapy in DLBCL. METHODS: CD5 immunohistochemistry (IHC) was performed on 404 DLBCLs to identify CD5 IHC+ and CD5 IHC- cases, which were subsequently characterized at the molecular level through mutational and transcriptional analyses. A 60-gene CD5 gene expression signature (CD5sig) was constructed using genes differentially expressed between CD5 IHC+ and CD5 IHC- non-germinal center B-cell-like (non-GCB DLBCL) DLBCLs. This CD5sig was applied to external DLBCL data sets, including pretreatment biopsies from patients enrolled in the PHOENIX study (n = 584) to define the extent to which the CD5sig could identify non-GCB DLBCLs that benefited from the addition of ibrutinib to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). RESULTS: CD5 expression was observed in 12% of non-GCB DLBCLs. CD5+ DLBCLs displayed transcriptional features of B-cell receptor (BCR) activation and were enriched for BCR-activating mutations known to correlate with BTKi sensitivity. However, most CD5+ DLBCLs lacked canonical BCR-activating mutations or were LymphGen-unclassifiable (LymphGen-Other). The CD5sig recapitulated these findings in multiple independent data sets, indicating its utility in identifying DLBCLs with genetic and nongenetic bases for BCR dependence. Supporting this notion, CD5sig+ DLBCLs derived a selective survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study, independent of LymphGen classification. CONCLUSION: CD5sig is a useful biomarker to identify DLBCLs vulnerable to BTKi-based therapies and complements current biomarker approaches by identifying DLBCLs with genetic and nongenetic bases for BTKi sensitivity.

Bruton's Tyrosine Kinase Inhibitors for Multiple Sclerosis Treatment: A New Frontier.

Multiple sclerosis (MS) can cause significant disability to patients via relapse-associated worsening and progression independent of relapses. The causes of neuronal and myelin damage can include lymphocyte-mediated inflammation and microglial activation. Bruton's tyrosine kinase (BTK) is an enzyme that mediates B cell activation and the proinflammatory phenotype of microglia. Inhibiting BTK provides a novel therapeutic target for MS but also has a complicated pharmacology based on binding specificity, CNS penetration, half-life, and enzyme inhibition characteristics. Multiple agents are being studied in phase 3 trials, and each agent will have unique efficacy and safety profiles that must be considered individually.

Signaling pathways regulating the extracellular digestion of lipoprotein aggregates by macrophages.

The interaction between aggregated low-density lipoprotein (agLDL) and macrophages in arteries plays a major role in atherosclerosis. Macrophages digest agLDL and generate free cholesterol in an extracellular, acidic, hydrolytic compartment known as the lysosomal synapse. Macrophages form a tight seal around agLDL through actin polymerization and deliver lysosomal contents into this space in a process termed digestive exophagy. Our laboratory has identified TLR4 activation of MyD88/Syk as critical for digestive exophagy. Here we use pharmacological agents and siRNA knockdown to characterize signaling pathways downstream of Syk that are involved in digestive exophagy. Syk activates Bruton's tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). We show that PLCγ2 and to a lesser extent BTK regulate digestive exophagy. PLCγ2 cleaves PI(4,5)P2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Soluble IP3 activates release of Ca2+ from the endoplasmic reticulum (ER). We demonstrate that Ca2+ release from the ER is upregulated by agLDL and plays a key role in digestive exophagy. Both DAG and Ca2+ activate protein kinase Cα (PKCα). We find that PKCα is an important regulator of digestive exophagy. These results expand our understanding of the mechanisms of digestive exophagy, which could be useful in developing therapeutic interventions to slow development of atherosclerosis.

Resisting the Resistance: Navigating BTK Mutations in Chronic Lymphocytic Leukemia (CLL).

Bruton's tyrosine kinase (BTK) plays a key role in the B-cell receptor (BCR) signaling pathway and confers anti-apoptotic and proliferative properties to malignant B-cells in chronic lymphocytic leukemia (CLL). Small molecule BTK inhibitors were designed to bind BTK's active site and block downstream signaling. These drugs have now been used in the treatment of thousands of patients with CLL, the most common form of leukemia in the western hemisphere. However, adverse effects of early generations of BTK inhibitors and resistance to treatment have led to the development of newer, more selective and non-covalent BTK inhibitors. As the use of these newer generation BTK inhibitors has increased, novel BTK resistance mutations have come to light. This review aims to discuss previously known and novel BTK mutations, their mechanisms of resistance, and their relationship with patient treatment. Also discussed here are future studies that are needed to investigate the underlying cause allowing these mutations to occur and how they incite resistance. New treatments on the horizon that attempt to maneuver around these resistance mutations can be met with new resistance mutations, creating an unmet need for patients with CLL. Novel therapies and combinations that address all forms of resistance are discussed.

Bruton's tyrosine kinase-bearing B cells and microglia in neuromyelitis optica spectrum disorder.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory autoimmune disease of the central nervous system that involves B-cell receptor signaling as well as astrocyte-microglia interaction, which both contribute to evolution of NMOSD lesions. MAIN BODY: Through transcriptomic and flow cytometry analyses, we found that Bruton's tyrosine kinase (BTK), a crucial protein of B-cell receptor was upregulated both in the blood and cerebrospinal fluid of NMOSD patients. Blockade of BTK with zanubrutinib, a highly specific BTK inhibitor, mitigated the activation and maturation of B cells and reduced production of causal aquaporin-4 (AQP4) autoantibodies. In a mouse model of NMO, we found that both BTK and pBTK expression were significantly increased in microglia. Transmission electron microscope scan demonstrated that BTK inhibitor ameliorated demyelination, edema, and axonal injury in NMO mice. In the same mice colocalization of GFAP and Iba-1 immunofluorescence indicated a noticeable increase of astrocytes-microglia interaction, which was alleviated by zanubrutinib. The smart-seq analysis demonstrated that treatment with BTK inhibitor instigated microglial transcriptome changes including downregulation of chemokine-related genes and genes involved in the top 5 biological processes related to cell adhesion and migration, which are likely responsible for the reduced crosstalk of microglia and astrocytes. CONCLUSIONS: Our results show that BTK activity is enhanced both in B cells and microglia and BTK inhibition contributes to the amelioration of NMOSD pathology. These data collectively reveal the mechanism of action of BTK inhibition and corroborate BTK as a viable therapeutic target.

A Comprehensive Review of Small-Molecule Inhibitors Targeting Bruton Tyrosine Kinase: Synthetic Approaches and Clinical Applications.

Bruton tyrosine kinase (BTK) is an essential enzyme in the signaling pathway of the B-cell receptor (BCR) and is vital for the growth and activation of B-cells. Dysfunction of BTK has been linked to different types of B-cell cancers, autoimmune conditions, and inflammatory ailments. Therefore, focusing on BTK has become a hopeful approach in the field of therapeutics. Small-molecule inhibitors of BTK have been developed to selectively inhibit its activity and disrupt B-cell signaling pathways. These inhibitors bind to the active site of BTK and prevent its phosphorylation, leading to the inhibition of downstream signaling cascades. Regulatory authorities have granted approval to treat B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), with multiple small-molecule BTK inhibitors. This review offers a comprehensive analysis of the synthesis and clinical application of conventional small-molecule BTK inhibitors at various clinical stages, as well as presents promising prospects for the advancement of new small-molecule BTK inhibitors.

Identification of the Axis ß-Catenin-BTK in the Dynamic Adhesion of Chronic Lymphocytic Leukemia Cells to Their Microenvironment.

In the microenvironment, cell interactions are established between different cell types to regulate their migration, survival and activation. ß-Catenin is a multifunctional protein that stabilizes cell-cell interactions and regulates cell survival through its transcriptional activity. We used chronic lymphocytic leukemia (CLL) cells as a cellular model to study the role of ß-catenin in regulating the adhesion of tumor cells to their microenvironment, which is necessary for tumor cell survival and accumulation. When co-cultured with a stromal cell line (HS-5), a fraction of the CLL cells adhere to stromal cells in a dynamic fashion regulated by the different levels of ß-catenin expression. In non-adherent cells, ß-catenin is stabilized in the cytosol and translocates into the nucleus, increasing the expression of cyclin D1. In adherent cells, the level of cytosolic ß-catenin is low but membrane ß-catenin helps to stabilize the adhesion of CLL to stromal cells. Indeed, the overexpression of ß-catenin enhances the interaction of CLL with HS-5 cells, suggesting that this protein behaves as a regulator of cell adhesion to the stromal component and of the transcriptional regulation of cell survival. Inhibitors that block the stabilization of ß-catenin alter this equilibrium and effectively disrupt the support that CLL cells receive from the cross-talk with the stroma.

Lysine-Reactive N-Acyl-N-aryl Sulfonamide Warheads: Improved Reaction Properties and Application in the Covalent Inhibition of an Ibrutinib-Resistant BTK Mutant.

The covalent inhibition of a target protein has gained widespread attention in the field of drug discovery. Most of the current covalent drugs utilize the high reactivity of cysteines toward modest electrophiles. However, there is a growing need for warheads that can target lysine residues to expand the range of covalently druggable proteins and to deal with emerging proteins with mutations resistant to cysteine-targeted covalent drugs. We have recently developed an N-acyl-N-alkyl sulfonamide (NASA) as a lysine-targeted electrophile. Despite its successful application, this NASA warhead suffered from instability in physiological environments, such as serum-containing medium, because of its high intrinsic reactivity. In this study, we sought to modify the structure of the NASA warhead and found that N-acyl-N-aryl sulfonamides (ArNASAs) are promising electrophiles for use in a lysine-targeted covalent inhibition strategy. We prepared a focused library of ArNASA derivatives with diverse structures and reactivity and identified several warhead candidates with suppressed hydrolysis-mediated inactivation and reduced nonspecific reactions with off-target proteins, without sacrificing the reactivity toward the target. These reaction properties enabled the improved covalent inhibition of intracellular heat shock protein 90 (HSP90) in the presence of serum and the development of the first irreversible inhibitor for ibrutinib-resistant Bruton's tyrosine kinase (BTK) bearing the C481S mutation. This study clearly demonstrated the use of a set of ArNASA warheads to create highly potent covalent drugs and highlighted the importance of enriching the current arsenal of lysine-reactive warheads.

Development of 7H-pyrrolo[2,3-d]pyrimidin-4-amine Derivatives Using QSARINS Tool as BTK Inhibitors for the Treatment of Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of the joints, leading to pain, swelling, and joint deformity. Effective management of RA involves the use of disease-modifying drugs that can slow down disease progression and alleviate symptoms. Among the potential targets for RA treatment is Bruton's tyrosine kinase (BTK), which plays a crucial role in B-cell signalling and contributes to the pathogenesis of RA. AIMS: QSARINS (QSAR-INSUBRIA) is software used for the development and validation of Quantitative Structure-Activity Relationship (QSAR) analysis. In the present work, this software was explored for pharmacophore optimization of the pyrrolo-pyrimidine nucleus for anti-rheumatoid activity. METHODS: A series of pyrrolo-pyrimidine derivatives were used to build the QSAR models. These models were generated to identify structural features that correlate significantly with the activity. We followed the assessment of statistical parameters to ensure thorough validation of all the QSAR models. The QSAR models demonstrating better statistical performance were selected, and descriptors of these models were analysed. RESULTS: The results showed that the QSAR models were highly statistically robust and exhibited a strong external predictive ability. Their structural features were also deduced. CONCLUSION: This QSAR study provided crucial information about the specific molecular features that can be used for the optimization of the pharmacophores. This research provides valuable insights into the structural features essential for BTK inhibition and paves the way for the design and development of novel anti-rheumatic agents targeting BTK in RA.

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-1ß 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.

Expression of Bruton´s tyrosine kinase in different type of brain lesions of multiple sclerosis patients and during experimental demyelination.

Background: Inhibition of Bruton's tyrosine kinase (BTK) is an emerging multiple sclerosis (MS) therapy. BTK inhibitors (BTKi) cross the blood-brain barrier and modulate B cells and microglia, major cellular players in active and chronic active lesions. Objective: To assess potential lesional and cellular targets of BTKi, we examined BTK expression in different type of MS white matter (WM) lesions, in unmanipulated CNS resident cells, and in a degenerative MS model associated with microglia activation in vivo. Methods: We examined BTK expression by next-generation RNA-sequencing in postmortem 25 control WM, 19 NAWM, 6 remyelinating, 18 active, 13 inactive and 17 chronic active lesions. Presence of B cells and microglia were examined by immunohistochemistry. CNS resident cells were isolated from the mouse brain by magnetic sorting. BTK expression was examined by quantitative PCR in isolated cells and dissected corpus callosum from mice treated with cuprizone (CPZ). Results: BTK expression was significantly increased in active and chronic active lesions with upregulated complement receptors and Fcγ receptors. Active lesions contained high number of perivascular B cells, microglia, and macrophages. Chronic active lesions were characterized by microglia/macrophages in the rim. Microglia expressed BTK at high level (120-fold) in contrast to other CNS cell types (2-4-fold). BTK expression was increasing during CPZ treatment reaching significance after stopping CPZ. Conclusion: Considering BTK expression in MS lesions and resident cells, BTKi may exert effect on B cells, microglia/macrophages in active lesions, and limit microglia activation in chronic active lesions, where tissue damage propagates.

Synthesis and application of small molecules approved for the treatment of lymphoma.

Lymphoma is a form of cancer that impacts the lymphatic system, which plays a crucial role in defending the body against infections and illnesses. It is characterized by the atypical proliferation of lymphocytes, a type of white blood cell, which can form tumors in the lymph nodes, bone marrow, spleen, etc. Lymphoma is usually treated using a combination of targeted therapy, chemotherapy, and radiation therapy. In recent years, there has been a growing interest in the development of new drugs to treat lymphoma, which has led to the discovery of several promising compounds. The primary targets for lymphoma treatment have been identified as Bruton's tyrosine kinase (BTK), phosphoinositide3-kinase (PI3K), histone deacetylase (HDAC), and DNA polymerase (POLA). This review aims to provide an overview of the clinical applications and synthesis of several notable drugs approved to treat lymphoma, to expedite the exploration of more potent novel medications for the management of lymphoma.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma.

The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

BTK inhibition potentiates anti-PD-L1 treatment in murine melanoma: potential role for MDSC modulation in immunotherapy.

Myeloid-derived suppressor cells (MDSC) have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species and the production of inhibitory cytokines. Our group has shown that signaling through Bruton's tyrosine kinase (BTK) is important for MDSC function. Ibrutinib is an orally administered targeted agent that inhibits BTK activation and is currently used for the treatment of B cell malignancies. Using a syngeneic murine model of melanoma, the effect of BTK inhibition with ibrutinib on the therapeutic response to systemic PD-L1 blockade was studied. BTK was expressed by murine MDSC and their activation was inhibited by ibrutinib. Ibrutinib was not directly cytotoxic to cancer cells in vitro, but it inhibited BTK activation in MDSC and reduced expression of inducible nitric oxide synthase (NOS2) and production of nitric oxide. Ibrutinib treatments decreased the levels of circulating MDSC in vivo and increased the therapeutic efficacy of anti-PD-L1 antibody treatment. Gene expression profiling showed that ibrutinib decreased Cybb (NOX2) signaling, and increased IL-17 signaling (upregulating downstream targets Mmp9, Ptgs2, and S100a8). These results suggest that further exploration of MDSC inhibition could enhance the immunotherapy of advanced melanoma.PrécisInhibition of Bruton's tyrosine kinase, a key enzyme in myeloid cellular function, improves therapeutic response to an anti-PD-L1 antibody in an otherwise fairly resistant murine melanoma model.

Exposure to Therapeutic BTK Inhibitors Induces Phenocopying of Btk29A Mutants in the Fruit Fly Drosophila melanogaster.

BACKGROUND: Bruton's tyrosine kinase (BTK) is a non-receptor type tyrosine kinase originally identified as the genetic signature responsible for X-linked agammaglobulinemia (XLA) when mutated. Its functional form is required for B lymphocyte maturation in both humans and mice, whereas loss-of-function causes a different form of developmental defect in the fruit fly, Drosophila melanogaster. METHODS: Ibrutinib and other therapeutic inhibitors of BTK have been extensively used to successfully treat various leukemias and lymphomas. Btk29A type 2 is the ortholog of BTK in the fruit fly. We show that feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants, i.e., failure in the fusion of left and right halves of the dorsal cuticles, partial loss of wing tissues and dysregulation of germ cell production. RESULTS: We have previously reported that Btk29A phosphorylates Drosophila Arm (ß-catenin), and ibrutinib reduces phosphorylation at Tyrosine142 of endogenously expressed ß-catenin in Cos7 cells transfected with Btk29A type 2 cDNA. CONCLUSIONS: Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.

Susceptibility to Cryptococcus neoformans Infection with Bruton's Tyrosine Kinase Inhibition.

Patients receiving the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of fungal infections. The objectives of this study were to determine if Cryptococcus neoformans infection severity was isolate dependent with BTK inhibition and whether blocking BTK impacted infection severity in a mouse model. We compared four clinical isolates from patients on ibrutinib to virulent (H99) and avirulent (A1-35-8) reference strains. BTK knockout (KO) and wild-type (WT) C57 mice and WT CD1 mice were infected by intranasal (i.n.), oropharyngeal aspiration (OPA), and intravenous (i.v.) routes. Infection severity was assessed by survival and fungal burden (CFU per gram of tissue). Ibrutinib (25 mg/kg) or vehicle was administered daily through intraperitoneal injections. In the BTK KO model, no isolate-dependent effect on fungal burden was observed, and infection severity was not significantly different from that of the WT with i.n., OPA, and i.v. routes. Ibrutinib treatment did not impact infection severity. However, when the four clinical isolates were compared to H99, two of these isolates were less virulent, with significantly longer survival and reduced rates of brain infection. In conclusion, C. neoformans infection severity in the BTK KO model does not appear to be isolate dependent. BTK KO and ibrutinib treatment did not result in significantly different infection severities. However, based on repeated clinical observations of increased susceptibility to fungal infections with BTK inhibitor therapy, further work is needed to optimize a mouse model with BTK inhibition to better understand the role that this pathway plays in susceptibility to C. neoformans infection.