Activation Of Bruton's Tyrosine Kinase Research Articles

Inhibition of Bruton's Tyrosine Kinase Activity Attenuates Hemorrhagic Shock-Induced Multiple Organ Dysfunction in Rats.

The aim of this study was to investigate (a) the potential of the Bruton's tyrosine kinase (BTK) inhibitors acalabrutinib and fenebrutinib to reduce multiple organ dysfunction syndrome (MODS) in acute (short-term and long-term follow-up) hemorrhagic shock (HS) rat models and (b) whether treatment with either acalabrutinib or fenebrutinib attenuates BTK, NF-κB and NLRP3 activation in HS. The MODS caused by an excessive systemic inflammatory response following trauma is associated with a high morbidity and mortality. The protein BTK is known to play a role in the activation of the NLRP3 inflammasome, which is a key component of the innate inflammatory response. However, its role in trauma-hemorrhage is unknown. Acute HS rat models were performed to determine the influence of acalabrutinib or fenebrutinib on MODS. The activation of BTK, NF-κB and NLRP3 pathways were analyzed by western blot in the kidney. We demonstrated that (a) HS caused organ injury and/or dysfunction and hypotension (post-resuscitation) in rats, while (b) treatment of HS-rats with either acalabrutinib or fenebrutinib attenuated the organ injury and dysfunction in acute HS models and (c) reduced the activation of BTK, NF- kB and NLRP3 pathways in the kidney. Our results point to a role of BTK in the pathophysiology of organ injury and dysfunction caused by trauma/hemorrhage and indicate that BTK inhibitors may be repurposed as a potential therapeutic approach for MODS after trauma and/or hemorrhage.

REC8 enhances stemness and promotes metastasis of colorectal cancer through BTK/Akt/β-catenin signaling pathway

Cancer/testis antigens (CTAs) are often aberrantly expressed in cancer stem cells (CSCs) which are responsible for tumor metastasis. Rec8 meiotic recombination protein (REC8), a member of CTAs, shares distinct roles in various cancers, while its contribution to CSCs and colorectal cancer (CRC) remains unclear. We found that overexpression of REC8 facilitated the migration and invasion of CRC cells (DLD-1 and SW480 cells) in vitro and promoted the liver metastasis of CRC in vivo. Moreover, REC8 is highly expressed in CRC stem-like cells and is required for the maintenance of CSC stemness. Mechanistic studies suggested that REC8 mediated through the activation of Bruton tyrosine kinase (BTK). Inhibition of BTK by ibrutinib not only suppressed the migration and invasion-promoting ability, but also declined the increased expression of p-BTK, p-Akt, β-catenin, and CSC markers upon REC8 overexpression. Importantly, high expression of REC8 in cancerous tissues was related to advanced clinical stage and lymph node metastasis of 62 CRC patients, and REC8 was enriched in the cancerous cells positive for CSC markers. Collectively, our results indicate that REC8 promotes CRC metastasis by increasing cell stemness through BTK/Akt/β-catenin pathway.

Favorable Modulation of Chimeric Antigen Receptor T Cells Safety and Efficacy By the Non-Covalent BTK Inhibitor Vecabrutinib

Favorable Modulation of Chimeric Antigen Receptor T Cells Safety and Efficacy By the Non-Covalent BTK Inhibitor Vecabrutinib

Final Results of a Phase 1/2 Study of SYK Inhibitor Entospletinib in Combination with Obinutuzumab in Patients with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL)

Final Results of a Phase 1/2 Study of SYK Inhibitor Entospletinib in Combination with Obinutuzumab in Patients with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL)

Discovery of 1-Amino-1H-imidazole-5-carboxamide Derivatives as Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitors.

Bruton's tyrosine kinase (BTK) inhibitors suppressing the aberrant activation of BTK have led to a paradigm shift in the therapy of B-cell malignancies. However, there is an urgent need to discover more selective covalent BTK inhibitors owing to the off-target adverse effects of the approved inhibitor, ibrutinib. Herein, we disclose the discovery and preliminary activity studies of novel BTK inhibitors carrying 1-amino-1H-imidazole-5-carboxamide as a hinge binder. The most potent BTK inhibitor 26 demonstrates impressive selectivity, favorable pharmacokinetic properties, and robust antitumor efficacy in vivo, which indicates its potential as a novel therapeutic option for B-cell lymphomas. Importantly, to the best of our knowledge, this is the first example of a 1-amino-1H-imidazole-5-carboxamide scaffold used as the hinge binder of kinase inhibitors, which will largely expand the chemical diversity of kinase inhibitors.

A mechanism for localized dynamics-driven activation in Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) plays a vital role in mature B-cell proliferation, development and function. Its inhibitors have gradually been applied for the treatment of many B-cell malignancies. However, because of treatment-associated drug resistance or low efficacy, it is urgent to develop new inhibitors and/or improve the efficacy of current inhibitors, where finding the intrinsic activation mechanism becomes the key to solve this problem. Here, we used BTK T474M mutation as a resistance model for inhibitors to study the mechanism of BTK activation and drug resistance by free molecular dynamics simulations. The results showed that the increase of kinase activity of T474M mutation is coming from the conformation change of the activation ring and ATP binding sites located in BTK N-terminus region. Specifically, the Thr474 mutation changed the structure of A-loop and stabilized the binding site of ATP, thus promoting the catalytic ability in the kinase domain. This localized dynamics-driven activation mechanism and resistance mechanism of BTK may provide new ideas for drug development in B-cell malignancies.

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.

CG '806, a Highly Potent First-in-Class Pan-FLT3/BTK Inhibitor, Targets Multiple Clusters of Kinases Operative in Diverse Subtypes of Acute Myeloid Leukemia

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/ Bruton's Tyrosine Kinase (BTK) multi-kinase inhibitor. This small molecule was designed to inhibit the wild type (WT) and C481S mutant forms of BTK, as well as the WT and mutant forms (including the internal tandem duplication, ITD) of the FLT3 receptor tyrosine kinase. Previously (2017 AACR Hematologic Malignancies abstract # 25 and #44), CG'806 was shown to have nM potency against FLT3 mutation-driven (including ITD and point mutations in tyrosine kinase domain) human acute myeloid leukemia (AML) primary patient cells, and in cultured human (eg. MV4-11 and MOLM-13) and mouse (Ba/F3) AML cell lines. CG'806 inhibited FLT3 signaling, induced apoptosis, and eradicated MV4-11 tumors with no observed toxicity in murine xenograft models. Because AML cells demonstrate extreme heterogeneity in gene expression and because FLT3 mutations account for no more than 30% of AML patients, the effect of CG'806 was further evaluated in AML cells without FLT3 mutations. Immunoblotting demonstrated that CG'806 potently decreased phospho-BTK, phospho-aurora kinases A/B/C and phospho-H3S10 without affecting FLT3 activity in AML cells having low/normal expression levels of homozygous WT FLT3 (eg. KG-1 and NOMO-1). Measurement of BrdU incorporation by flow cytometry demonstrated that CG'806 induced G2/M cell cycle arrest or polyploidy and subsequent apoptosis in FLT3 WT AML cell lines. Interestingly, CG'806 was found to induce G0/G1 cell cycle arrest but not polyploidy in FLT3-ITD driven MV4-11 and MOLM-13 cells with no effect on activity of BTK or aurora kinases at concentrations that completely inhibited FLT3 signaling (FLT3/STAT5/ERK). These findings indicate that the molecular mechanism of cell killing by CG'806 is context-dependent and varies in different AML subtypes. Evaluation of enzyme inhibitory activity (IC50) against 583 kinase enzymes, as well as binding affinities (Kd) with 483 kinase proteins, revealed pM to low nM IC50 and Kd values for CG'806 against the FLT3, BTK, AURK, c-Src, c-Met, c-Ret, DDR, TRK and STE/MAPK families of kinases. The ability of CG'806 to target multiple families or clusters of kinases is responsible for its broad efficacy in different AML subtypes driven by different genomic aberrations. Together, these findings illustrate the ability of CG'806 to inhibit entire FLT3 family of kinases, BTK and the AURK families and pathways that are operative in AML, thereby supporting the development of CG'806 for treatment of patients with FLT3-ITD and other subpopulations of patients with AML. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Pooled Analysis of Safety Data from Clinical Trials Evaluating Acalabrutinib Monotherapy in Hematologic Malignancies

Pooled Analysis of Safety Data from Clinical Trials Evaluating Acalabrutinib Monotherapy in Hematologic Malignancies

PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura

PRN1008 is an oral, reversible covalent inhibitor of Bruton's tyrosine kinase (BTK) in clinical development for the treatment of multiple autoimmune diseases. BTK is an essential signaling element downstream of the B cell receptor (BCR), Fc-gamma receptor and Fc-epsilon receptor pathways. BTK activation is critical for B cell activation and maturation. BTK also regulates antibody mediated activation of other immune cells, such as macrophages, neutrophils and mast cells through Fc receptor signaling.Treatment with PRN1008 in vitro profoundly inhibited B cell activation and blocked antibody mediated activation of immune cells via Fc receptor signaling. In preclinical studies, PRN1008 demonstrated a significant dose-dependent reduction of platelet-loss in an anti-CD41 induced mouse model of immune thrombocytopenia. In addition, PRN1008 showed rapid and significant anti-inflammatory effects in an antibody driven rat arthus model and in spontaneous autoantibody-mediated canine pemphigus foliaceus. The preclinical data suggest that PRN1008 could reduce platelet destruction via inhibition of autoantibody/Fc-gamma receptor signaling in splenic macrophages, and also affect autoantibody generation through effects on B-cell activation, to diminish platelet loss in ITP.Platelets express high levels of BTK, however alternative signaling pathways exist which bypass BTK signaling to retain normal platelet functions. The effect of PRN1008 on platelet function was assessed in vitro in both normal healthy volunteer and ITP patient platelets using a standard panel of platelet agonists. In contrast to ibrutinib, treatment with PRN1008 at clinically relevant concentrations did not impact collagen-induced platelet aggregation in either normal or ITP patient platelet samples, or interfere with responses to all other platelet agonists tested. In the clinic, PRN1008 has been well tolerated. Thrombocytopenia, bleeding or bruising signals have not been reported. An analysis of platelet counts from the ongoing 12 week study of pemphigus vulgaris (NCT02704429) showed no evidence of an effect of PRN1008 on platelet counts in the normal range.Principia Biopharma is developing PRN1008 for the treatment of autoimmune diseases including ITP, blistering skin diseases such as pemphigus vulgaris and other immunologic disorders where autoantibodies and/or B-cell signaling are pathogenic. DisclosuresLangrish:Principia Biopharma: Employment. Bradshaw:Principia Biopharma: Employment. Owens:Principia Biopharma: Employment. Campbell:Principia Biopharma: Employment. Francesco:Principia Biopharma: Employment. Karr:Principia Biopharma: Employment. Murray:Principia Biopharma: Employment. Quesenberry:Principia Biopharma: Employment. Smith:Principia Biopharma: Consultancy. Taylor:Principia Biopharma: Consultancy. Zhu:Principia Biopharma: Employment. Nunn:Principia Biopharma: Employment. Gourlay:Principia Biopharma: Employment.

CG '806, a First-in-Class Non-Covalent Pan-FLT3/BTK Inhibitor, Exerts Superior Potency Against B-Cell Malignant Cells

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/Bruton's tyrosine kinase (BTK) multi-kinase inhibitor that potently inhibits wild type (WT) and mutant forms of FLT3 and BTK. As described previously (2017 AACR Hematologic Malignancies abstract #25 and #44), CG'806 inhibits on FLT3-ITD-driven acute myeloid leukemia (AML) and other forms of AML. Because CG'806 targets the WT BTK (IC50 = 8 nM) and the C481S-BTK (IC50 = 2.5 nM), which is a mutant form resistant to ibrutinib and other covalent BTK inhibitors, this small molecule was profiled for its actions on various types of B-cell malignancies in which B cell receptor (BCR) and BTK signaling are reported to play pivotal pathogenic roles. Against 10 malignant B-cell lines, while CG'806 reduced the level of BTK-Y223 phosphorylation, it was less potent than ibrutinib. In contrast, CG'806 was 50-6,000 times more potent (lower IC50) in the ability to kill the malignant B cells across all tested cell lines evaluated by MTS-based assays of cell proliferation and inhibition of colony formation. Hence, the efficacy of killing malignant B cells by CG'806 and by ibrutinib did not correlate with the degree of BTK inhibition. Pathway analyses (KinomeScan and immunoblotting) demonstrated that CG'806, in addition to inhibiting intracellular BTK, potently inhibited the activity of aurora kinases A, B and C, phospho-H3S10, FLT3 and ERK that are differentially expressed and operative in various cell lines representing mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), B-acute lymphocytic leukemia (B-ALL) and Burkitt's lymphoma. Indeed, measuring BrdU incorporation and Annexin V by flow cytometry, CG'806 directly induced polyploidy and triggered apoptosis in malignant B cells at picomolar (pM) to low nanomolar (nM) concentrations, while ibrutinib potently inhibited BTK activity but was unable to induce apoptosis at lower than micromolar (µM) concentrations. These data distinguish CG'806 from ibrutinib and other covalent and non-covalent BTK inhibitors, and illustrate that CG'806 has activity across a wide range of malignant B cell lines including those intolerant, refractory or resistant (due to the presence of a BTK-C481S mutation) to ibrutinib and other BTK inhibitors. [Display omitted] DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Predicting Mechanisms of Ibrutinib-Resistance in Chronic Lymphocytic Leukemia (CLL) Using Computational-Based Modeling for Personalized Therapy with Clinical Validation

Background: Bruton's tyrosine kinase (BTK), a mediator of B cell receptor signaling, has been effectively exploited as a therapeutic target in Chronic lymphocytic leukemia (CLL). Ibrutinib is a BTK inhibitor that has shown excellent efficacy in treatment-naïve, relapsed/refractory, and high-risk CLL. However, concerns regarding ibrutinib resistance have emerged and characterizing the underlying mechanisms may aid in identifying patients with increased risk of resistance/disease progression. Moreover, understanding the interplay between determinants of ibrutinib resistance may reveal alternate therapeutic targets. Using a computational biology modeling (CBM) simulation-based method, we reverse-engineered the salient molecular architecture of a CLL patient progressing on ibrutinib to identify potential drug combinations to overcome drug resistance and induce remission.Methods: For validation of the computational biological modeling (CBM) software (Cellworks group), we selected a cohort of 15 ibrutinib resistant CLL patients from previously published studies (Table 1). Genomic data derived from whole exome sequencing (WES), targeted next-generation sequencing (NGS), copy number variation (CNV), and/or karyotype data were used in this study. All available genomic data for each profile was entered into the CBM, which generates a disease-specific protein network map using PubMed and other online resources. Disease-biomarkers that are unique to each patient were identified within the protein network maps by using CBM technology. Ibrutinib was selected from a digital drug library and simulated on every relapsed CLL patient case. Drug impact was assessed by quantitatively measuring the drug's effect on a cell growth score, which is a composite of cell proliferation, viability, and apoptosis, along with the simulated impact on each disease-specific biomarker score. Each patient-specific map was digitally screened to identify any Ibrutinib resistance mechanism(s).Results: In our modeled patient set, 4 different resistance mechanisms were identified across 14/15 ibrutinib- refractory CLL cases. 1) BTK C481S mutation (n=4) impacts ibrutinib binding affinity; 2) PLCG2 mutations (n=4) lead to activation of PRKCB and AKT1 signaling; 3) CARD11 mutation (n=2) results in constitutive activation of BTK downstream effectors; and 4) decreased G-protein coupled receptor (GPCR) pathway signaling, an upstream activator of BTK (n=5), was found to be a novel resistance mechanism identified by CBM. Knockdown (KD) of ALOX5AP, ARRB1 and CYSLTR2 reduced GPCR pathway signaling, resulting in reduced BTK activity (Table 1). After validating the CBM algorithm, we rendered a digital avatar for a relapsed-refractory CLL patient (Del17p+); progressing on ibrutinib. The patient had rapidly escalating ALC and lymphadenopathy; with the largest node measuring ~16cm. In addition to p53 loss, simulation modeling accounted for BTK C481S mutation (BAF 0.69) expressed in the patients tumor cells. CBM predicted venetoclax would be the most effective therapeutic for the patient due to several upstream signaling pathways converging on Bcl-2. Indeed, addition of venetoclax to the ongoing ibrutinib monotherapy dramatically reduced the patients ALC (from 113 to 3.3 x 109/L) by day 4 post-treatment, accompanied by reduction in lymphadenopathy (left axillary node decreased to ~3cm). Ibrutinib was discontinued within 1 month of starting the combination, with venetoclax monotherapy continued per dosing guidelines thereafter. Notably, the patient maintained remission status on venetoclax monotherapy alone.Conclusions: Computational modeling was used to identify ibrutinib resistance mechanisms in CLL patients. Using this novel approach, we identified an effective therapy (venetoclax) for a high-risk CLL patient who was progressing on ibrutinib. CBM-based approaches can be used to predict alternative therapeutic regimens for drug-refractory patients, targeting the disease-specific biomarkers unique to each patient. Altogether, our study demonstrates proof-of-principle on the use of computational predictive algorithms to simulate multiple protein-interaction networks operational in a tumor cell to exploit individualized therapeutic vulnerabilities. [Display omitted] DisclosuresYsebaert:Janssen: Consultancy, Research Funding, Speakers Bureau. Kumar:Cellworks: Employment. Singh:Cellworks Research India Pvt. Ltd: Employment. Ullal:Cellworks Research India: Employment. Bhargav:Cellworks: Employment. Azam:cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

The HCK/BTK inhibitor KIN-8194 is active in MYD88-driven lymphomas and overcomes mutated BTKCys481 ibrutinib resistance

AbstractActivating mutations in MYD88 promote malignant cell growth and survival through hematopoietic cell kinase (HCK)–mediated activation of Bruton tyrosine kinase (BTK). Ibrutinib binds to BTKCys481 and is active in B-cell malignancies driven by mutated MYD88. Mutations in BTKCys481, particularly BTKCys481Ser, are common in patients with acquired ibrutinib resistance. We therefore performed an extensive medicinal chemistry campaign and identified KIN-8194 as a novel dual inhibitor of HCK and BTK. KIN-8194 showed potent and selective in vitro killing of MYD88-mutated lymphoma cells, including ibrutinib-resistant BTKCys481Ser-expressing cells. KIN-8194 demonstrated excellent bioavailability and pharmacokinetic parameters, with good tolerance in rodent models at pharmacologically achievable and active doses. Pharmacodynamic studies showed sustained inhibition of HCK and BTK for 24 hours after single oral administration of KIN-8194 in an MYD88-mutated TMD-8 activated B-cell diffuse large B-cell lymphoma (ABC DLBCL) and BCWM.1 Waldenström macroglobulinemia (WM) xenografted mice with wild-type BTK (BTKWT)– or BTKCys481Ser-expressing tumors. KIN-8194 showed superior survival benefit over ibrutinib in both BTKWT- and BTKCys481Ser-expressing TMD-8 DLBCL xenografted mice, including sustained complete responses of >12 weeks off treatment in mice with BTKWT-expressing TMD-8 tumors. The BCL_2 inhibitor venetoclax enhanced the antitumor activity of KIN-8194 in BTKWT- and BTKCys481Ser-expressing MYD88-mutated lymphoma cells and markedly reduced tumor growth and prolonged survival in mice with BTKCys481Ser-expressing TMD-8 tumors treated with both drugs. The findings highlight the feasibility of targeting HCK, a key driver of mutated MYD88 pro-survival signaling, and provide a framework for the advancement of KIN-8194 for human studies in B-cell malignancies driven by HCK and BTK.

Acalabrutinib: a highly selective, potent Bruton tyrosine kinase inhibitor for the treatment of chronic lymphocytic leukemia

Inhibiting the activity of Bruton tyrosine kinase (BTK) prevents the activation of the B‐cell receptor (BCR) signaling pathway, which in turn prevents both B‐cell activation and BTK‐mediated activation of downstream survival pathways. Acalabrutinib is an orally available, highly selective, next-generation inhibitor of BTK. Based on the results of two key phase 3 trials (ELEVATE-TN in patients with previously untreated chronic lymphocytic leukemia [CLL] and ASCEND in patients with relapsed or refractory CLL), which demonstrated superior progression-free survival while maintaining favorable tolerability, acalabrutinib was granted US Food and Drug Administration (FDA) approval in 2019 for the treatment of patients with CLL. Acalabrutinib appears to offer similar efficacy but a significantly improved tolerability profile to first-generation agents. Acalabrutinib is a good candidate to combine with other anti-cancer therapies, including B-cell lymphoma 2 inhibitors and monoclonal antibodies, a factor that may help to further improve clinical outcomes in CLL.

Relevance of the Bruton Tyrosine Kinase as a Target for COVID-19 Therapy.

The outbreak of the novel coronavirus disease 2019 (COVID-19) has emerged as one of the biggest global health threats worldwide. As of October 2020, more than 44 million confirmed cases and more than 1,160,000 deaths have been reported globally, and the toll is likely to be much higher before the pandemic is over. There are currently little therapeutic options available and new potential targets are intensively investigated. Recently, Bruton tyrosine kinase (BTK) has emerged as an interesting candidate. Elevated levels of BTK activity have been reported in blood monocytes from patients with severe COVID-19, compared with those from healthy volunteers. Importantly, various studies confirmed empirically that administration of BTK inhibitors (acalabrutinib and ibrutinib) decreased the duration of mechanical ventilation and mortality rate for hospitalized patients with severe COVID-19. Herein, we review the current information regarding the role of BTK in severe acute respiratory syndrome coronavirus 2 infections and the suitability of its inhibitors as drugs to treat COVID-19. The use of BTK inhibitors in the management of COVID-19 shows promise in reducing the severity of the immune response to the infection and thus mortality. However, BTK inhibition may be contributing in other ways to inhibit the effects of the virus and this will need to be carefully studied.

Chemoproteomic Profiling of an Ibrutinib Analogue Reveals its Unexpected Role in DNA Damage Repair.

Ibrutinib is an FDA-approved drug to treat B-lymphoid malignancies, which functions mechanistically as a covalent inhibitor for Bruton's tyrosine kinase (BTK). During the course of screening more potent and selective BTK inhibitors, we discovered that MM2-48, an ibrutinib analogue that contains the alkynyl amide functional group in place of the acrylamide warhead, exhibits a much stronger cytotoxicity. Comparative chemoproteomic profiling of the targets of ibrutinib and MM2-48 revealed that the alkynyl amide warhead exhibits much higher reactivity in proteomes. Unexpectedly, MM2-48 covalently targets a functional cysteine in a BRCA2 and CDKN1A-interacting protein, BCCIP, and significantly inhibits DNA damage repair. Our findings suggest that simultaneous inhibition of BTK activity and DNA damage repair might be a more effective therapeutic strategy for combating B-cell malignancies.

Targeting phosphatidylinositol 3 kinase-β and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma

AbstractDiffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse after a complete response or are refractory to therapy. To survive, the activated B-cell (ABC) subtype of DLBCL relies upon B-cell receptor signaling, which can be modulated by the activity of Bruton tyrosine kinase (BTK). Targeting BTK with ibrutinib, an inhibitor, provides a therapeutic approach for this subtype of DLBCL. However, non-Hodgkin lymphoma is often resistant to ibrutinib or acquires resistance soon after exposure. We explored how this resistance develops. We generated 3 isogenic ibrutinib-resistant DLBCL cell lines and investigated the deregulated pathways known to be associated with tumorigenic properties. Reduced levels of BTK and enhanced phosphatidylinositol 3-kinase (PI3K)/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of PI3K-β expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-β/δ. Treatment with the selective PI3K-β/δ dual inhibitor KA2237 reduced both tumorigenic properties and survival-based PI3K/AKT/mTOR signaling of these ibrutinib-resistant cells. In addition, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited metabolic growth. This study elucidates the compensatory upregulated PI3K/AKT axis that emerges in ibrutinib-resistant cells.

Inhibition of Bruton tyrosine kinase by acalabrutinib dampens lipopolysaccharide/galactosamine-induced hepatic damage

Bruton tyrosine kinase (BTK) sits at the crossroads of adaptive and innate immunities. Nevertheless, the detailed role of BTK activation in hepatic inflammatory disorders is still elusive to date. Accordingly, we investigated the impact of blocking BTK activation by acalabrutinib (ACB) on lipopolysaccharide/galactosamine (LPS/D-GaIN)-induced deleterious manifestations in the liver. This was achieved by pretreating mice with ACB (6, 12 or 24 mg/kg, oral) 2 h before challenge with LPS/D-GaIN (70 μg/kg and 700 mg/kg, respectively, i.p.) for 6 h. The results showed that ACB (6 and 12 mg/kg) (i) curbed LPS/D-GaIN-induced rise in biochemical (serum ALT, AST and LDH) and histological (necrosis, degeneration and congestion scores) indices of hepatocellular injury; (ii) attenuated LPS/D-GaIN-induced elevation in parameters of hepatocellular apoptosis (cleaved caspase 3) and proliferation (PCNA); and (iii) importantly, mitigated LPS/D-GaIN-induced recruitment and infiltration of the inflammatory cells to the liver evidenced by lowering elevated serum MCP-1 concentration and hepatic F4/80 immunostaining. These effects were linked to ACB dose-dependent inhibition of NF-κB nuclear translocation that subsequently reduced LPS/D-GaIN-mediated release of TNF-α, IL-1β and IL-22 in the blood circulation. However, a dose of 12 mg/kg of ACB elevated the hepatic TNF-α, IL-1β and IL-22 concentrations that arose from a compensatory activation of ERK and JNK. Inhibition of BTK also attenuated LPS/D-GaIN-induced overexpression of CD98, which is another contributor alongside cytokines for monocyte recruitment. Therapeutically, targeting BTK by ACB is an efficient approach for hitting multiple points with one agent that can dampen hepatocellular injury, death, immune cell recruitment and inflammation cascade.

Driving Potency with Rotationally Stable Atropisomers: Discovery of Pyridopyrimidinedione-Carbazole Inhibitors of BTK.

Bruton's tyrosine kinase (BTK) has been shown to play a key role in the pathogenesis of autoimmunity. Therefore, the inhibition of the kinase activity of BTK with a small molecule inhibitor could offer a breakthrough in the clinical treatment of many autoimmune diseases. This Letter describes the discovery of BMS-986143 through systematic structure-activity relationship (SAR) development. This compound benefits from defined chirality derived from two rotationally stable atropisomeric axes, providing a potent and selective single atropisomer with desirable efficacy and tolerability profiles.

Abstract IA13: Treatment of COVID-19 pulmonary failure by targeting BTK

Abstract Pulmonary failure is the main cause of mortality related to COVID-19 infection. Up to 80% of patients hospitalized for COVID-19 infection require supplemental oxygenation, of whom 30-40% may require mechanical ventilation. SARS-CoV-2 binds via the ACE2-receptor that is highly expressed on alveolar type II (ATII) cells in the lung. ATII cells constitute 5-15% of the lung epithelium. While alveolar type I cells are highly adapted for gas exchange, alveolar type II cells have a specialized role in innate immune response. ATII cells express Toll receptors (TLRs) and can trigger inflammatory cytokines and chemoattractants in response to pathogens that recruit and activate other immune cells, including macrophages and neutrophils. We and others previously showed that BTK, and its upstream activator HCK, were involved in TLR-mediated signaling. Both BTK and HCK are triggered by MYD88, a TLR-adaptor protein that signals for all Toll receptors except TLR3 in response to viral and bacterial pathogens, including coronaviruses. ATII cells express TLRs, as do alveolar macrophages that coordinate inflammatory responses with ATII cells. As components of TLR/MYD88 signaling, BTK and HCK can drive inflammatory cytokine production through ERK1/2. The potential for BTK inhibitors to abrogate lung injury and death was demonstrated in an experimental model wherein mice challenged with a lethal intranasal inoculum of a mouse-adapted strain of H1N1 influenza virus were protected against lung injury. Control mice developed respiratory failure, along with histologic and CT findings consistent with lung injury in sharp contrast to the mice that received ibrutinib. Control mice also lost weight and died, whereas those treated with ibrutinib recovered their weight after a brief loss and all survived. Mice treated with ibrutinib also showed decreased inflammatory cell infiltration as well as proinflammatory cytokines in lung tissues that included proinflammatory and chemoattractant cytokines such as IL-1β, IL-6, KC/CXCL1, TNFα, and MCP-1 observed in SARS-Cov-1 and SARS-CoV-2 patients. In a series of Waldenstrom’s macroglobulinemia (WM) patients who were on full-dose ibrutinib (420 mg/day) and contracted COVID-19, none experienced dyspnea. In this series, one WM patient who contracted COVID-19 on reduced-dose ibrutinib (140 mg/day) experienced dyspnea and became hypoxic when ibrutinib was stopped and required mechanical ventilation. He was started on full-dose ibrutinib and showed rapid improvement, was extubated the next day, and did not require any oxygen supplementation two days later. Consistent with his rapid course of improvement on ibrutinib, his C-reactive protein level showed a marked decrease. In a subsequent and larger experience, Roschewski and colleagues administered acalabrutinib to 19 COVID-19 patients, 11 on supplemental oxygenation and 8 on mechanical ventilation. Eight of 11 (73%) and 2/8 (25%) on supplemental oxygenation and mechanical ventilation were discharged on room air, respectively. Ex vivo analysis of peripheral blood samples showed attenuation of activated monocyte BTK expression and IL-6 production following acalabrutinib. Serially collected blood samples from patients with chronic lymphocytic leukemia (CLL), WM, and chronic graft-versus-host disease (cGVHD) on ibrutinib monotherapy also showed marked reductions in proinflammatory and chemoattractant cytokines that greatly overlapped with those reported elevated in the plasma of SARS-Cov-1 and SARS-COV-2 patients, and in ACE2+ cells from lung tissue of SARS-CoV-1 patients. In the iLLUMINATE randomized study, CLL subjects treated with ibrutinib immediately prior to infusion with obinutuzumab also showed significantly decreased levels of inflammatory cytokines associated with infusion related reactions (a cytokine release syndrome). These findings are consistent with a shift from an M1 to M2 polarized macrophage response following ibrutinib and are supported by preclinical and clinical studies showing dependence of macrophage lineage commitment on BTK function. BTK-inhibitors may therefore provide protection against lung injury and even improve pulmonary function in hypoxic patients with COVID-19. Randomized clinical trials to examine the benefit of BTK-inhibitors in COVID-19 patients in pulmonary distress and/or accompanying corollary studies to identify response biomarkers have been initiated: NCT04439006, NCT04382586, NCT04346199, NCT04380688. Selected