Bruton's Tyrosine Kinase Protein Research Articles

Novel 5-Substituted Oxindole Derivatives as Bruton's Tyrosine Kinase Inhibitors: Design, Synthesis, Docking, Molecular Dynamics Simulation, and Biological Evaluation.

Bruton's tyrosine kinase (BTK) is a non-RTK cytoplasmic kinase predominantly expressed by hemopoietic lineages, particularly B-cells. A new oxindole-based focused library was designed to identify potent compounds targeting the BTK protein as anticancer agents. This study used rational approaches like structure-based pharmacophore modeling, docking, and ADME properties to select compounds. Molecular dynamics simulations carried out at 20 ns supported the stability of compound 9g within the binding pocket. All the compounds were synthesized and subjected to biological screening on two BTK-expressing cancer cell lines, RAMOS and K562; six non-BTK cancer cell lines, A549, HCT116 (parental and p53-/-), U2OS, JURKAT, and CCRF-CEM; and two non-malignant fibroblast lines, BJ and MRC-5. This study resulted in the identification of four new compounds, 9b, 9f, 9g, and 9h, possessing free binding energies of -10.8, -11.1, -11.3, and -10.8 kcal/mol, respectively, and displaying selective cytotoxicity against BTK-high RAMOS cells. Further analysis demonstrated the antiproliferative activity of 9h in RAMOS cells through selective inhibition of pBTK (Tyr223) without affecting Lyn and Syk, upstream proteins in the BCR signaling pathway. In conclusion, we identified a promising oxindole derivative (9h) that shows specificity in modulating BTK signaling pathways.

Preclinical Efficacy and Mechanism for a Novel BTK-Protacs Against Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL) that exhibits diverse clinical courses and biological contexts. Given the significance of Bruton tyrosine kinase (BTK) as a therapeutic target in MCL, this study aimed to design a range of BTK-PROTACs. The objective was to develop efficient BTK-PROTACs capable of degrading BTK proteins, inhibiting the proliferation of MCL cells, and overcoming resistance to both covalent and non-covalent BTK inhibitors in both in vivo and in vitro settings. Methods:A series of BTK-PROTACs were gradually screened by Western blot and MTS assays, and the best active drug was finally determined. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. RNA sequencing identified differentially expressed genes and important signaling pathways that influenced by BTK-PROTACs. BTKi-resistant cell lines were constructed to evaluate the ability of BTK-PROTACs to degrade BTK protein and suppress the proliferation of MCL cells. R esults: BTK-PROTACs C23 demonstrated effective degradation of BTK protein and inhibition of tumor cell growth across various MCL cell lines. Additionally, C23 exhibited the ability to induce cell apoptosis, activate the ubiquitin pathway, and inhibit the B-cell receptor (BCR), NF-κB, and PI3K-AKT-mTOR signaling pathways. Importantly, C23 effectively degraded BTK in both Z138-BTK C481S and Z138-BTK L528W cells, suggesting its potential to overcome resistance to both covalent and non-covalent BTK inhibitors. In our in vivo experiments using MCL xenograft NSG mouse models, BTK-PROTACs C23 significantly impeded tumor proliferation in Z138-BTK WT, Z138-BTK C481S, and Z138-BTK L528W MCL models. Additionally, C23 induced the degradation of BTK in tumor tissues. Notably, HE staining revealed no significant adverse effects of BTK-PROTACs C23 on normal organs and tissues. Moreover, routine blood tests and liver function tests showed no impairment of hematopoietic or hepatic function caused by BTK-PROTACs C23. C onclusion: In this study, we successfully designed and synthesized BTK-PROTACs C23, which demonstrated a remarkable ability to degrade BTK protein and effectively suppress the proliferation of MCL cells both in vitro and in vivo. Importantly, BTK-PROTACs C23 showed promising potential to overcome resistance to both covalent and non-covalent BTK inhibitors.

Initial Findings from a First-in-Human Phase 1a/b Trial of NX-5948, a Selective Bruton's Tyrosine Kinase (BTK) Degrader, in Patients with Relapsed/Refractory B Cell Malignancies

Introduction: Although BTK inhibitors (BTKi) are effective therapeutics in the treatment of B cell malignancies, emerging BTK resistance mutations in chronic lymphocytic leukemia (CLL), as well as potential growth-promoting kinase-independent scaffolding function of BTK, present a need for improved or new approaches. NX-5948 is a novel, orally administered small molecule that induces specific BTK protein degradation by the cereblon E3 ligase complex without degradation of other cereblon neo-substrates. Importantly, NX-5948 induces degradation of wild-type and mutant forms of BTK in B-cells [Noviski et al. 2023] at sub-nanomolar potencies and exhibits potent tumor growth inhibition in TMD8 xenograft models that contain either wild-type BTK or BTKi-resistant mutations [Robbins 2021]. Here we provide the first disclosure of preliminary safety and efficacy findings from a Phase 1a trial of NX-5948 in patients with relapsed/refractory B cell malignancies. Methods: NX-5948-301 is a Phase 1, first-in-human, dose-escalation and cohort-expansion trial evaluating the safety, tolerability, and clinical activity of NX-5948 in relapsed/refractory CLL and various subtypes of non-Hodgkin's lymphoma (NHL). Key eligibility criteria: ≥2 prior lines of therapy; measurable or other evaluable disease per indication-specific response criteria; Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0-1. Phase 1a (dose escalation) evaluates safety and tolerability of NX-5948 via a standard 3+3 dose escalation in patients with relapsed/refractory B cell malignancies. Approximately 110 patients (30 in Phase 1a, 80 in Phase 1b) may be enrolled and treated until confirmed disease progression or unacceptable toxicity. Endpoints include dose-limiting toxicities (DLTs); treatment-emergent adverse events (TEAEs); deaths; changes in safety parameters; objective response rate per disease-specific response criteria. Phase 1b (dose expansion) will include up to four expansion cohorts. Results: As of June 9, 2023, 14 patients were enrolled in Phase 1a and received NX-5948 at 50 mg (n=7), 100 mg (n=4), or 200 mg (n=3) orally once daily. Median age was 65 (range 46-79) years; female/male ratio 28.6%/71.4%; white 92.9%; ECOG PS 0/1 21.4%/78.6%; primary diagnoses were CLL (n=4), diffuse large B cell lymphoma (DLBCL, n=4), mantle cell lymphoma (MCL, n=3), marginal zone lymphoma (MZL, n=2), and follicular lymphoma (FL, n=1). Median number of prior therapies was 4.5 (range 2-10), which included: for CLL - BTKi (n=4/4) and BCL2 inhibitor (n=3/4); for NHL - BTKi (n=5/10), bispecific antibody (n=3/10), and CAR-T (n=2/10). NX-5948 was well tolerated with no DLTs and no TEAEs resulting in drug discontinuation or dose reduction. In addition, there were no NX-5948-related grade ≥3 TEAEs or related serious adverse events. The most common TEAEs were purpura/contusion (57.1%, all below grade 3), nausea (35.7%), and thrombocytopenia (35.7%). No atrial fibrillation/flutter or hypertension was reported. Median duration of treatment was 2.8 (range 0.5-9.6) months with 9/14 patients remaining on treatment. Current data indicate that NX-5948 exhibits dose-dependent pharmacokinetics (PK) and a half-life of ~24 hours, supporting once daily dosing (Figure a). Rapid, robust and sustained BTK degradation was observed in all patients, regardless of absolute BTK starting level, tumor type, or NX-5948 dose (Figure b). Of three evaluable patients with CLL receiving the lowest dose of 50 mg, early signs of clinical activity were observed including one confirmed partial response (PR; at 8 and 16 weeks) and 2 patients with stable disease (SD; at 8 weeks). Further treatment responses will be reported at the time of presentation. Summary/conclusion: Current findings in this heavily pre-treated population of patients with CLL and NHL are encouraging and indicate that NX-5948 is safe and well tolerated and has clinical activity, supporting continuation of its development in CLL and NHL. NX-5948 also exhibits dose-proportional PK, resulting in rapid, robust and sustained BTK degradation. Additional data with higher dose levels and longer treatment duration will be presented at the meeting.

Clinical features and mutational analysis of X-linked agammaglobulinemia patients in Malaysia.

Bruton's tyrosine kinase (BTK) is a cytoplasmic protein involved in the B cell development. X-linked agammaglobulinemia (XLA) is caused by mutation in the BTK gene, which results in very low or absent B cells. Affected males have markedly reduced immunoglobulin levels, which render them susceptible to recurrent and severe bacterial infections. Methods: Patients suspected with X-linked agammaglobulinemia were enrolled during the period of 2010-2018. Clinical summary, and immunological profiles of these patients were recorded. Peripheral blood samples were collected for monocyte BTK protein expression detection and BTK genetic analysis. The medical records between January 2020 and June 2023 were reviewed to investigate COVID-19 in XLA. Twenty-two patients (from 16 unrelated families) were molecularly diagnosed as XLA. Genetic testing revealed fifteen distinct mutations, including four splicing mutations, four missense mutations, three nonsense mutations, three short deletions, and one large indel mutation. These mutations scattered throughout the BTK gene and mostly affected the kinase domain. All mutations including five novel mutations were predicted to be pathogenic or deleterious by in silico prediction tools. Genetic testing confirmed that eleven mothers and seven sisters were carriers for the disease, while three mutations were de novo. Flow cytometric analysis showed that thirteen patients had minimal BTK expression (0-15%) while eight patients had reduced BTK expression (16-64%). One patient was not tested for monocyte BTK expression due to insufficient sample. Pneumonia (n=13) was the most common manifestation, while Pseudomonas aeruginosa was the most frequently isolated pathogen from the patients (n=4). Mild or asymptomatic COVID-19 was reported in four patients. This report provides the first overview of demographic, clinical, immunological and genetic data of XLA in Malaysia. The combination of flow cytometric assessment and BTK genetic analysis provides a definitive diagnosis for XLA patients, especially with atypical clinical presentation. In addition, it may also allow carrier detection and assist in genetic counselling and prenatal diagnosis.

Discovery of Ibrutinib-based BTK PROTACs with in vivo anti-inflammatory efficacy by inhibiting NF-κB activation

As a critical upstream regulator of nuclear factor-κB (NF-κB) activation, Bruton's tyrosine kinase (BTK) has been identified to be an effective therapeutic target for the treatment of acute or chronic inflammatory diseases. Herein, we describe the design, synthesis and structure-activity-relationship analysis of a novel series of Ibrutinib-based BTK PROTACs by recruiting Cereblon (CRBN) ligase. Among them, compound 15 was identified as the most potent degrader with a DC50 of 3.18 nM, significantly better than the positive control MT802 (DC50 of 63.31 nM). Compound 15 could also degrade BTK protein in Lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and suppress the mRNA expression and secretion of proinflammatory cytokines such as IL-1β and IL-6 by inhibiting NF-κB activation. Furthermore, compound 15 reduced inflammatory responses in a mouse zymosan-induced peritonitis (ZIP) model. Our findings demonstrated for the first time that targeting BTK degradation by PROTACs might be an alternative option for the treatment of inflammatory disorders, and compound 15 represents one of the most efficient BTK PROTACs (DC50 = 3.18 nM; Dmax = 99.90%; near 100% degradation at 8 h) reported so far and could serve as a lead compound for further investigation as an anti-inflammatory agent.

Simulation-Based Research on Phytoconstituents of Embelia ribes Targeting Proteins with Pathophysiological Implications in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a heterogeneous inflammatory disease with an autoimmune origin and an incompletely elucidated pathophysiological mechanism. RA pharmacotherapy is based on chemically or biologically active substances that provide clinical alleviation and remission, but the disease is still incurable. As a result, there remains a need for significant therapeutic development, and adjuvant therapies may play an essential role in the search for novel RA treatment strategies. The aim of the present study was to investigate potential phytocompounds and phytocompound derivates as RA treatment agents, using in silico methodologies. In this regard, five phytoconstituents identified in different structures of Embelia ribes were evaluated by in silico methods for their potential action on target proteins of therapeutic interest in RA. The methodology involved identifying the phytocompound with the highest binding toward the target protein via molecular docking using AutoDock Vina 1.5.7, followed by a ligand-based virtual screening based on the structure of the most promising phytocompound using SwissSimilarity. This process led to the identification of ligands that are not currently utilized in medical practice, but that might have the potential to be used in the management of RA after further extensive experimental endorsements. ZINC000004024651 showed the highest binding affinity for the Bruton's tyrosine kinase protein, followed by ZINC000000434197 for p38 mitogen-activated protein kinases, ZINC000087606977 for interleukin-1 receptor-associated kinase 4, and ZINC000014728393 for matrix metallopeptidase 9, the latter two showing higher affinity than the co-crystallized compound. The relatively high affinities to target proteins and the pharmacokinetic data obtained by in silico studies using SwisADME suggest a first step for the inclusion of promising new compounds in various more advanced studies, leading to the evaluation of efficacy and safety profiles.

Discovery of Novel Bruton's Tyrosine Kinase PROTACs with Enhanced Selectivity and Cellular Efficacy.

Bruton's tyrosine kinase (BTK) is a target for treating B-cell malignancies and autoimmune diseases, and several BTK inhibitors are already approved for use in humans. Heterobivalent BTK protein degraders are also in development, based on the premise that proteolysis targeting chimeras (PROTACs) may provide additional therapeutic benefits. However, most BTK PROTACs are based on the BTK inhibitor ibrutinib raising concerns about their selectivity profiles, given the known off-target effects of ibrutinib. Here, we disclose the discovery and in vitro characterization of BTK PROTACs based on the selective BTK inhibitor GDC-0853 and the cereblon recruitment ligand pomalidomide. PTD10 is a highly potent BTK degrader (DC50 0.5 nM) that inhibited cell growth and induced apoptosis at lower concentrations than the two parent molecules, as well as three previously reported BTK PROTACs, and had improved selectivity compared to ibrutinib-based BTK PROTACs.

Base editor-mediated correction of a BTK Mutation in XLA Patient Hematopoietic Stem Cells restores Human B cell development

IntroductionX-linked agammaglobulinemia (XLA) due to Bruton's tyrosine kinase (BTK) mutations causes lack of circulating mature B lymphocytes (<1%), lack of serum immunoglobulins (IgG/A/M) and increased susceptibility to infections. Correction of BTK mutation in hematopoietic stem/progenitor cells (HSPC) should restore B cell development and humoral immunity. Here, we develop a novel approach that leverages adenine base editors (ABEs) to correct HSPCs from a XLA patient. Materials and methodsPeripheral blood HSPCs were collected from an XLA BTK:c.46C>T (p.Gln16Ter) patient and healthy donors (HD) after G-CSF/plerixafor mobilization. HSPCs were cultured ex vivo (StemSpan™ SFEM II with SCF, FLT3L, TPO) for 2 days before base editing (BE) with mRNA-encoded ABEs (CELLSCRIPT LLC) and gRNAs (Synthego) including ABE8e-SpRY with a gRNA that positions the target adenine in nucleotide 6 of the target site (A6), and ABE8e-SpG or ABE8e-SpCas9-NG with gRNA A7. BE-HSPCs were transplanted two days later into NSG mice conditioned with busulfan (20 mg/kg). Analyses were performed at 12, 16 weeks and animals sacrificed at 20 weeks to assess engraftment and B cell immune reconstitution. ResultsHSPCs BE with ABE8e-SpRY/gRNA A6(SpRY/A6) achieved highest correction at 86.6% BTKc.46C >T and restored BTK protein expression in ex vivo differentiated (14 days, DMEM+G-CSF) CD33+ myeloid cells (Fig.1A) which restored peripheral blood (PB) CD19+CD20+ B cells to HD level (Fig.1C). Phenotype analysis of PB confirmed predominantly mature naïve B cells in BE comparable with HD instead and mostly transitional B cells in mice with naïve-XLA cells (Fig.1F) with CD179a+ that corresponded to Pre-B cells (Fig.1G). Analysis of bone marrow confirmed restored immature (BE: 16.3 vs naïve:1.0, p = 0.001) and mature B cells (BE: 1.2 vs naïve:0.11 p = 0.002) in mice with BE-HSPCs to HD levels while naïve mice comprised of mostly Pre-B-II cells (mean BE: 64.7% vs naïve:81.3%, p = 0.0006). ConclusionsOur studies demonstrate a novel base editing strategy for a XLA missense mutation that overcame the bone marrow arrest of B cells to restore mature B cells in humanized NSG peripheral blood. These findings demonstrate preclinical efficacy of an ABE-based treatment for BTK. The highly efficient-specific correction with base editing holds great promise for gene therapy for XLA disease. [Display omitted]

Abstract CT128: Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies

Abstract BACKGROUND: BTK, a non-receptor kinase in the B-cell receptor signaling pathway, plays an essential role in B-cell development and differentiation. BTK is critical for malignant B-cell survival. Oral BTK inhibitors (BTKis), standard treatments for patients (pts) with B-cell malignancies, prevent proliferation and induce apoptosis of B cells. Currently approved BTKis covalently bind to BTK residue C481, irreversibly inhibiting phosphorylation of downstream kinases and blocking B-cell activation. Because these agents are often given continuously, their use may lead to clinical resistance and unacceptable toxicities. Indeed, acquired resistance to covalent BTKis is common and caused by the expansion of clones with a cysteine-to-serine mutation at residue 481. A novel strategy being developed to overcome BTKi resistance, is PROTAC-induced degradation of BTK. PROTACs are small molecules with one domain that binds the target protein linked to another domain that binds to ubiquitin E3 ligase, resulting in ubiquitin-dependent proteasome degradation of the target protein. HSK29116 is a small BTK PROTAC molecule that is currently undergoing clinical investigation for the treatment of B-cell malignancies. In preclinical studies, HSK29116 overcame drug resistance caused by the C481S BTK mutation. Also, HSK29116 has demonstrated inhibition of B-cell proliferation without measurable loss of other BTK-family kinase activities. This kinase selectivity may limit some of the off-target toxicities seen with currently available BTKis. METHODS: This first-in-human, multicenter, open-label, phase 1 study (NCT04861779) began recruiting in 2021 to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of oral HSK29116. Eligible pts are ≥18 years old with diagnosis of a B-cell malignancy that has relapsed or become refractory to standard therapy (≥2 prior systemic therapies, which may have included a BTKi) and for which no other treatments known to provide clinical benefit exist. Pts must have an Eastern Cooperative Oncology Group performance status ≤2 and life expectancy &amp;gt;3 months. Phase 1a includes dose escalation to identify dose-limiting toxicities and establish the maximum tolerated dose and/or recommended dose of HSK29116 for a phase 1b dose expansion, initiating in the US. Approximately 36 pts will be enrolled in phase 1a and 90 pts in phase 1b (≥40 pts must have previously received covalent BTKis). The primary endpoint is safety. Secondary endpoints include PK and investigator-assessed overall response rate, duration of response, time to response, and progression-free survival. Exploratory analyses will evaluate the relationship between HSK29116 anti-tumor activity and BTK gene mutations, as well as BTK protein degradation effects of HSK29116. Descriptive statistics will be used to summarize all data. Citation Format: Jianyong Li, Wei Xu, Pangke Yan, Yong Cao, Mengyue Hu, William Daley. Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT128.

Rare deleterious mutations in Bruton’s tyrosine kinase as biomarkers for ibrutinib-based therapy: an in silico insight

Squamous cell carcinoma (SCC) is the second most common type of skin cancer caused by malignant keratinocytes. Multiple studies have shown that protein mutations have a significant impact on the development and progression of cancer, including SCC. We attempted to decode the effect of single amino acid mutations in the Bruton's tyrosine kinase (BTK) protein in this study. Molecular dynamic (MD) simulations were performed on selected deleterious mutations of the BTK protein, revealing that the variants adversely affect the protein, indicating that they may contribute to the prognosis of SCC by making the protein unstable. Then, we investigated the interaction between the protein and its mutants with ibrutinib, a drug designed to treat SCC. Even though the mutations have deleterious effects on protein structure, they bind to ibrutinib similarly to their wild type counterpart. This study demonstrates that the effect of detected missense mutations is unfavorable and can result in function loss, which is severe for SCC, but that ibrutinib-based therapy can still be effective on them, and the mutations can be used as biomarkers for Ibrutinib-based treatment. Seven different computational techniques were used to compute the effect of SAVs in accordance with the experimental requirements of this study. To understand the differences in protein and mutant dynamics, MD simulation and trajectory analysis, including RMSD, RMSF, PCA, and contact analysis, were performed. The free binding energy and its decomposition for each protein-drug complex were determined using docking, MM-GBSA, MM-PBSA, and interaction analysis (wild and mutants).

Novel BTK Mutations Conferring Resistance to Non-Covalent BTK Inhibitors and Alternative Treatment Strategy

Inhibitors of Bruton tyrosine kinase (BTK) denote an important class of molecules for treatment of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. While resistance to covalent BTK inhibitors (BTKi) is predominantly mediated by BTK mutations at cysteine 481 and activating PLCG2 mutations, mechanisms of resistance to non-covalent BTKi are not fully characterized. Recently, Wang E et al. (NEJM 2022) identified novel BTK mutations in patients resistant to the non-covalent BTKi pirtobrutinib. Here，we generated and comprehensively characterized BTK and PLCG2 mutations conferring resistance to ibrutinib and five different non-covalent BTKi namely pirtobrutinib (LOXO-305), vecabrutinib (SNS-062), nemtabrutinib (ARQ-531), fenebrutinib (GDC-0853), and RN-486. Long-term in vitro dose escalation method was performed to generate resistance using multiple parallel lines of mantle cell lymphoma REC-1 cells. REC-1 were selected due to their high sensitivity to BTKi. Targeted next generation sequencing was used to identify acquired mutations in BTK and PLCG2. While all the six lines of REC-1 cells treated long term with ibrutinib gained the C481F BTK mutation, in cells resistant to the non-covalent BTKi we identified six different mutations in BTK (variant allele frequency (VAF) 22% to 99%) and three mutations in PLCG2 (VAF 10%, 30% and 53%). Four out of six lines of REC-1 cells resistant to vecabrutinib acquired BTK G409R mutation while one line acquired a L845F PLCG2 mutation. Three different BTK mutations, L528S, G480R and D539H were found in three independent lines of REC-1 cells resistant to fenebrutinib. RN-486-resistant REC-1 cells acquired G480R and V416L BTK mutations in two out of three different lines. One of the six pirtobrutinib-resistant lines acquired a BTK A428D mutation, while two other resistant lines gained R727L and S1079R PLCG2 mutations. Interestingly, mutations in three BTK residues A428, L528, and V416 reported to mediate pirtobrutinib resistance in CLL patients were also identified in the REC-1 cell lines that acquired resistance to pirtobrutinib, fenebrutinib, and RN-486, respectively. Of note, nemtabrutinib-resistant REC-1 cells, despite the increased IC50, did not acquire a BTK or PLCG2 mutation, indicating the role of other mechanisms in the generation of resistance. To functionally analyze BCR signaling capabilities of the different BTK mutant REC-1 cells, we performed measurement of intracellular [Ca2+] ([Ca2+]i) upon stimulation with anti-IgM. Except for A428D, all BTK mutant REC-1 cell lines showed a similar or enhanced [Ca2+]i compared to wild type (WT) cells. Western blot analyses showed decreased anti-IgM-mediated activation of BTK in all the different BTK mutant cell lines compared to WT. However, the downstream activation of phospho-AKT was enhanced in all the mutant lines, similar to earlier reports, while phospho-ERK levels changed marginally. Moreover, by studying sensitivity of the BTK mutated cells to a panel of nine different covalent and non-covalent BTKi, we identified G409R and D539H mutants to be sensitive to covalent BTKi while the other BTK mutants were resistant to both covalent and non-covalent BTKi. The BCL2 inhibitor venetoclax is an important treatment option for CLL, however, the response to venetoclax in the context of non-covalent BTKi resistance is unknown. Flow cytometry based BH3 profiling assay was performed to study apoptotic priming in the BTK mutants. Except for G409R, all BTK mutants showed increased apoptotic priming to BAD peptide exposure, indicating enhanced BCL2 dependency compared to WT. In line with this, the non-covalent BTKi resistant REC-1 cells showed enhanced sensitivity to venetoclax in vitro while G409R BTK mutant had a similar IC50 as WT. In summary, we identified novel BTK protein kinase domain mutations that confer resistance to non-covalent BTKi. Our findings highlight the importance of sequencing either all exons or at least the kinase domain (Exons 13 to 19) of BTK in the context of progression under non-covalent BTKi treatment. We also found that cells harboring these novel BTK mutations showed differential sensitivity to the covalent vs. non-covalent BTKi. We further demonstrate the potential of venetoclax as follow up treatment upon resistance to non-covalent BTKi. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CLL-129 BRUIN CLL-313: A Phase 3 Open-Label, Randomized Study of Pirtobrutinib Versus Bendamustine Plus Rituximab in Untreated Patients With Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (Trial in Progress)

Covalent Bruton's Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative, and the majority of patients will require additional treatment. Covalent BTKi shares pharmacologic liabilities (e.g., low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover such as accelerating CLL/SLL, ultimately manifesting as acquired resistance in some patients. To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. Determine whether pirtobrutinib is superior to bendamustine plus rituximab in treatment naïve CLL/SLL patients with retained 17p. BRUIN CLL-313 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus bendamustine plus rituximab (BR) in treatment naïve CLL/SLL patients with retained 17p. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by IGHV mutation status (mutated vs unmutated), and Rai stage (low/intermediate vs high). Patients in the BR arm are eligible to crossover to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 and confirmed by an independent review committee (IRC). Global; community hospitals, academic medical centers. Eligible patients are adults with confirmed diagnosis of CLL/SLL and who require therapy per iwCLL 2018 criteria. Key exclusion criteria include CNS involvement by CLL/SLL, Richter's transformation to DLBCL, prolymphocytic leukemia or Hodgkin lymphoma any time pre-enrollment, presence of 17p deletion, prior systemic therapy for CLL/SLL, and significant cardiovascular disease. Oral pirtobrutinib monotherapy versus bendamustine plus rituximab. The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an independent review committee. Secondary endpoints include investigator-assessed PFS, overall survival (OS), overall response rate (ORR), duration of response (DoR), safety and tolerability, and patient-reported outcomes. The global study is currently enrolling patients (NCT05023980). Trial in Progress. Trial in Progress.

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.

Zanubrutinib in lymphoproliferative disorders: a comprehensive review.

The availability of Bruton tyrosine kinase (BTK) inhibitors has brought about a paradigm shift in the treatment of patients with B-cell lymphomas and chronic lymphocytic leukemia. BTK was clinically validated as a target by the efficacy of the first-in-class inhibitor ibrutinib. The extended survival conferred by BTK inhibitors has brought long-term tolerability to the foreground. To minimize toxicities thought to be attributable to off-target kinase inhibition, a next generation of BTK inhibitors with greater selectivity was developed. In the United States, zanubrutinib, a next-generation BTK inhibitor, has been approved for treating adults with mantle cell lymphoma who have received at least one prior therapy, for adults with Waldenström macroglobulinemia, and for adults with relapsed or refractory marginal zone lymphoma who have received at least one anti-CD20–based therapy. Because few head-to-head comparative trials of BTK inhibitors have so far been reported, no BTK ‘inhibitor of choice’ can be identified. Zanubrutinib has promising efficacy in its approved indications and appears to have reduced cardiac toxicities, particularly atrial fibrillation, which may influence the choice of BTK inhibitor treatment by prescribers. Further studies are needed to inform on optimal treatment sequencing of zanubrutinib and its combination with other agents. Here, we summarize existing clinical evidence for its efficacy and safety in mantle cell lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, and other B-lymphoproliferative indications.Plain Language Summary Zanubrutinib is a drug that was shown to effectively treat cancer of B cells without causing excessive serious side effects Patients with certain B-cell malignancies (cancers of white blood cells) benefit from treatment with Bruton tyrosine kinase (BTK) inhibitors, drugs that block the BTK protein and keep cancer from growing and spreading. Patients experience extended survival with ibrutinib, the first-generation BTK inhibitor approved by US Food and Drug Administration (FDA); however, one in five patients quit treatment because of harmful side effects. Ibrutinib-related side effects such as increased risk of bleeding, atrial fibrillation (abnormal heart rhythm), and high blood pressure are thought to be caused by ibrutinib blocking other proteins besides the intended target protein BTK. To reduce these side effects, zanubrutinib, a next-generation BTK inhibitor, was designed to block BTK more specifically than ibrutinib. Results of clinical studies on zanubrutinib treatment appear promising in patients with several types of B-cell malignancies, including mantle cell lymphoma (MCL), Waldenström macroglobulinemia (WM), marginal zone lymphoma (MZL), chronic lymphocytic leukemia, and small lymphocytic lymphoma. There are not yet enough clinical data to determine which BTK inhibitor is most effective in treating B-cell malignancies without causing harmful side effects. Early data from the phase 3 ALPINE clinical study suggest that zanubrutinib works better than ibrutinib, and fewer patients experience side effects and quit treatment. Zanubrutinib is currently approved for use for treatment of adult patients with MCL who have received at least one prior therapy, for adults with WM, and for adults with MZL who have received at least one anti-CD20–based therapy.

BTK-inhibitor drug covalent binding to lysine in human serum albumin using LC-MS/MS

Irreversible Bruton's tyrosine kinase (BTK) inhibitor drugs are designed to bind covalently to a free-thiol cysteine in the BTK protein active site. However, these reactive drugs bind to off-target proteins as well. In this study, seven BTK-inhibitor drugs containing acrylamide warheads were incubated with human serum albumin (HSA) and analyzed using an LC-MS/MS peptide mapping approach to determine the amino acid sites of drug covalent binding. Significant adduction at the free-thiol cysteine of HSA was only observed for two of the drugs. However, significant adduction was observed for at least four lysine residues. This is just a small percentage of the 59 total lysine residues in HSA. These four lysine residues are likely partially buried, accessible to the drugs, and exist at least partially in a neutral state. The levels of adduction observed in the in-vitro experimental conditions are only indicative of a relative propensity for adduction with the individual lysine residues of HSA, and are not in-vivo predictions. Widespread off-target lysine binding could impact clearance and bioavailability for irreversible inhibitor drugs. However, the extent of the impact on clearance may be limited in comparison to conjugation with glutathione.

BRUIN CLL-321: A Phase 3 Open-Label, Randomized Study of Pirtobrutinib Versus Investigator's Choice of Idelalisib Plus Rituximab or Bendamustine Plus Rituximab in BTK Inhibitor Pretreated Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (Trial in Progress)

Background: Covalent Bruton Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative and many patients will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage especially in rapidly proliferating tumors with high BTK protein turnover such as accelerating CLL/SLL, ultimately manifesting as acquired resistance in some patients. To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. In the phase 1/2 BRUIN study, pirtobrutinib achieved pharmacokinetic exposures that exceeded its BTK IC96 at trough, was well tolerated and demonstrated promising efficacy in CLL/SLL patients regardless of prior therapy, number of prior lines of therapy, or BTK C481 mutation status (Mato et al. Lancet 2021;397,10277:892-901). The purpose of this randomized phase 3 study is to demonstrate the superiority of continued BTK pathway inhibition with pirtobrutinib compared to other available therapies in patients with BTKi-treated CLL/SLL.Study Design and Methods: BRUIN CLL-321 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus investigator's choice of idelalisib plus rituximab or bendamustine plus rituximab in patients with CLL/SLL who have been treated with a prior covalent BTKi. Prior therapy with venetoclax is permitted. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by 17p deletion (yes/no) and prior venetoclax (yes/no). Patients receiving investigator's choice are eligible to crossover to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 (determined by IRC).Eligible patients are adults aged ≥18 years with a diagnosis of CLL/SLL who require therapy per iwCLL 2018 criteria and who have received prior covalent BTKi. Unlimited number of lines of prior therapy are allowed. Key exclusion criteria include CNS involvement by CLL/SLL, Richter transformation any time pre-enrollment, a major bleeding event on prior covalent BTKi and history of allogeneic stem cell transplant (SCT) or autologous SCT or chimeric antigen receptor (CAR) T-cell therapy within 60 days of randomization.The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an independent review committee (IRC). Secondary endpoints include overall survival (OS), overall response rate (ORR), duration of response (DoR), safety and tolerability, and patient reported outcomes. The global study is currently enrolling patients (NCT04666038). DisclosuresSharman: BeiGene: Consultancy; BMS: Consultancy; AstraZeneca: Consultancy; TG Therapeutics: Consultancy; Lilly: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy; Centessa: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy. Jurczak: Abbvie, AstraZeneca, BeiGene, Celtrion, Celgene, Debbiopharm, Epizyme, Incyte, Janssen, Loxo Oncology, Merck, Mei Pharma, Morphosys, Novo Nordisk, Roche, Sandoz, Takeda, TG Therapeutics: Research Funding; Astra Zeneca, BeiGene, Janssen, Loxo Oncology, Sandoz, Roche,: Membership on an entity's Board of Directors or advisory committees. Coombs: LOXO: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria; Genentech: Honoraria; MEI Pharma: Honoraria. Hill: Loxo Oncology at Lilly: Current Employment. Wang: Loxo Oncology at Lilly: Current Employment. Ku: Loxo Oncology at Lilly: Current Employment, Current holder of stock options in a privately-held company. Guntur: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Shahda: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Leow: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Ghia: Gilead: Consultancy, Research Funding; Celgene/Juno/BMS: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; ArQule/MSD: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sunesis: Research Funding. Mato: Janssen: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Nurix: Research Funding; MSKCC: Current Employment; Adaptive Biotechnologies: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Johnson and Johnson: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; AstraZeneca: Consultancy; Genmab: Research Funding; LOXO: Consultancy, Research Funding; Acerta/AstraZeneca: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; TG Therapeutics: Consultancy, Other: DSMB, Research Funding; DTRM BioPharma: Consultancy, Research Funding; Genentech: Consultancy, Research Funding.

BRUIN CLL-313: A Phase 3 Open-Label, Randomized Study of Pirtobrutinib Versus Bendamustine Plus Rituximab in Untreated Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (Trial in Progress)

Background: Covalent Bruton's Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative and the majority of patients will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover such as accelerating CLL/SLL, ultimately manifesting as acquired resistance in some patients. To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. In a phase 1/2 BRUIN study, pirtobrutinib achieved pharmacokinetic exposures that exceeded its BTK IC96 at trough, was well tolerated, and demonstrated promising efficacy in CLL/SLL patients regardless of prior therapy, number of prior lines of therapy, or BTK C481 mutation status (Mato et al. Lancet 2021;397, 10277:892-901).Study Design and Methods: BRUIN CLL-313 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus bendamustine plus rituximab (BR) in treatment naïve CLL/SLL patients with retained 17p. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by IGHV mutation status (mutated vs unmutated), and Rai stage (low/intermediate vs high). Patients in the BR arm are eligible to crossover to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 and confirmed by an independent review committee (IRC).Eligible patients are adults with confirmed diagnosis of CLL/SLL and who require therapy per iwCLL 2018 criteria. Key exclusion criteria include CNS involvement by CLL/SLL, Richter transformation to DLBCL, prolymphocytic leukemia or Hodgkin lymphoma any time pre-enrollment, presence of 17p deletion, prior systemic therapy for CLL/SLL, and significant cardiovascular disease.The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an IRC. Secondary endpoints include investigator-assessed PFS, overall survival (OS), overall response rate (ORR), duration of response (DoR), safety and tolerability, and patient reported outcomes. The global study is currently enrolling patients. DisclosuresJurczak: Abbvie, AstraZeneca, BeiGene, Celtrion, Celgene, Debbiopharm, Epizyme, Incyte, Janssen, Loxo Oncology, Merck, Mei Pharma, Morphosys, Novo Nordisk, Roche, Sandoz, Takeda, TG Therapeutics: Research Funding; Astra Zeneca, BeiGene, Janssen, Loxo Oncology, Sandoz, Roche,: Membership on an entity's Board of Directors or advisory committees. Dartigeas: Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Other: travel grants/Congress; Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: travel grants/Congress; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: travel grants/Congress. Coscia: Gilead: Honoraria; AbbVie: Honoraria, Other; Janssen: Honoraria, Other, Research Funding; AstraZeneca: Honoraria. Wang: Eli Lilly and Company: Current Employment, Current equity holder in publicly-traded company. Bao: Loxo Oncology at Lilly: Current Employment; Genentech: Ended employment in the past 24 months. Leow: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Shahda: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Zinzani: Eusapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck Sharp & Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ADC Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen Cilag: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics: Honoraria, Speakers Bureau; Celtrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Portola: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau.

BRUIN MCL-321: A Phase 3 Open-Label, Randomized Study of Pirtobrutinib Versus Investigator Choice of BTK Inhibitor in Patients with Previously Treated, BTK Inhibitor Naïve Mantle Cell Lymphoma (Trial in Progress)

BRUIN MCL-321: A Phase 3 Open-Label, Randomized Study of Pirtobrutinib Versus Investigator Choice of BTK Inhibitor in Patients with Previously Treated, BTK Inhibitor Naïve Mantle Cell Lymphoma (Trial in Progress)

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].

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)