Bruton's Tyrosine Kinase Research Articles

Oncotherapy resistance explained by Darwinian and Lamarckian models.

Cell and antibody therapies directed against surface molecules on B cells, e.g., CD19-targeting chimeric antigen receptor T cells (CD19 CAR-T), are now standard for patients with chemorefractory B cell acute lymphoblastic leukemias and other B cell malignancies. However, early relapse rates remain high. In this issue of the JCI, Aminov, Giricz, and colleagues revealed that leukemia cells resisting CD19-targeted therapy had reduced CD19 but also low CD22 expression and were sensitive to Bruton's tyrosine kinase and/or MEK inhibition. Overall, their observations support the evolution of resistance following a Lamarckian model: the oncotherapy directly elicits adaptive, resistance-conferring reconfigurations, which are then inherited by daughter cells as epigenetic changes. The findings prompt reflection also on the broader role of epigenetics in decoupling of replication from lineage differentiation activation by the B cell lineage master transcription factor hub. Such oncogenesis and resistance mechanisms, being predictable and epigenetic, offer practical opportunities for intervention, potentially non-cross-resistant and safe vis-à-vis present cytotoxic and CAR-T treatments.

CNS Aspergillosis and Cryptococcosis with Cytomegalovirus Pneumonia in a Patient with Chronic Lymphocytic Leukemia Treated with Acalabrutinib.

Bruton's tyrosine kinase inhibitors (BTKis) are the preferred treatment for chronic lymphocytic leukemia (CLL). Despite their therapeutic benefits, these targeted agents have been associated with an increased risk of invasive infections. We describe a 68-year-old male who developed multiple bacterial, fungal and viral infections while on treatment with acalabrutinib. To our knowledge, this is the first reported case of concomitant CNS infections with Cryptococcus neoformans and Aspergillus fumigatus, along with cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1) pneumonia while on acalabrutinib. This case adds to the scarce literature of fungal and bacterial infections associated with acalabrutinib, raising the suspicion that infection risk is a medication class effect for BTKis.

Richter transformation acquiring PLCG2 mutation during Bruton tyrosine kinase inhibitors treatment

Richter transformation acquiring <i>PLCG2</i> mutation during Bruton tyrosine kinase inhibitors treatment

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

The t(1;19) translocation, which codes for the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre-B cell receptor (preBCR+) phenotype. Relapse in E2A-PBX1+ ALL patients frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL.Using unbiased shRNA library screening approaches, we identified Bruton's tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, combination of dasatinib with BTK inhibitors (BTKi) (ibrutinib, acalabrutinib or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced PLCG2 and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, reducing particularly CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs.In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse E2A-PBX1+/preBCR+ ALL in most of performed assays, and the combination of dasatinib and BTKi is very effective in reducing CNS-infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Investigating bleeding adverse events associated with BTK inhibitors in the food and drug administration adverse event reporting system (FAERS)

ABSTRACT Background This study analyzed the bleeding adverse events (AEs) resulting from the treatment of B-cell lymphoma with Bruton tyrosine kinase (BTK) inhibitors, according to reports in the US Food and Drug Administration’s Adverse Event Reporting System (FAERS) Methods Bleeding AEs associated with BTK inhibitors (including ibrutinib, zanubrutinib, and acalabrutinib) from the first quarter of 2013 to the third quarter of 2023 were extracted. Reporting odds ratio (ROR) and proportional reporting ratio (PRR) were reported. Preferred Terms (PTs) of Medical Dictionary for Regulatory Activities (MedDRA) terms were mapped to System Organ Class terms (SOC) terms and analyzed bleeding AEs associated with three BTK inhibitors. Results A total of 463 cases of bleeding AEs were included. Contusion, subcutaneous hemorrhage, hematuria, and cerebral hemorrhage were included in PTs. Blood urine was present and subdural hematoma were also reported. The incidence of bleeding AEs was higher with ibrutinib (Case number = 10,696) than with zanubrutinib (Case number = 213) and acalabrutinib (Case number = 314). Conclusion Our findings indicate that bleeding AEs linked to BTK inhibitors in various conditions underscore the need for cautious clinical decision-making, particularly in nervous system disorders, injuries, poisoning, surgical complications, vascular disorders, and others.

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib’s effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1–10 µM and 25 mg/kg) and in combination with doxil (10–100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell–cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a − 53% versus − 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib’s ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

Managing multiple sclerosis in individuals aged 55 and above: a comprehensive review

Multiple Sclerosis (MS) management in individuals aged 55 and above presents unique challenges due to the complex interaction between aging, comorbidities, immunosenescence, and MS pathophysiology. This comprehensive review explores the evolving landscape of MS in older adults, including the increased incidence and prevalence of MS in this age group, the shift in disease phenotypes from relapsing-remitting to progressive forms, and the presence of multimorbidity and polypharmacy. We aim to provide an updated review of the available evidence of disease-modifying treatments (DMTs) in older patients, including the efficacy and safety of existing therapies, emerging treatments such as Bruton tyrosine kinase (BTKs) inhibitors and those targeting remyelination and neuroprotection, and the critical decisions surrounding the initiation, de-escalation, and discontinuation of DMTs. Non-pharmacologic approaches, including physical therapy, neuromodulation therapies, cognitive rehabilitation, and psychotherapy, are also examined for their role in holistic care. The importance of MS Care Units and advance care planning are explored as a cornerstone in providing patient-centric care, ensuring alignment with patient preferences in the disease trajectory. Finally, the review emphasizes the need for personalized management and continuous monitoring of MS patients, alongside advocating for inclusive study designs in clinical research to improve the management of this growing patient demographic.

Abstract CT156: A Phase I study evaluating AC676, an innovative BTK chimeric degrader, in patients with relapsed and refractory B-cell malignancies

Abstract Study Background Bruton tyrosine kinase (BTK) is a key signaling molecule that plays a central role in B-cell receptor transduction. The development of drugs that inhibit BTK has transformed the management of patients with B-cell malignancies. However, therapy using covalent BTK inhibitors such as ibrutinib, and non-covalent inhibitors such as pirtobrutinib, can still result in resistance, primarily due to the development of mutations in BTK including residues C481 and L528. AC676 was designed as a chimeric degrader to target and degrade BTK using Accutar's proprietary Protein-Protein Interaction Targeting Chimeras (PPI-TAC) platform. By effectively linking a BTK ligand to the cereblon E3-ligase recruiting ligand, AC676 brings BTK in proximity to cereblon, thereby inducing subsequent ubiquitination and degradation of BTK. AC676 degrades BTK proteins irrespective of mutations; including C481, kinase dead L528 and others, and thus may be effective for the treatment of patients who progress on both covalent and non-covalent BTK inhibitors. Notably, it is also effective in cell lines expressing gain of function PLCG2 mutants, suggesting that it removes BTK’s scaffolding function. AC676 does not degrade cereblon neo-substrates, so neutropenia is not expected to be an on-target effect. This abstract describes an ongoing first in human Phase 1 trial of AC676. Study Description AC676-001 is a Phase 1 dose-escalation study of AC676 administered orally once daily as monotherapy in patients with relapsed and refractory B-cell malignancies. Approximately 60 patients may be enrolled. Eligible patients must be ≥ 18 years, and have one of the following histologically confirmed relapsed or refractory disease types: Chronic lymphocytic leukemia and small lymphocytic lymphoma, Diffuse large B cell lymphoma - non-GCB subtype, Follicular lymphoma, Mantle cell lymphoma, Marginal zone lymphoma, Lymphoplasmacytic lymphoma including Waldenstrom macroglobulinemia. Patients must have received at least two prior systemic therapies or have no other standard of care therapies to provide significant clinical benefit. Patients must have measurable disease per disease-specific response criteria and Eastern Cooperative Oncology Group performance status≤ 1. Dose-escalation will begin with an accelerated titration phase, followed by a standard 3+3 phase. AC676 is administered orally once daily on a 28 day per cycle schedule at doses ranging from 50mg to 600mg. The primary objective of the study is to evaluate the safety and tolerability of AC676. Secondary objectives include the evaluation of anti-tumor activity and the pharmacokinetic profile following single and multiple doses. Study enrollment began in April 2023 with three sites currently open in the United States (NCT05780034). Citation Format: Jennifer Woyach, Michael Tees, Nancy Mota, Gladys Brown, Su Young Kim, Manish R. Patel. A Phase I study evaluating AC676, an innovative BTK chimeric degrader, in patients with relapsed and refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT156.

Covalent docking-driven virtual screening of extensive small-molecule libraries against Bruton tyrosine kinase for the identification of highly selective and potent novel therapeutic candidates

Bruton tyrosine kinases (BTKs) play critical roles in various diseases, including chronic lymphatic leukemia (CLL), Waldenström Macroglobulinemia, Marginal Zone Lymphoma, Mantle Cell Lymphoma (MCL), and Graft Versus Host diseases. BTKs are a family of tyrosine kinases involved in B lymphocyte signal transduction, development, and maturation. Their overexpression can lead to cancer as they are essential for the activation of the B Cell Receptor (BCR) signaling pathway. Blocking the activation of BTKs presents a promising approach for treating CLL. This study was centered around the identification of small-molecule therapeutics that have an impact on human BTK. The covalently bound Ibrutinib molecule, recognized for its ability to inhibit BTK, was used as the query molecule. IUPAC text files containing molecular fragments of Ibrutinib were employed to virtually screen five different libraries comprising small-molecules, resulting in the screening of over 2.4 million synthesized compounds. Covalent docking simulations were applied to the selected small-molecules obtained through text mining from databases. Potent hit molecules capable of inhibiting BTKs through virtual screening algorithms were identified, paving the way for novel therapeutic strategies in the treatment of CLL.

The effect of Bruton’s tyrosine kinase (BTK) inhibitor in the eosinophilic asthma model of mouse

Bruton’s Tyrosine kinase (BTK) plays a pivotal role as the key mediator in B cell signaling. Recent research has revealed that it is also expressed in cells critical to asthma development, such as T cells, and eosinophils. This study aims to investigate the potential of BTK inhibitor in eosinophilic asthma mouse model. BALB/c mice were sensitized with ovalbumin (OVA) via intraperitoneal injections and followed by OVA nebulizations. The mice were treated with 250 µg/ml or 500 µg/ml of ibrutinib before the second intraperitoneal injection and the first nebulization. Two days after the last OVA challenge, airway hyperresponsiveness (AHR) was assessed with methacholine, and differential cell count in bronchoalveolar lavage fluid (BALF) was performed. The cytokines were measured in BALF, and serum OVA-specific IgE and IgG antibody levels were evaluated by ELISA. The inhibitory effect of ibrutinib was also evaluated in splenic mononuclear cells, mast cells, eosinophils, and T cells in vitro. Treatment with ibrutinib significantly attenuated AHR and airway inflammation, compared to the OVA-induced positive control. The treatment also reduced IL-4, IL-5, IL-13 and IFN-γ cytokine levels and suppressed OVA-specific IgE and IgG production compared to the OVA-induced positive control. Additionally, ibrutinib decreased beta-hexosaminidase release from mast cells, type 2 cytokine productions from mononuclear cells and T cells, and eosinophilic activation markers in vitro. The results of this study suggest that ibrutinib treatment could exert anti-allergic effects by inactivating B cells and other BTK-expressing cells. Further studies are needed to investigate the potential therapeutic effect of ibrutinib on allergic diseases.

Challenges associated with the use of Bruton's tyrosine kinase inhibitors: A life‑saving therapy for chronic lymphocytic leukemia (Review)

Challenges associated with the use of Bruton's tyrosine kinase inhibitors: A life‑saving therapy for chronic lymphocytic leukemia (Review)

Brentuximab Vedotin Plus Ibrutinib in Relapsed and Refractory Hodgkin Lymphoma

IntroductionBrentuximab vedotin (BV) is an antibody-drug conjugate that delivers monomethyl auristatin E (MMAE) to CD30+ cells and is safe and effective in relapsed/refractory (r/r) Hodgkin lymphoma (HL). Although most patients respond to BV, only a minority will obtain a complete response (CR), and almost all patients eventually progress.Ibrutinib is a Bruton's tyrosine kinase (BTK) inhibitor highly active in multiple subtypes of non-Hodgkin lymphoma; limited data exist regarding its use in HL. It irreversibly inhibits interleukin-2-inducible kinase (ITK) with Th1 based immune responses. As we previously observed preclinical synergy between ibrutinib and BV, we hypothesized ibrutinib may enhance the antitumor activity of BV in HL. We designed and conducted a phase II trial of ibrutinib plus BV in patients with R/R HL, and herein report the final primary analysis of safety and efficacy. MethodsThis was a multicenter phase II trial with a lead-in cohort in patients with r/r HL. Eligibility criteria included age ≥ 15 years with r/r HL after at least one prior line of therapy. Treatment consisted of 1.8 mg/kg BV intravenously every 3 weeks and ibrutinib 560 mg PO daily (420 mg PO daily in the lead-in cohort). Prior BV was allowed if patients were not refractory. The primary endpoint was the CR rate according to Lugano 2014. Secondary endpoints included toxicities, overall response rate (ORR), and duration of response (DOR). Results39 patients were enrolled onto the study, of which 67% were male; the median age was 33 (range: 17-71). 38% had extranodal disease at baseline, 51% had advanced stage disease, 51% were refractory to the prior therapy, and 21% had prior BV. Of 36 patients who were evaluable for response, the CR rate was 33% and ORR 64%; median DOR was 25.5 months. 13 patients proceeded to autologous transplant and 3 patients proceeded to allogeneic transplant for consolidation after response. The most common adverse events were nausea (67%), peripheral neuropathy (62%), diarrhea (59%), fatigue (46%), thrombocytopenia (46%), headache (41%), rash (41%), elevated ALT (38%), anemia (36%), vomiting (36%), abdominal pain (33%), fever (33%), and hypertension (33%). Six patients experienced unacceptable toxicity, defined as Gr 3/4 non-hematologic toxicity or non-resolving Gr 3/4 hematologic toxicity including one patient who died of multiorgan failure from suspected COVID-19 infection during cycle 1. DiscussionThe combination of BV and ibrutinib was active in r/r HL; however, given significant toxicity, it cannot be recommended for future development. MicroAbstractBrentuximab vedotin (BV) is highly active in Hodgkin lymphoma (HL) and is approved as monotherapy for relapsed/refractory (r/r) disease; nonetheless, depth and duration of response to BV monotherapy are limited. Ibrutinib is a Bruton's tyrosine kinase (BTK) inhibitor but also inhibits other tyrosine kinases including interleukin-2-inducible kinase (ITK) and has multiple immunomodulatory effects. We conducted a phase II trial (NCT02744612) of BV with ibrutinib in patients with r/r HL and enrolled 39 patients, 36 of whom were evaluable for response. The primary endpoint was CR with a promising rate considered to be 50%. The median follow-up time was 29.4 months, and the CR rate was 33% and ORR 64%; 2-year OS and PFS were 92% and 36%, respectively while the 2-year CIR was 64%. 6 patients experienced unacceptable toxicity defined as grade 3+ non-hematologic toxicity or grade 3+ hematologic toxicity not resolving to grade 1-2 within 7 days. Ultimately, combination therapy did not appear to be more effective than BV monotherapy but conferred additional toxicity.

Mouse models of CLL: in vivo modeling of disease initiation, progression, and transformation to Richter transformation

Chronic lymphocytic leukemia (CLL) is a highly complex disease characterized by the proliferation of CD5+ B cells in lymphoid tissues. Current modern treatments have brought significant clinical benefits to CLL patients. However, there are still unmet needs. Patients relapse on Bruton's tyrosine kinase inhibitors and BCL2 inhibitors and often develop more aggressive diseases including Richter transformation (RT), an incurable complication of up to ∼10% patients. This evidence underscores the need for improved immunotherapies, combination treatment strategies, and predictive biomarkers. A mouse model that can recapitulate human CLL disease and certain components of the tumor immune microenvironment represents a promising preclinical tool for such purposes. In this review, we provide an overview of CRISPR-engineered and xenograft mouse models utilizing either cell lines, or primary CLL cells suitable for studies of key events driving the disease onset, progression and transformation of CLL. We also review how CRISPR/Cas9 established mouse models carrying loss-of-function lesions allow one to study key mutations driving disease progression. Finally, we discuss how next generation humanized mice might improve to generation of faithful xenograft mouse models of human CLL.

A multicenter study of venetoclax-based treatment for patients with Richter transformation of chronic lymphocytic leukemia

AbstractPatients with chronic lymphocytic leukemia (CLL) who develop Richter transformation (RT) have a poor prognosis when treated with chemoimmunotherapy regimens used for de novo diffuse large B-cell lymphoma. Venetoclax, a B-cell lymphoma 2 protein inhibitor, has single-agent efficacy in patients with RT and is potentially synergistic with chemoimmunotherapy. In this multicenter, retrospective study, we evaluated 62 patients with RT who received venetoclax-based treatment outside of a clinical trial, in combination with a Bruton tyrosine kinase inhibitor (n = 28), R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone; n = 13), or intensive chemoimmunotherapy other than R-CHOP (n = 21). The best overall and complete response rates were 36% and 25%, 54% and 46%, and 52% and 38%, respectively. The median progression-free and overall survival estimates for the same treatment groups were 4.9 and 14.3 months, 14.9 months and not reached, and 3.3 and 9 months, respectively. CLL with del(17p) was associated with a lower complete response rate in the total cohort (odds ratio [OR], 0.15; 95% confidence interval [CI], 0.04-0.6; P = .01) and venetoclax-naïve subgroup (OR, 0.13; 95% CI, 0.02-0.66; P = .01). TP53-mutated CLL was associated with a lower complete response rate (OR, 0.15; 95% CI, 0.03-0.74; P = .02) and shorter progression-free survival (hazard ratio, 3.1; 95% CI, 1.21-7.95; P = .02) only in the venetoclax-naïve subgroup. No other clinical or baseline characteristics, including prior venetoclax treatment for CLL, showed statistically significant association with outcomes. Grade 3/4 neutropenia and thrombocytopenia events were most frequent with intensive chemoimmunotherapy + venetoclax; grade 3/4 infection rates were similar across treatment groups. In this difficult-to-treat patient population with RT, venetoclax-based combination regimens achieved high response rates, with durable remission and survival observed in a subset of patients.

Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy for Richter Transformation: An International, Multicenter, Retrospective Study.

Outcomes for Richter transformation (RT) are poor with current therapies. The efficacy and safety of anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T) for RT are not established. We performed an international multicenter retrospective study of patients with RT who received CAR-T. Patient, disease, and treatment characteristics were summarized using descriptive statistics, and modeling analyses were used to determine association with progression-free survival (PFS) and overall survival (OS). PFS and OS were estimated from the date of CAR-T infusion. Sixty-nine patients were identified. The median age at CAR-T infusion was 64 years (range, 27-80). Patients had a median of four (range, 1-15) previous lines of therapy for CLL and/or RT, including previous Bruton tyrosine kinase inhibitor and/or BCL2 inhibitor therapy in 58 (84%) patients. The CAR-T product administered was axicabtagene ciloleucel in 44 patients (64%), tisagenlecleucel in 17 patients (25%), lisocabtagene maraleucel in seven patients (10%), and brexucabtagene autoleucel in one patient (1%). Eleven patients (16%) and 25 patients (37%) experienced grade ≥3 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, respectively. The overall response rate was 63%, with 46% attaining a complete response (CR). After a median follow-up of 24 months, the median PFS was 4.7 months (95% CI, 2.0 to 6.9); the 2-year PFS was 29% (95% CI, 18 to 41). The median OS was 8.5 months (95% CI, 5.1 to 25.4); the 2-year OS was 38% (95% CI, 26 to 50). The median duration of response was 27.6 months (95% CI, 14.5 to not reached) for patients achieving CR. CAR-T demonstrates clinical efficacy for patients with RT.

Bruton tyrosine kinase inhibitors in multiple sclerosis: evidence and expectations.

Despite availability of high-efficacy therapies for multiple sclerosis (MS), many patients experience significant disability worsening due to limited effects of currently available drugs on central nervous system (CNS)-compartmentalized inflammation. Bruton tyrosine kinase (BTK) is an intracellular signaling molecule involved in regulation of maturation, survival, migration, and activation of B cells and microglia, which are central players in the immunopathogenesis of progressive MS. Therefore, CNS-penetrant BTK inhibitors may better prevent disease progression by targeting immune cells on both sides of the blood-brain barrier. This review gives an overview on the preliminary results of clinical trials. Currently, the efficacy and safety of six BTK inhibitors are being evaluated in clinical trials in patients with relapsing and progressive MS. Evobrutinib, tolebrutinib and fenebrutinib have shown efficacy and safety in relapsing MS in phase 2 studies, and evobrutinib and tolebrutinib in their extension studies up to 3-5 years. However, evobrutinib failed to distinguish itself from the comparator drug teriflunomide in reduction of relapse rate (primary end point) in two phase 3 studies in relapsing MS. Inhibition of BTK has emerged as a promising therapeutic approach to target the CNS-compartmentalized inflammation. Results from phase 3 clinical trials will shed light on differences in efficacy and safety of BTK inhibitors and its potential role in the future MS landscape.

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.

A prospective study of zanubrutinib, a Bruton tyrosine kinase inhibitor, in relapsed/refractory idiopathic multicentric Castleman disease.

Relapsed and refractory (R/R) idiopathic multicentric Castleman disease (iMCD) is a clinical challenge with no standard treatment. In this preliminary clinical trial, we investigated the efficacy and safety profiles of a Bruton tyrosine kinase inhibitor (BTKi), zanubrutinib, in patients with R/R iMCD. The primary endpoint was theoverall response rate at Week 12according to theCastleman Disease Collaborative Network (CDCN) response criteria. The trial was terminated early due to a lack of treatment response in the first enrolled 5 patients. Although 3 patients achieved symptomatic response, none of the 5 patients had an overall response by Week 12. One patient had progressive disease and the other 4 had stable disease. The study drug was well tolerated without grade 2 or higher adverse events. Our findings suggest that BTKi therapy is not effective for iMCD, and further attempts at single-agent therapy with zanubrutinib or other BTKis for iMCD should be consideredwith caution and probably avoided. This trial was registered at www.clinialtrials.gov as #NCT04743687.

Bruton Tyrosine Kinase Inhibition: an Effective Strategy to Manage Waldenström Macroglobulinemia.

The treatment of Waldenström macroglobulinemia (WM) has evolved over the past decade. With the seminal discoveries of MYD88 and CXCR warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) mutations in WM cells, our understanding of the disease biology and treatment has improved. The development of a new class of agents, Bruton tyrosine kinase inhibitors (BTKi), has substantially impacted the treatment paradigm of WM. Herein, we review the current and emerging BTKi and the evidence for their use in WM. Clinical trials have established the role of covalent BTKi in the treatment of WM. Their efficacy is compromised among patients who harbor CXCR4WHIM mutation or MYD88WT genotype. The development of BTKC481 mutation-mediated resistance to covalent BTKi may lead to disease refractoriness. Novel, non-covalent, next-generation BTKi are emerging, and preliminary results of the early phase clinical trials show promising activity in WM, even among patients refractory to a covalent BTKi. Covalent BTK inhibitors have demonstrated meaningful outcomes in treatment-naïve (TN) and relapsed refractory (R/R) WM, particularly among those harboring the MYD88L265P mutation. The next-generation BTKi demonstrate improved selectivity, resulting in a more favorable toxicity profile. In WM, BTKi are administered until progression or the development of intolerable toxicity. Consequently, the potential for acquired resistance, the emergence of cumulative toxicities, and treatment-related financial burden are critical challenges associated with the continuous therapy approach. By circumventing BTK C481 mutations that alter the binding site to covalent BTKi, the non-covalent BTKi serve as alternative agents in the event of acquired resistance. Head-to-head comparative trials with the conventional chemoimmunotherapies are lacking. The findings of the RAINBOW trial (NCT046152), comparing the dexamethasone, rituximab, and cyclophosphamide (DRC) regimen to the first-generation, ibrutinib are awaited, but more studies are needed to draw definitive conclusions on the comparative efficacy of chemoimmunotherapy and BTKi. Complete response is elusive with BTKi, and combination regimens to improve upon the efficacy and limit the treatment duration are also under evaluation in WM.

Abstract 6044: EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases

Abstract Heterobifunctional kinase degraders (HKDs) are an important new class of therapeutic modalities, of which the first have recently entered clinical trials. HKDs are small molecules that comprise a kinase-of-interest binding moiety and an E3 ligase binding moiety connected via a spacer. HKDs are excellently suited to target kinases that have a scaffolding function, or that are resistant against conventional inhibitors. A HKD brings a kinase and an E3 ligase in proximity forming a ternary complex leading to ubiquitin-dependent catalytic degradation of the kinase of interest. This ternary complex formation is unique for each kinase which can make HKDs highly specific. To discover novel HKDs, we have developed a platform called Energetically Privileged Ligands (EPriLs), which is based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket with near-covalent affinity, and allows binding to kinases that have acquired known inhibitor resistance mutations. The macrocyclic structure allows attachment of E3 ligase ligands with multiple exit vectors. With the EPriL platform, we have developed novel HKDs for various clinically relevant kinase targets, including Bruton’s Tyrosine Kinase (BTK). BTK inhibitor drugs are used as first line treatment for chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and as second line treatment for mantle cell lymphoma (MCL). Despite their great success in the clinic, many patients on BTK inhibitors relapse eventually, mostly because of the emergence of resistance mutations in the BTK protein. These include BTKC481S, which abrogates binding of covalent drugs, and BTKT474I, BTKL528W and BTKV416L which preclude binding of non-covalent inhibitors like pirtobrutinib. HKDs can combat resistance by degrading such BTK variants, and by removing the scaffold function of BTK. BTK degraders such as NX-5948 and NX-2127 are currently in clinical Phase 1. To show the applicability of the EPriL platform for the discovery of novel BTK degraders, we screened a library of more than 100 EPriL HKDs for biochemical binding to BTK, and tested their propensity to induce degradation of HiBiT-tagged BTKWT, BTKC481S and BTKT474I in HEK293 cell lines. The degrading activity of hits was verified through western blot on the BTK-dependent DLBCL cell line TMD8. The best degraders - based on potency and efficacy - were tested in cell proliferation assays on a panel of TMD8 BTK mutant cell lines. In these lines, endogenous BTK was mutated by CRISPR to BTKC481S, BTKT474I, BTKL528W or BTKV416L. Several BTK degraders had potent (&amp;lt; 20 nM IC50) antiproliferative activity on most TMD8 lines, including difficult-to-target variants such as BTKV416L and BTKL528W. The unique binding mode of the EPriL scaffold thus translates to broad activity on resistance variants, while its selectivity ensures minimal degradation of off-target tyrosine kinases as observed for other degraders. Citation Format: Michelle Muller, Jos de Man, Joost C. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G. van Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6044.