Bruton's Tyrosine Kinase Pathway Research Articles

CLL-114 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)

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. Covalent BTKi share pharmacologic liabilities (e.g., low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, 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 equally low nM potency, was developed. Demonstrate the superiority of continued BTK pathway inhibition with pirtobrutinib compared with other available therapies in patients with BTKi-treated CLL/SLL. BRUIN CLL-321 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus the 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. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by 17p deletion (yes/no) and prior venetoclax (yes/no). Patients receiving the investigator's choice are eligible to cross over to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 (determined by an independent review committee [IRC]). Global; community hospitals, academic medical centers. 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. An unlimited number of lines of prior therapy is allowed. Key exclusion criteria include CNS involvement by CLL/SLL, Richter transformation at any time pre-enrollment, a major bleeding event on prior covalent BTKi, and a history of allogeneic or autologous stem cell transplant or chimeric antigen receptor (CAR) T-cell therapy within 60 days of randomization. Pirtobrutinib monotherapy versus investigator's choice of idelalisib plus rituximab or bendamustine plus rituximab. The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an IRC. Secondary endpoints include overall survival, overall response rate, duration of response, safety and tolerability, and patient-reported outcomes. The global study is currently enrolling patients (NCT04666038). This study is a trial in progress. This study is a trial in progress.

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.

A Novel BCL-2/BCL-Xl Dual Inhibitor Overcomes Ibrutinib-Resistance Conferred By the Upregulation of Integrin Pathway and BCL-Xl

Introduction Ibrutinib, a first-in-class oral covalent inhibitor of Bruton's tyrosine kinase (BTK), has been FDA-approved for the treatment of mantle cell lymphoma, chronic lymphocytic leukemia (CLL) and Waldenström's macroglobulinemia. In CLL, 80% overall response rate (OR) as a monotherapy or 96% OR in the combination with BCL-2 selective inhibitor venetoclax demonstrates a promising regimen for CLL patients. However, ibrutinib only yields to a modest (38%) OR in follicular lymphoma (FL) trial, especially doing poorly in patients with NF-kB constitutive activation via CARD11 mutation. To better understand ibrutinib resistance mechanism, we established a resistance model using sensitive follicular lymphoma cell line DOHH2, and characterized the gene expression profile and signaling pathway alteration, and tested potential approach to overcome the resistance. Methods 1. DOHH2-IR (ibrutinib resistant) cells were established by exposing to the increasing concentrations of the drug over 3 months and confirmed by cell viability assays. 2. RNA-seq was performed to reveal the transcriptome alteration between DOHH2 and DOHH2-IR cells. 3. Cell sensitivity to BCL-2/BCL-xL inhibitors was assessed by CTG assays. Results Parental DOHH2 and resistant DOHH2-IR cells were first confirmed ibrutinib sensitivity/resistance by CTG assay, which showed an IC50 of 0.074 uM for DOHH2, but 160 fold greater for DOHH2-IR at 12.19 uM. To decipher the mechanisms underlying the induced ibrutinib resistance, whole-transcriptome RNA-seq was performed on parental and resistant cells. Unsupervised hierarchical clustering showed significant alterations in gene expression signatures. Among them, 4,925 genes were significantly up-regulated and 3,727 genes were down-regulated. Kyoto Encyclopedia of Genes and Genome (KEGG) analysis showed that the components of BCR signaling pathway had been significantly downregulated in the resistant cells, so were those in pro-survival NF-kB and mTOR pathways, anti-apoptotic BCL-2 and BFL-1 genes, and TP53 pathway; in contrast, BCL-xL, MAP kinase and extracellular matrix (ECM)-receptor interaction genes, especially those integrin family genes, were increased (Figure 1). The upregulation of BCL-xL gene expression prompted us to investigate if inhibiting BCL-xL would resume cell sensitivity to killing. We thus treated cells with APG-1252-M1 (ester hydrolyzed active metabolite of APG-1252 for in vitro study), a novel BCL-2/BCL-xL dual inhibitor developed by Ascentage. In comparison, we also treated cells with BCL-2 selective inhibitor APG-2575, which is also a proprietary compound by Ascentage, and its reference compound ABT-199. APG-2575 and ABT-199 partially restored cell death in the resistant cells, with IC50s of 0.65 uM and 1.19 uM, respectively, which were greater than parental cells with IC50s of approximate 0.05 uM. However, the dual inhibitor APG-1252-M1 effectively killed DOHH2-IR cells with an IC50 of 0.017 uM, and cell death was potently elicited within 24 hours (Figure 2). This finding is consistent with RNA-seq data, that BCL-2 is downregulated, while BCL-xL is induced during ibrutinib resistant selection. Conclusions In an ibrutinib-resistant DOHH2 model, we show that reprogram of BTK and apoptosis pathway is associated with ibrutinib resistance. Instead of mutating BTK or its downstream PLCg2 gene in CLL, resistant cells have selected to bypass the pathway; and unexpectedly, NF-kB pathway is also downregulated in DOHH2-IR, which is contrast to previously report that it is one of resistant mechanisms for ibrutinib. Evading apoptosis is one of the key mechanisms in which cancer cells can survive or become resistant to therapies. Integrin family proteins or ECM - receptor interactions also play important roles in drug resistance. Further studies are needed to understand how the components of integrin pathway cross-talk with BCL-2 family proteins. However, the potent killing activity conferred by APG-1252 confirms that BCL-xL upregulation is key in this FL ibrutinib resistance model. Collectively, the results suggest that the combination of ibrutinib and BCL-2/BCL-xL dual inhibitor may produce deeper and longer duration response in follicular lymphoma patients. Disclosures Deng: Ascentage Pharma Group: Employment. Mao:Ascentage Pharma Group: Employment. Huang:Ascentage Pharma Group: Employment. Rui:Ascentage Pharma Group: Employment. Wang:Ascentage Pharma Group: Employment. Fang:Ascentage Pharma Group: Employment. Yang:Ascentage Pharma Group: Employment. Zhai:Ascentage Pharma Group: Employment.

Bruton's Tyrosine Kinase Inhibition Attenuates the Cardiac Dysfunction Caused by Cecal Ligation and Puncture in Mice.

Sepsis is one of the most prevalent diseases in the world. The development of cardiac dysfunction in sepsis results in an increase of mortality. It is known that Bruton's tyrosine kinase (BTK) plays a role in toll-like receptor signaling and NLRP3 inflammasome activation, two key components in the pathophysiology of sepsis and sepsis-associated cardiac dysfunction. In this study we investigated whether pharmacological inhibition of BTK (ibrutinib 30 mg/kg and acalabrutinib 3 mg/kg) attenuates sepsis associated cardiac dysfunction in mice. 10-week old male C57BL/6 mice underwent CLP or sham surgery. One hour after surgery mice received either vehicle (5% DMSO + 30% cyclodextrin i.v.), ibrutinib (30 mg/kg i.v.), or acalabrutinib (3 mg/kg i.v.). Mice also received antibiotics and an analgesic at 6 and 18 h. After 24 h, cardiac function was assessed by echocardiography in vivo. Cardiac tissue underwent western blot analysis to determine the activation of BTK, NLRP3 inflammasome and NF-κB pathway. Serum analysis of 33 cytokines was conducted by a multiplex assay. When compared to sham-operated animals, mice subjected to CLP demonstrated a significant reduction in ejection fraction (EF), fractional shortening (FS), and fractional area change (FAC). The cardiac tissue from CLP mice showed significant increases of BTK, NF-κB, and NLRP3 inflammasome activation. CLP animals resulted in a significant increase of serum cytokines and chemokines (TNF-α, IL-6, IFN-γ, KC, eotaxin-1, eotaxin-2, IL-10, IL-4, CXCL10, and CXCL11). Delayed administration of ibrutinib and acalabrutinib attenuated the decline of EF, FS, and FAC caused by CLP and also reduced the activation of BTK, NF-κB, and NLRP3 inflammasome. Both ibrutinib and acalabrutinib significantly suppressed the release of cytokines and chemokines. Our study revealed that delayed intravenous administration of ibrutinib or acalabrutinib attenuated the cardiac dysfunction associated with sepsis by inhibiting BTK, reducing NF-κB activation and the activation of the inflammasome. Cytokines associated with sepsis were significantly reduced by both BTK inhibitors. Acalabrutinib is found to be more potent than ibrutinib and could potentially prove to be a novel therapeutic in sepsis. Thus, the FDA-approved BTK inhibitors ibrutinib and acalabrutinib may be repurposed for the use in sepsis.

Identification of a Genetic Signature Enriching for Response to Ibrutinib in Relapsed/Refractory Follicular Lymphoma in the Dawn Phase 2 Trial

Identification of a Genetic Signature Enriching for Response to Ibrutinib in Relapsed/Refractory Follicular Lymphoma in the Dawn Phase 2 Trial

Activity of Bruton's tyrosine-kinase inhibitor ibrutinib in patients with CD117-positive acute myeloid leukaemia: a mechanistic study using patient-derived blast cells

Roughly 80% of patients with acute myeloid leukaemia have high activity of Bruton's tyrosine-kinase (BTK) in their blast cells compared with normal haemopoietic cells, rendering the cells sensitive to the oral BTK inhibitor ibrutinib in vitro. We aimed to develop the biological understanding of the BTK pathway in acute myeloid leukaemia to identify clinically relevant diagnostic information that might define a subset of patients that should respond to ibrutinib treatment. We obtained acute myeloid leukaemia blast cells from unselected patients attending our UK hospital between Feb 19, 2010, and Jan 20, 2014. We isolated primary acute myeloid leukaemia blast cells from heparinised blood and human peripheral blood mononuclear cells to establish the activity of BTK in response to CD117 activation. Furthermore, we investigated the effects of ibrutinib on CD117-induced BTK activation, downstream signalling, adhesion to primary bone-marrow mesenchymal stromal cells, and proliferation of primary acute myeloid leukaemia blast cells. We used the Mann-Whitney U test to compare results between groups. We obtained acute myeloid leukaemia blast cells from 29 patients. Ibrutinib significantly inhibited CD117-mediated proliferation of primary acute myeloid leukaemia blast cells (p=0·028). CD117 activation increased BTK activity by inducing phosphorylated BTK in patients with CD117-positive acute myeloid leukaemia. Furthermore, ibrutinib inhibited CD117-induced activity of BTK and downstream kinases at a concentration of 100 nM or more. CD117-mediated adhesion of CD117-expressing blast cells to bone-marrow stromal cells was significantly inhibited by Ibrutinib at 500 nM (p=0·028) INTERPRETATION: As first-in-man clinical trials of ibrutinib in patients with acute myeloid leukaemia commence, the data suggest not all patients will respond. Our findings show that BTK has specific pro-tumoural biological actions downstream of surface CD117 activation, which are inhibited by ibrutinib. Accordingly, we propose that patients with acute myeloid leukaemia whose blast cells express CD117 should be considered for forthcoming clinical trials of ibrutinib. Worldwide Cancer Research, The Big C, UK National Institutes for Health Research, the Humane Research Trust, the Department of Higher Education and Research of the Libyan Government, and Norwich Research Park.

Ibrutinib: A New Frontier in the Treatment of Chronic Lymphocytic Leukemia by Bruton’s Tyrosine Kinase Inhibition

Chronic lymphocytic leukemia (CLL) is characterized by progressive accumulation of nonfunctional mature B cells in blood, bone marrow and lymphoid tissues. In the last decade, our understanding of CLL and consequently our diagnostic and therapeutic approaches have changed dramatically. Conventional fludarabine based chemotherapy has led to improved disease response and longer survival in young patients with CLL. However its application in elderly patients has been restricted by substantial myelosuppression and infection. Treatment of CLL is now moving towards targeted therapy. The success of new class of agents such as monoclonal antibodies, proteasome inhibitors and immunomodulatory derivatives has sparked further search for treatment agents with novel targets to inhibit. The B cell receptor activating pathway involving the Bruton’s tyrosine kinase (BTK) is crucial in B cell production and maintenance and is an attractive therapeutic target. Ibrutinib is an oral covalent inhibitor of the BTK pathway that induces apoptosis of B cells. Early phase studies with Ibrutinib either as a single agent or in combination regimens have shown promising results with an excellent safety profile in patients with high-risk, refractory or relapsed CLL and elderly treatment-naïve patients. This review summarizes the current knowledge of Ibrutinib in the treatment of CLL.

Abstract LB-303: Small molecule inhibitor of the BTK pathway disrupts BCR signaling and demonstrates antitumor efficacy in a xenograft model.

Abstract Despite the recent advances made in the treatment and management of B cell malignancies, these diseases are not curable and overall survival is limited. Bruton's Tyrosine Kinase (BTK) is a member of the Tec family of intracellular kinases first identified for its signaling via the B-cell Receptor (BCR) and its role in the immune system. More recently, BTK was found to play an important role in B cell malignancies and select solid tumors. Preclinical and clinical results with selective irreversible BTK inhibitors provide validation for BTK as a therapeutic target in B cell malignancies. Aiming to leverage the contribution of the BTK signaling pathway to tumor growth, and its role in progression and drug resistance, we have developed a series of relatively selective, reversible, small molecule BTK inhibitors and evaluated their activity in enzyme, cell-based, and in vivo studies. Data obtained with the orally available MKC4659 compound illustrates our findings. In biochemical assays, compound MKC4659 demonstrated a relatively select targeting profile focused on a narrow group of PTKs including significant activity against BTK with IC50 less than 25 nM. In cellular assays the compound demonstrated significant in vitro potency against B cell lymphoma cell lines, inhibiting the growth of several B cell tumor cell lines including ones unresponsive to currently known BTK inhibitors. Importantly, MKC4659 showed a differential effect on B cell lymphoma, with no significant activity detected in control cells lacking detectable BTK expression. In vitro mode of action studies demonstrated that MKC4659 induces apoptosis and PARP cleavage in B cell lymphoma but not in control cells. Assays evaluating the in vitro on-target effect of compounds showed significant inhibition of the B cell receptor-mediated activation of the BTK pathway. In addition to inhibiting the phosphorylation of BTK, MKC4659 inhibited the phosphorylation of PLCγ in several B cell lymphoma cell lines. With in vitro potency demonstrated, and PK and ADMET profiles amenable to in vivo dosing, MKC4659 was evaluated for in vivo efficacy in a xenograft model of B cell lymphoma. In vivo dosing of MKC4659 inhibited growth of DOHH2 xenograft tumors in a dose dependent manner. In summary, our team has identified BTK pathway inhibitors with demonstrated on-target and anti-tumor activity in cellular assays, and efficacy in a preclinical model of B cell malignancy. This effort provides a platform for compound development and evaluation for the treatment of hematologic malignancies. Optimization efforts on the MKC4659 series are ongoing and have yielded potent and drug-like preclinical candidates that are now moving into advanced animal studies. Citation Format: Mary Faris, Uriel M. Malyankar, Victor Tam, Colleen Schweitzer, Diljeet Joea, Alexis Mollard, Bret Stephens, Steven L. Warner, David J. Bearss, Qingping Zeng. Small molecule inhibitor of the BTK pathway disrupts BCR signaling and demonstrates antitumor efficacy in a xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2013-LB-303

Ibrutinib: an evidence-based review of its potential in the treatment of advanced chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable course, and remains an incurable disease. Frequent relapses and eventual resistance to fludarabine characterize symptomatic CLL and portends a dismal prognosis for patients. Growing evidence has shown that signaling pathways such as the B cell receptor and NFkB are implicated in the survival and proliferation of the CLL cells which are ultimately associated with persistence of the disease. The Bruton’s tyrosine kinase pathway regulates downstream activation of the B cell receptor and has emerged as an attractive target. Ibrutinib inhibits the Bruton’s tyrosine kinase pathway, and consequently induces apoptosis of B cells. Phase I and II studies have shown impressive response rates with an excellent safety profile in patients with refractory/relapsed CLL and elderly treatment-naïve CLL patients. This paper reviews the preclinical and clinical data for ibrutinib when used in the treatment of CLL. Recent studies showing the benefit of combination therapy using ibrutinib, monoclonal antibodies, and chemoimmunotherapy are also discussed.

Bruton's tyrosine kinase (BTK) is a binding partner for hypoxia induced mitogenic factor (HIMF/FIZZ1) and mediates myeloid cell chemotaxis

Hypoxia induced mitogenic factor (HIMF) is a member of the FIZZ/resistin/RELM family of proteins that we have shown to have potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. In the current report, we identified Bruton's tyrosine kinase (BTK) as a functional HIMF binding partner through glutathione S-transferase (GST)-HIMF pull-down studies and mass spectrometry. Using primary cultured HIMF-stimulated murine bone marrow cells, we demonstrated that HIMF causes redistribution of BTK to the leading edge of the cells. HIMF stimulation induced BTK autophosphorylation, which peaked at 2.5 min. A transwell migration assay showed that treatment with recombinant murine HIMF induced migration of primary cultured bone marrow cells that was completely blocked by the BTK inhibitor, LFM-A13. Our results demonstrate BTK as the first known functional binding partner of the HIMF/FIZZ family of proteins and that HIMF acts as a chemotatic molecule in stimulating the migration of myeloid cells through activation of the BTK pathway.