ABSTRACT
BACKGROUND: Exposure to antibiotics predisposes to dysbiosis and Clostridioides difficile infection (CDI) that can be severe, recurrent (rCDI), and life-threatening. Nonselective drugs that treat CDI and perpetuate dysbiosis are associated with rCDI, in part due to loss of microbiome-derived secondary bile acid (SBA) production. Ridinilazole is a highly selective drug designed to treat CDI and prevent rCDI. METHODS: In this phase 3 superiority trial, adults with CDI, confirmed with a stool toxin test, were randomized to receive 10 days of ridinilazole (200â mg twice daily) or vancomycin (125â mg 4 times daily). The primary endpoint was sustained clinical response (SCR), defined as clinical response and no rCDI through 30 days after end of treatment. Secondary endpoints included rCDI and change in relative abundance of SBAs. RESULTS: Ridinilazole and vancomycin achieved an SCR rate of 73% versus 70.7%, respectively, a treatment difference of 2.2% (95% CI: -4.2%, 8.6%). Ridinilazole resulted in a 53% reduction in recurrence compared with vancomycin (8.1% vs 17.3%; 95% CI: -14.1%, -4.5%; P = .0002). Subgroup analyses revealed consistent ridinilazole benefit for reduction in rCDI across subgroups. Ridinilazole preserved microbiota diversity, increased SBAs, and did not increase the resistome. Conversely, vancomycin worsened CDI-associated dysbiosis, decreased SBAs, increased Proteobacteria abundance (â¼3.5-fold), and increased the resistome. CONCLUSIONS: Although ridinilazole did not meet superiority in SCR, ridinilazole greatly reduced rCDI and preserved microbiome diversity and SBAs compared with vancomycin. These findings suggest that treatment of CDI with ridinilazole results in an earlier recovery of gut microbiome health. Clinical Trials Registration.Ri-CoDIFy 1 and 2: NCT03595553 and NCT03595566.
Subject(s)
Anti-Bacterial Agents , Clostridioides difficile , Clostridium Infections , Gastrointestinal Microbiome , Vancomycin , Humans , Vancomycin/therapeutic use , Vancomycin/adverse effects , Clostridium Infections/drug therapy , Clostridium Infections/microbiology , Male , Female , Middle Aged , Double-Blind Method , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/adverse effects , Aged , Clostridioides difficile/drug effects , Gastrointestinal Microbiome/drug effects , Adult , Treatment Outcome , Metabolome/drug effects , Oxadiazoles/therapeutic use , Oxadiazoles/adverse effects , Dysbiosis/chemically induced , Benzimidazoles , PyridinesABSTRACT
Background: Ibrutinib, a first-in-class, once-daily inhibitor of Bruton's tyrosine kinase (BTK), is approved in the US and EU for the treatment of various B-cell malignancies. In clinical studies, BTK inhibitors have been associated with increased bleeding risk, which may result from BTK inhibition in platelets.Methods: To better understand the mechanism of ibrutinib in bleeding events, we isolated platelet-rich plasma from healthy donors (n = 8) and donors with conditions associated with impaired platelet function or with potentially increased bleeding risk (on hemodialysis, taking aspirin, or taking warfarin; n = 8 each cohort) and used light transmission aggregometry to assess platelet aggregation in vitro after exposure to escalating concentrations of ibrutinib, spanning and exceeding the pharmacologic range of clinical exposure.Results: Platelet aggregation was induced by agonists of 5 major platelet receptors: adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP6), ristocetin, collagen, or arachidonic acid (AA). Platelet aggregation induced by ADP, TRAP6, ristocetin, and AA was not meaningfully inhibited by the maximal concentrations of ibrutinib (10â µM). In contrast, collagen-induced platelet aggregation was dose-dependently inhibited by ibrutinib in all donor cohorts (maximum aggregation % with 10â µM ibrutinib, -64% to -83% of agonist activity compared to control agonist samples but without ibrutinib).Conclusion: These results confirm prior reports and support a mechanistic role for the inhibition of collagen-induced platelet aggregation in bleeding events among susceptible individuals receiving ibrutinib therapy.
Subject(s)
Blood Platelets/drug effects , Platelet Aggregation Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Adenine/analogs & derivatives , Adult , Aged , Female , Humans , Male , Middle Aged , Piperidines , Platelet Aggregation Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Tissue Donors , Young AdultABSTRACT
OBJECTIVES: Bruton's tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) are expressed by human platelets. These kinases participate in platelet activation through the collagen receptor glycoprotein VI and may perform overlapping functions. In clinical studies, BTK inhibitors (ibrutinib, acalabrutinib, tirabrutinib, zanubrutinib) have been associated with increased bleeding risk, which may result from inhibition of BTK alone or of both BTK and TEC, although the role of TEC in bleeding risk remains unclear. METHODS: Here, in vitro catalytic and binding activities of ibrutinib and acalabrutinib were determined with four assay systems. Platelet aggregation assays determined inhibitor potency and its relationship to selectivity between BTK and TEC. RESULTS: Neither inhibitor was substantially more selective for BTK over TEC. The potencies at which BTK inhibitors suppressed platelet aggregation correlated with the potencies in on-target BTK assays, including those in cells. At clinically relevant plasma concentration, ibrutinib, acalabrutinib, and tirabrutinib inhibited collagen-induced platelet aggregation to a similar extent, despite differing in vitro IC50 s. CONCLUSIONS: Our results suggest BTK inhibition is the primary driver for inhibition of platelet aggregation. The subtle differences between these inhibitors suggest only randomized, double-blind, placebo-controlled clinical studies can fully address the bleeding risks of different BTK inhibitors.
ABSTRACT
Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the most prevalent B-lymphocyte neoplasms in which abnormal activation of the Bruton tyrosine kinase (BTK)-mediated B-cell receptor signaling pathway contributes to pathogenesis. Ibrutinib is an oral covalent BTK inhibitor that has shown some efficacy in both indications. To improve ibrutinib efficacy through combination therapy, we first investigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms of resistance. Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL patients who experienced poorer response to ibrutinib had higher BCL2 gene expression. We further demonstrated synergistic growth suppression by ibrutinib and ABT-199 in multiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma cell lines. The combination of both drugs also reduced colony formation, increased apoptosis, and inhibited tumor growth in a TMD8 xenograft model. A synergistic combination effect was also found in ibrutinib-resistant cells generated by either genetic mutation or drug treatment. Together, these findings suggest a potential clinical benefit from ibrutinib and ABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246-56. ©2017 AACR.
Subject(s)
Drug Resistance, Neoplasm/genetics , Lymphoma, Follicular/drug therapy , Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Animals , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphoma, Follicular/genetics , Lymphoma, Follicular/pathology , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Piperidines , Protein Kinase Inhibitors/administration & dosage , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , Signal Transduction/drug effects , Sulfonamides/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
Targeting B-cell receptor (BCR) signaling is a successful therapeutic strategy in mature B-cell malignancies. Precursor BCR (pre-BCR) signaling, which is critical during normal B lymphopoiesis, also plays an important role in pre-BCR+ B cell acute lymphoblastic leukemia (B-ALL). Here, we investigated the activity and mechanism of action of the BTK inhibitor ibrutinib in preclinical models of B-ALL. Pre-BCR+ ALL cells were exquisitely sensitive to ibrutinib at therapeutically relevant drug concentrations. In pre-BCR+ ALL, ibrutinib thwarted autonomous and induced pre-BCR signaling, resulting in deactivation of PI3K/Akt signaling. Ibrutinib modulated the expression of pre-BCR regulators (PTPN6, CD22, CD72, and PKCß) and substantially reduced BCL6 levels. Ibrutinib inhibited ALL cell migration toward CXCL12 and beneath marrow stromal cells and reduced CD44 expression. CRISPR-Cas9 gene editing revealed that both BTK and B lymphocyte kinase (BLK) are relevant targets of ibrutinib in pre-BCR+ ALL. Consequently, in mouse xenograft models of pre-BCR+ ALL, ibrutinib treatment significantly prolonged survival. Combination treatment of ibrutinib with dexamethasone or vincristine demonstrated synergistic activity against pre-BCR+ ALL. These data corroborate ibrutinib as a promising targeted agent for pre-BCR+ ALL and highlight the importance of ibrutinib effects on alternative kinase targets.
Subject(s)
Antineoplastic Agents/pharmacology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , src-Family Kinases/antagonists & inhibitors , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Flow Cytometry , Gene Expression Profiling , Gene Knockout Techniques , Humans , Mice , Piperidines , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Protein-Tyrosine Kinases/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Diffuse large B cell lymphoma (DLBCL) is a heterogeneous lymphoma and the most common subtype of non-Hodgkin lymphoma, accounting for roughly 30% of newly diagnosed cases in the United States. DLBCL can be separated into the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, with distinct gene expression profiles, oncogenic aberrations, and clinical outcomes. ABC-DLBCL is characterized by chronically active B-cell receptor (BCR) signaling that can be modulated by Bruton's tyrosine kinase (BTK) activity. Thus, BTK serves as an attractive therapeutic target in this type of B-cell malignancy. Ibrutinib, a first-in-class, orally available covalent BTK inhibitor, has demonstrated clinical activity in several B-cell leukemias and lymphomas. A phase 1/2 clinical trial of single-agent ibrutinib in relapsed and refractory DLBCL patients revealed an overall response rate of 37% in ABC-DLBCL patients. However, responses to kinase-directed therapies are often limited by emerging resistance mechanisms that bypass the therapeutic target. Here we report the discovery of point mutations within the kinase PIM1 that reduce sensitivity to ibrutinib in ABC-DLBCL. These mutations stabilize PIM1 and affect upstream regulators and downstream targets of NF-κB signaling. The introduction of mutant PIM1 into an ABC-DLBCL cell line, TMD8, increased colony formation and decreased sensitivity to ibrutinib. In addition, ibrutinib-resistant cell lines generated by prolonged ibrutinib exposure in vitro upregulated PIM1 expression, consistent with a role for PIM1 in antagonizing ibrutinib activity. The combination of a pan-PIM inhibitor with ibrutinib synergistically inhibited proliferation in vitro and tumor growth in vivo. Together, these data provide a rationale for combining BTK and PIM1 inhibition in the treatment of ABC-DLBCL.
ABSTRACT
Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (<100 nmol/L); in addition, the IC50s were lower than that of lapatinib and dacomitinib. Inhibition of cell growth by ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2+ MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinib's unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835-44. ©2016 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Adenine/analogs & derivatives , Animals , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , ErbB Receptors/metabolism , Female , Humans , Mice , Piperidines , Receptor, ErbB-2/metabolism , Signal Transduction/drug effects , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
UNLABELLED: Pancreas ductal adenocarcinoma (PDAC) has one of the worst 5-year survival rates of all solid tumors, and thus new treatment strategies are urgently needed. Here, we report that targeting Bruton tyrosine kinase (BTK), a key B-cell and macrophage kinase, restores T cell-dependent antitumor immune responses, thereby inhibiting PDAC growth and improving responsiveness to standard-of-care chemotherapy. We report that PDAC tumor growth depends on cross-talk between B cells and FcRγ(+) tumor-associated macrophages, resulting in T(H)2-type macrophage programming via BTK activation in a PI3Kγ-dependent manner. Treatment of PDAC-bearing mice with the BTK inhibitor PCI32765 (ibrutinib) or by PI3Kγ inhibition reprogrammed macrophages toward a T(H)1 phenotype that fostered CD8(+) T-cell cytotoxicity, and suppressed PDAC growth, indicating that BTK signaling mediates PDAC immunosuppression. These data indicate that pharmacologic inhibition of BTK in PDAC can reactivate adaptive immune responses, presenting a new therapeutic modality for this devastating tumor type. SIGNIFICANCE: We report that BTK regulates B-cell and macrophage-mediated T-cell suppression in pancreas adenocarcinomas. Inhibition of BTK with the FDA-approved inhibitor ibrutinib restores T cell-dependent antitumor immune responses to inhibit PDAC growth and improves responsiveness to chemotherapy, presenting a new therapeutic modality for pancreas cancer.
Subject(s)
Cell Communication/immunology , Immune System/cytology , Immune System/pathology , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/metabolism , Protein-Tyrosine Kinases/metabolism , Agammaglobulinaemia Tyrosine Kinase , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Biomarkers , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Disease Models, Animal , Disease Progression , Humans , Leukocytes/immunology , Leukocytes/metabolism , Macrophage Activation , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Knockout , Myeloid Cells/immunology , Myeloid Cells/metabolism , Pancreatic Neoplasms/genetics , Receptors, IgG/metabolism , Signal TransductionABSTRACT
PURPOSE: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment. EXPERIMENTAL DESIGN: Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed. RESULTS: Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4(+)T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4(+)T cells in vitro Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells. CONCLUSIONS: In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. See related commentary by Bachireddy and Wu, p. 1547.
Subject(s)
Antineoplastic Agents/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Protein Kinase Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Tumor Microenvironment/drug effects , Adenine/analogs & derivatives , Aged , Aged, 80 and over , Animals , Antineoplastic Agents/pharmacology , Bone Marrow/pathology , Cell Communication/drug effects , Cell Communication/immunology , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Movement/immunology , Cytokines/blood , Cytokines/metabolism , Disease Models, Animal , Female , Humans , Immunophenotyping , Inflammation Mediators/blood , Inflammation Mediators/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Middle Aged , Piperidines , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/metabolismABSTRACT
The two major subtypes of diffuse large B cell lymphoma (DLBCL)--activated B cell-like (ABC) and germinal center B cell-like (GCB)--arise by distinct mechanisms, with ABC selectively acquiring mutations that target the B cell receptor (BCR), fostering chronic active BCR signaling. The ABC subtype has a â¼40% cure rate with currently available therapies, which is worse than the rate for GCB DLBCL, and highlights the need for ABC subtype-specific treatment strategies. We hypothesized that ABC, but not GCB, DLBCL tumors would respond to ibrutinib, an inhibitor of BCR signaling. In a phase 1/2 clinical trial that involved 80 subjects with relapsed or refractory DLBCL, ibrutinib produced complete or partial responses in 37% (14/38) of those with ABC DLBCL, but in only 5% (1/20) of subjects with GCB DLBCL (P = 0.0106). ABC tumors with BCR mutations responded to ibrutinib frequently (5/9; 55.5%), especially those with concomitant myeloid differentiation primary response 88 (MYD88) mutations (4/5; 80%), a result that is consistent with in vitro cooperation between the BCR and MYD88 pathways. However, the highest number of responses occurred in ABC tumors that lacked BCR mutations (9/29; 31%), suggesting that oncogenic BCR signaling in ABC does not require BCR mutations and might be initiated by non-genetic mechanisms. These results support the selective development of ibrutinib for the treatment of ABC DLBCL.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptors, Antigen, B-Cell/physiology , Signal Transduction/drug effects , Adenine/analogs & derivatives , Adult , Aged , Base Sequence , CD79 Antigens/genetics , Female , Humans , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/mortality , Male , Middle Aged , Molecular Sequence Data , Mutation , Myeloid Differentiation Factor 88/genetics , PiperidinesABSTRACT
Monoclonal antibodies can block cellular interactions that negatively regulate T-cell immune responses, such as CD80/CTLA-4 and PD-1/PD1-L, amplifying preexisting immunity and thereby evoking antitumor immune responses. Ibrutinib, an approved therapy for B-cell malignancies, is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway, which is critical to the survival of malignant B cells. Interestingly this drug also inhibits ITK, an essential enzyme in Th2 T cells and by doing so it can shift the balance between Th1 and Th2 T cells and potentially enhance antitumor immune responses. Here we report that the combination of anti-PD-L1 antibody and ibrutinib suppresses tumor growth in mouse models of lymphoma that are intrinsically insensitive to ibrutinib. The combined effect of these two agents was also documented for models of solid tumors, such as triple negative breast cancer and colon cancer. The enhanced therapeutic activity of PD-L1 blockade by ibrutinib was accompanied by enhanced antitumor T-cell immune responses. These preclinical results suggest that the combination of PD1/PD1-L blockade and ibrutinib should be tested in the clinic for the therapy not only of lymphoma but also in other hematologic malignancies and solid tumors that do not even express BTK.
Subject(s)
Cell Cycle Checkpoints/drug effects , Immunity, Cellular/drug effects , Neoplasms, Experimental/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Th1 Cells/immunology , Th2 Cells/immunology , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Animals , Antibodies, Neoplasm/pharmacology , B7-H1 Antigen , Cell Line, Tumor , Mice , Mice, Inbred BALB C , Neoplasms, Experimental/immunology , Neoplasms, Experimental/pathology , Piperidines , Th1 Cells/pathology , Th2 Cells/pathologyABSTRACT
BACKGROUND: Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. METHODS: We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. RESULTS: We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. CONCLUSIONS: Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).
Subject(s)
Drug Resistance, Neoplasm/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Phospholipase C gamma/genetics , Point Mutation , Protein-Tyrosine Kinases/genetics , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Aged , Binding Sites/genetics , Exome , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Middle Aged , Phospholipase C gamma/metabolism , Piperidines , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptors, Antigen, B-Cell/metabolism , Recurrence , Sequence Analysis, DNAABSTRACT
The function and role of Bruton's tyrosine kinase (BTK) in human B cell development was demonstrated by its association with X-linked agammaglobulinemia (XLA) manifested by a substantial reduction in immunoglobulins and B cells. BTK has a crucial role in pre-B cell receptor (BCR) and BCR signaling during normal B cell development and activation. Aberrant BCR signaling is associated with autoimmune diseases, such as rheumatoid arthritis (RA). In addition, BTK is also expressed in myeloid cell populations, including monocytes, macrophages, neutrophils and mast cells. These innate cells infiltrate the synovial cavity and produce inflammatory cytokines, aggravating arthritic symptoms. In myeloid cell populations, BTK functions downstream of the Fcγ receptors (FcγR) and FcÉ receptors (FcÉR). In the absence of BTK, FcR-mediated functions, such as cytokine production, are impaired. In addition, Xid mice, which have a mutation in BTK, have decreased susceptibility to developing collagen-induced arthritis (CIA). Given that BTK is involved in multiple signaling pathways downstream of the BCR and FcR, it is an attractive therapeutic target for RA.
Subject(s)
Arthritis, Rheumatoid/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Agammaglobulinaemia Tyrosine Kinase , Animals , Arthritis, Rheumatoid/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Humans , Pre-B Cell Receptors/immunology , Protein Kinase Inhibitors/immunology , Protein-Tyrosine Kinases/immunology , Signal Transduction/drug effects , Signal Transduction/immunologyABSTRACT
BACKGROUND: Chemoimmunotherapy has led to improved numbers of patients achieving disease response, and longer overall survival in young patients with chronic lymphocytic leukaemia; however, its application in elderly patients has been restricted by substantial myelosuppression and infection. We aimed to assess safety and activity of ibrutinib, an orally administered covalent inhibitor of Bruton tyrosine kinase (BTK), in treatment-naive patients aged 65 years and older with chronic lymphocytic leukaemia. METHODS: In our open-label phase 1b/2 trial, we enrolled previously untreated patients at clinical sites in the USA. Eligible patients were aged at least 65 years, and had symptomatic chronic lymphocytic leukaemia or small lymphocytic lymphoma requiring therapy. Patients received 28 day cycles of once-daily ibrutinib 420 mg or ibrutinib 840 mg. The 840 mg dose was discontinued after enrolment had begun because comparable activity of the doses has been shown. The primary endpoint was the safety of the dose-fixed regimen in terms of frequency and severity of adverse events for all patients who received treatment. This study is registered with ClinicalTrials.gov, number NCT01105247. FINDINGS: Between May 20, 2010, and Dec 18, 2012, we enrolled 29 patients with chronic lymphocytic leukaemia and two patients with small lymphocytic lymphoma. Median age was 71 years (range 65-84), and 23 (74%) patients were at least 70 years old. Toxicity was mainly of mild-to-moderate severity (grade 1-2). 21 (68%) patients had diarrhoea (grade 1 in 14 [45%] patients, grade 2 in three [10%] patients, and grade 3 in four [13%] patients). 15 (48%) patients developed nausea (grade 1 in 12 [39%] patients and grade 2 in three [10%] patients). Ten (32%) patients developed fatigue (grade 1 in five [16%] patients, grade 2 in four [13%] patients, and grade 3 in one [3%] patient). Three (10%) patients developed grade 3 infections, although no grade 4 or 5 infections occurred. One patient developed grade 3 neutropenia, and one developed grade 4 thrombocytopenia. After a median follow-up of 22.1 months (IQR 18.4-23.2), 22 (71%) of 31 patients achieved an objective response (95% CI 52.0-85.8); four patients (13%) had a complete response, one patient (3%) had a nodular partial response, and 17 (55%) patients had a partial response. INTERPRETATION: The safety and activity of ibrutinib in elderly, previously untreated patients with symptomatic chronic lymphocytic leukaemia, or small lymphocytic lymphoma is encouraging, and merits further investigation in phase 3 trials. FUNDING: Pharmacyclics, Leukemia and Lymphoma Society, D Warren Brown Foundation, Mr and Mrs Michael Thomas, Harry Mangurian Foundation, P50 CA140158 to Prof J C Byrd MD.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Aged , Aged, 80 and over , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Male , Piperidines , Pyrazoles/adverse effects , Pyrimidines/adverse effectsABSTRACT
Bone-resorbing osteoclasts play an essential role in normal bone homeostasis, as well as in various bone disorders such as osteoporosis and rheumatoid arthritis. Previously we showed that the Tec family of tyrosine kinases is essential for the differentiation of osteoclasts and the inhibition of Btk is a promising strategy for the prevention of the bone loss in osteoclast-associated bone disorders. Here we demonstrate that an orally available Btk inhibitor, ibrutinib (PCI-32765), suppresses osteoclastic bone resorption by inhibiting both osteoclast differentiation and function. Ibrutinib downregulated the expression of NFATc1, the key transcription factor for osteoclastogenesis, and disrupted the formation of the actin ring in mature osteoclasts. In addition, genome-wide screening revealed that Btk regulates the expression of the genes involved in osteoclast differentiation and function in both an NFATc1-dependent and -independent manner. Finally, we showed that ibrutinib administration ameliorated the bone loss that developed in a RANKL-induced osteoporosis mouse model. Thus, this study suggests ibrutinib to be a promising therapeutic agent for osteoclast-associated bone diseases.
Subject(s)
Bone Resorption/drug therapy , Bone Resorption/prevention & control , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/administration & dosage , Pyrazoles/therapeutic use , Pyrimidines/administration & dosage , Pyrimidines/therapeutic use , Adenine/analogs & derivatives , Administration, Oral , Agammaglobulinaemia Tyrosine Kinase , Animals , Bone Resorption/complications , Bone Resorption/genetics , Cell Differentiation/drug effects , Disease Models, Animal , Female , Gene Expression Regulation/drug effects , Mice , Mice, Inbred C57BL , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoblasts/pathology , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoclasts/pathology , Osteoporosis/complications , Osteoporosis/drug therapy , Osteoporosis/pathology , Osteoporosis/prevention & control , Piperidines , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RANK LigandABSTRACT
Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eµ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Protein-Tyrosine Kinases/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Adult , Agammaglobulinaemia Tyrosine Kinase , Animals , Apoptosis/drug effects , Apoptosis/immunology , Cell Survival/drug effects , Cell Survival/immunology , Disease Models, Animal , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Piperidines , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Signal Transduction/immunologyABSTRACT
Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib treatments, as seen with other inhibitors of the B-cell receptor (BCR) pathway. In a phase 1 study of ibrutinib, we noted similar effects in patients with mantle cell lymphoma (MCL). Here, we characterize the patterns and phenotypes of cells mobilized among patients with MCL and further investigate the mechanism of this effect. Peripheral blood CD19(+)CD5(+) cells from MCL patients were found to have significant reduction in the expression of CXCR4, CD38, and Ki67 after 7 days of treatment. In addition, plasma chemokines such as CCL22, CCL4, and CXCL13 were reduced 40% to 60% after treatment. Mechanistically, ibrutinib inhibited BCR- and chemokine-mediated adhesion and chemotaxis of MCL cell lines and dose-dependently inhibited BCR, stromal cell, and CXCL12/CXCL13 stimulations of pBTK, pPLCγ2, pERK, or pAKT. Importantly, ibrutinib inhibited migration of MCL cells beneath stromal cells in coculture. We propose that BTK is essential for the homing of MCL cells into lymphoid tissues, and its inhibition results in an egress of malignant cells into peripheral blood. This trial was registered at www.clinicaltrials.gov as #NCT00114738.
Subject(s)
Antineoplastic Agents/therapeutic use , B-Lymphocytes/drug effects , Chemotaxis, Leukocyte/drug effects , Lymphoma, Mantle-Cell/blood , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Adenine/analogs & derivatives , Antigens, CD19/biosynthesis , B-Lymphocytes/metabolism , Blotting, Western , CD5 Antigens/biosynthesis , Cell Adhesion/drug effects , Flow Cytometry , Humans , Lymphoma, Mantle-Cell/drug therapy , Piperidines , Protein Kinase Inhibitors/therapeutic useABSTRACT
Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.
Subject(s)
Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Th1 Cells/drug effects , Adenine/analogs & derivatives , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/enzymology , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Jurkat Cells , Leishmaniasis, Cutaneous/drug therapy , Leishmaniasis, Cutaneous/immunology , Leukemia/drug therapy , Leukemia/immunology , Listeriosis/drug therapy , Listeriosis/immunology , Lymphocyte Activation/drug effects , Mice , Piperidines , Primary Cell Culture , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Th1 Cells/cytology , Th1 Cells/enzymology , Th2 Cells/cytology , Th2 Cells/drug effects , Th2 Cells/enzymologyABSTRACT
BACKGROUND: Bruton's tyrosine kinase (BTK) is a mediator of the B-cell-receptor signaling pathway implicated in the pathogenesis of B-cell cancers. In a phase 1 study, ibrutinib, a BTK inhibitor, showed antitumor activity in several types of non-Hodgkin's lymphoma, including mantle-cell lymphoma. METHODS: In this phase 2 study, we investigated oral ibrutinib, at a daily dose of 560 mg, in 111 patients with relapsed or refractory mantle-cell lymphoma. Patients were enrolled into two groups: those who had previously received at least 2 cycles of bortezomib therapy and those who had received less than 2 complete cycles of bortezomib or had received no prior bortezomib therapy. The primary end point was the overall response rate. Secondary end points were duration of response, progression-free survival, overall survival, and safety. RESULTS: The median age was 68 years, and 86% of patients had intermediate-risk or high-risk mantle-cell lymphoma according to clinical prognostic factors. Patients had received a median of three prior therapies. The most common treatment-related adverse events were mild or moderate diarrhea, fatigue, and nausea. Grade 3 or higher hematologic events were infrequent and included neutropenia (in 16% of patients), thrombocytopenia (in 11%), and anemia (in 10%). A response rate of 68% (75 patients) was observed, with a complete response rate of 21% and a partial response rate of 47%; prior treatment with bortezomib had no effect on the response rate. With an estimated median follow-up of 15.3 months, the estimated median response duration was 17.5 months (95% confidence interval [CI], 15.8 to not reached), the estimated median progression-free survival was 13.9 months (95% CI, 7.0 to not reached), and the median overall survival was not reached. The estimated rate of overall survival was 58% at 18 months. CONCLUSIONS: Ibrutinib shows durable single-agent efficacy in relapsed or refractory mantle-cell lymphoma. (Funded by Pharmacyclics and others; ClinicalTrials.gov number, NCT01236391.)
Subject(s)
Lymphoma, Mantle-Cell/drug therapy , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Adenine/analogs & derivatives , Administration, Oral , Adult , Agammaglobulinaemia Tyrosine Kinase , Aged , Aged, 80 and over , Drug Resistance, Neoplasm , Female , Humans , Lymphocyte Count , Lymphoma, Mantle-Cell/mortality , Male , Middle Aged , Piperidines , Protein Kinase Inhibitors/adverse effects , Pyrazoles/adverse effects , Pyrimidines/adverse effects , Recurrence , Survival AnalysisABSTRACT
BACKGROUND: The treatment of relapsed chronic lymphocytic leukemia (CLL) has resulted in few durable remissions. Bruton's tyrosine kinase (BTK), an essential component of B-cell-receptor signaling, mediates interactions with the tumor microenvironment and promotes the survival and proliferation of CLL cells. METHODS: We conducted a phase 1b-2 multicenter study to assess the safety, efficacy, pharmacokinetics, and pharmacodynamics of ibrutinib (PCI-32765), a first-in-class, oral covalent inhibitor of BTK designed for treatment of B-cell cancers, in patients with relapsed or refractory CLL or small lymphocytic lymphoma. A total of 85 patients, the majority of whom were considered to have high-risk disease, received ibrutinib orally once daily; 51 received 420 mg, and 34 received 840 mg. RESULTS: Toxic effects were predominantly grade 1 or 2 and included transient diarrhea, fatigue, and upper respiratory tract infection; thus, patients could receive extended treatment with minimal hematologic toxic effects. The overall response rate was the same in the group that received 420 mg and the group that received 840 mg (71%), and an additional 20% and 15% of patients in the respective groups had a partial response with lymphocytosis. The response was independent of clinical and genomic risk factors present before treatment, including advanced-stage disease, the number of previous therapies, and the 17p13.1 deletion. At 26 months, the estimated progression-free survival rate was 75% and the rate of overall survival was 83%. CONCLUSIONS: Ibrutinib was associated with a high frequency of durable remissions in patients with relapsed or refractory CLL and small lymphocytic lymphoma, including patients with high-risk genetic lesions. (Funded by Pharmacyclics and others; ClinicalTrials.gov number, NCT01105247.).