Publications by authors named "Betty Y Chang"

29 Publications

  • Page 1 of 1

Effects of ibrutinib on in vitro platelet aggregation in blood samples from healthy donors and donors with platelet dysfunction.

Hematology 2020 Dec;25(1):112-117

Department of Medicine, McMaster University, Hamilton, Canada.

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. To better understand the mechanism of ibrutinib in bleeding events, we isolated platelet-rich plasma from healthy donors ( = 8) and donors with conditions associated with impaired platelet function or with potentially increased bleeding risk (on hemodialysis, taking aspirin, or taking warfarin;  = 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. 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 (10 µM). In contrast, collagen-induced platelet aggregation was dose-dependently inhibited by ibrutinib in all donor cohorts (maximum aggregation % with 10 μM ibrutinib, -64% to -83% of agonist activity compared to control agonist samples but without ibrutinib). 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.
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http://dx.doi.org/10.1080/16078454.2020.1730080DOI Listing
December 2020

The effect of Bruton's tyrosine kinase (BTK) inhibitors on collagen-induced platelet aggregation, BTK, and tyrosine kinase expressed in hepatocellular carcinoma (TEC).

Eur J Haematol 2018 Jul 20. Epub 2018 Jul 20.

Pharmacyclics, LLC, an AbbVie Company, Sunnyvale, CA, USA.

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 IC 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.
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http://dx.doi.org/10.1111/ejh.13148DOI Listing
July 2018

Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma.

Mol Cancer Ther 2017 07 20;16(7):1246-1256. Epub 2017 Apr 20.

Research Department, Pharmacyclics LLC, an AbbVie Company, Sunnyvale, California.

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 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 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. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0555DOI Listing
July 2017

Ibrutinib inhibits pre-BCR B-cell acute lymphoblastic leukemia progression by targeting BTK and BLK.

Blood 2017 03 28;129(9):1155-1165. Epub 2016 Dec 28.

Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX.

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 PKCβ) 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.
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http://dx.doi.org/10.1182/blood-2016-06-722900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374732PMC
March 2017

The role of PIM1 in the ibrutinib-resistant ABC subtype of diffuse large B-cell lymphoma.

Am J Cancer Res 2016 1;6(11):2489-2501. Epub 2016 Nov 1.

Department of Research, Pharmacyclics, LLC, an AbbVie Company Sunnyvale, CA, USA.

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 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 and tumor growth . Together, these data provide a rationale for combining BTK and PIM1 inhibition in the treatment of ABC-DLBCL.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126268PMC
November 2016

Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth.

Mol Cancer Ther 2016 12 27;15(12):2835-2844. Epub 2016 Sep 27.

Research Department, Pharmacyclics LLC, an AbbVie Company, Sunnyvale, California.

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 ICs 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.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0923DOI Listing
December 2016

Bruton Tyrosine Kinase-Dependent Immune Cell Cross-talk Drives Pancreas Cancer.

Cancer Discov 2016 Mar 29;6(3):270-85. Epub 2015 Dec 29.

Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, Oregon. Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.

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.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783268PMC
March 2016

Disruption of in vivo Chronic Lymphocytic Leukemia Tumor-Microenvironment Interactions by Ibrutinib--Findings from an Investigator-Initiated Phase II Study.

Clin Cancer Res 2016 Apr 9;22(7):1572-82. Epub 2015 Dec 9.

Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland.

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.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-1965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818677PMC
April 2016

Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma.

Nat Med 2015 Aug 20;21(8):922-6. Epub 2015 Jul 20.

Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA.

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 ∼40% 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.
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http://dx.doi.org/10.1038/nm.3884DOI Listing
August 2015

Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK.

Proc Natl Acad Sci U S A 2015 Mar 17;112(9):E966-72. Epub 2015 Feb 17.

Division of Oncology, Department of Medicine, Stanford University, Stanford, CA 94305-5151; and

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.
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http://dx.doi.org/10.1073/pnas.1500712112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352777PMC
March 2015

Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib.

N Engl J Med 2014 Jun 28;370(24):2286-94. Epub 2014 May 28.

From the Division of Hematology, Department of Internal Medicine (J.A.W., T.-M.L., A.S.R., S.M.J., K.A.B., A.L., A.J.J., J.C. Byrd), the Department of Biomedical Informatics (H.G.O., A.S.Y.), and the Department of Pathology (G.L.), Ohio State University, Columbus; the Department of Medicine, Division of Hematology-Oncology, Weill Cornell Medical College, New York (R.R.F.); the Division of Molecular Genetics, German Cancer Research Center, Heidelberg (M.Z., P.L.), and the Department of Internal Medicine III, University of Ulm, Ulm (S.S.) - both in Germany; Pharmacyclics, Sunnyvale, CA (L.X., D.H.-H.L., S.M.S., D.F.J., J.J.B., B.Y.C.); the Duke Cancer Institute, Duke University, Durham, NC (S.S.D., J.Z.); the Division of Hematology-Oncology, Department of Medicine, Hofstra North Shore-LIJ School of Medicine, New Hyde Park, NY (J.C. Barrientos); and Janssen Research and Development, Beerse, Belgium (M.V.).

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.).
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http://dx.doi.org/10.1056/NEJMoa1400029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144824PMC
June 2014

Bruton's tyrosine kinase inhibitors for the treatment of rheumatoid arthritis.

Drug Discov Today 2014 Aug 12;19(8):1200-4. Epub 2014 Apr 12.

Pharmacyclics, Inc., 999 East Arques Avenue, Sunnyvale, CA 94085, USA. Electronic address:

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 Fcɛ receptors (FcɛR). 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.
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http://dx.doi.org/10.1016/j.drudis.2014.03.028DOI Listing
August 2014

Ibrutinib as initial therapy for elderly patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma: an open-label, multicentre, phase 1b/2 trial.

Lancet Oncol 2014 Jan 10;15(1):48-58. Epub 2013 Dec 10.

Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA.

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.
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http://dx.doi.org/10.1016/S1470-2045(13)70513-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134524PMC
January 2014

The orally available Btk inhibitor ibrutinib (PCI-32765) protects against osteoclast-mediated bone loss.

Bone 2014 Mar 4;60:8-15. Epub 2013 Dec 4.

Department of Cell Signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549, Japan; Department of Immunology, Graduate School of Medicine, The University of Tokyo, Japan; Japan Science and Technology Agency (JST), Explorative Research for Advanced Technology (ERATO) Program, Takayanagi Osteonetwork Project, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bone.2013.11.025DOI Listing
March 2014

Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL).

Blood 2014 Feb 5;123(8):1207-13. Epub 2013 Dec 5.

Division of Hematology, Department of Internal Medicine and Comprehensive Cancer Center, The Ohio State University, Columbus, OH;

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.
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http://dx.doi.org/10.1182/blood-2013-07-515361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931190PMC
February 2014

Egress of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients.

Blood 2013 Oct 12;122(14):2412-24. Epub 2013 Aug 12.

Research Department, Pharmacyclics, Inc., Sunnyvale, CA;

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.
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http://dx.doi.org/10.1182/blood-2013-02-482125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790509PMC
October 2013

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes.

Blood 2013 Oct 25;122(15):2539-49. Epub 2013 Jul 25.

Department of Internal Medicine, Division of Hematology.

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.
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http://dx.doi.org/10.1182/blood-2013-06-507947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795457PMC
October 2013

Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia.

N Engl J Med 2013 Jul 19;369(1):32-42. Epub 2013 Jun 19.

Division of Hematology, Department of Internal Medicine, Ohio State University, Columbus, OH 43210, USA.

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.).
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http://dx.doi.org/10.1056/NEJMoa1215637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772525PMC
July 2013

Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma.

N Engl J Med 2013 Aug 19;369(6):507-16. Epub 2013 Jun 19.

Department of Lymphoma and Myeloma, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

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.)
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http://dx.doi.org/10.1056/NEJMoa1306220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513941PMC
August 2013

Modulating proximal cell signaling by targeting Btk ameliorates humoral autoimmunity and end-organ disease in murine lupus.

Arthritis Res Ther 2012 Nov 8;14(6):R243. Epub 2012 Nov 8.

Introduction: Systemic lupus erythematosus is a chronic autoimmune disease characterized by an abundance of autoantibodies against nuclear antigens. Bruton's tyrosine kinase (Btk) is a proximal transducer of the BCR signal that allows for B-cell activation and differentiation. Recently, selective inhibition of Btk by PCI-32765 has shown promise in limiting activity of multiple cells types in various models of cancer and autoimmunity. The aim of this study was to determine the effect of Btk inhibition by PCI-32765 on the development of lupus in lupus-prone B6.Sle1 and B6.Sle1.Sle3 mice.

Methods: B6.Sle1 or B6.Sle1.Sle3 mice received drinking water containing either the Btk inhibitor PCI-32765 or vehicle for 56 days. Following treatment, mice were examined for clinical and pathological characteristics of lupus. The effect of PCI-32765 on specific cell types was also investigated.

Results: In this study, we report that Btk inhibition dampens humoral autoimmunity in B6.Sle1 monocongenic mice. Moreover, in B6.Sle1.Sle3 bicongenic mice that are prone to severe lupus, Btk inhibition also dampens humoral and cellular autoimmunity, as well as lupus nephritis.

Conclusions: These findings suggest that partial crippling of cell signaling in B cells and antigen presenting cells (APCs) may be a viable alternative to total depletion of these cells as a therapeutic modality for lupus.
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http://dx.doi.org/10.1186/ar4086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674619PMC
November 2012

Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies.

J Clin Oncol 2013 Jan 8;31(1):88-94. Epub 2012 Oct 8.

Stanford University Medical Center, Stanford, CA 94305-5821, USA.

Purpose: Survival and progression of mature B-cell malignancies depend on signals from the B-cell antigen receptor, and Bruton tyrosine kinase (BTK) is a critical signaling kinase in this pathway. We evaluated ibrutinib (PCI-32765), a small-molecule irreversible inhibitor of BTK, in patients with B-cell malignancies.

Patients And Methods: Patients with relapsed or refractory B-cell lymphoma and chronic lymphocytic leukemia received escalating oral doses of ibrutinib. Two schedules were evaluated: one, 28 days on, 7 days off; and two, once-daily continuous dosing. Occupancy of BTK by ibrutinib in peripheral blood was monitored using a fluorescent affinity probe. Dose escalation proceeded until either the maximum-tolerated dose (MTD) was achieved or, in the absence of MTD, until three dose levels above full BTK occupancy by ibrutinib. Response was evaluated every two cycles.

Results: Fifty-six patients with a variety of B-cell malignancies were treated over seven cohorts. Most adverse events were grade 1 and 2 in severity and self-limited. Dose-limiting events were not observed, even with prolonged dosing. Full occupancy of the BTK active site occurred at 2.5 mg/kg per day, and dose escalation continued to 12.5 mg/kg per day without reaching MTD. Pharmacokinetic data indicated rapid absorption and elimination, yet BTK occupancy was maintained for at least 24 hours, consistent with the irreversible mechanism. Objective response rate in 50 evaluable patients was 60%, including complete response of 16%. Median progression-free survival in all patients was 13.6 months.

Conclusion: Ibrutinib, a novel BTK-targeting inhibitor, is well tolerated, with substantial activity across B-cell histologies.
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http://dx.doi.org/10.1200/JCO.2012.42.7906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505166PMC
January 2013

Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma.

Blood 2012 Aug 11;120(9):1877-87. Epub 2012 Jun 11.

LeBow Institute for Myeloma Therapeutics and Jerome Lipper Center for Multiple Myeloma Research, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

Bruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF-induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17 nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5 nM) and MM patients. It decreased SDF-1-induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P < .03) and MM cell-induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.
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http://dx.doi.org/10.1182/blood-2011-12-396853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433091PMC
August 2012

The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia.

Blood 2012 Mar 25;119(11):2590-4. Epub 2012 Jan 25.

Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α(4)β(1)-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.
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http://dx.doi.org/10.1182/blood-2011-11-390989DOI Listing
March 2012

The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells.

Arthritis Res Ther 2011 Jul 13;13(4):R115. Epub 2011 Jul 13.

Pharmacyclics, Inc, Research Department, Sunnyvale, CA 94085-4521, USA.

Introduction: The aim was to determine the effect of the Bruton tyrosine kinase (Btk)-selective inhibitor PCI-32765, currently in Phase I/II studies in lymphoma trials, in arthritis and immune-complex (IC) based animal models and describe the underlying cellular mechanisms.

Methods: PCI-32765 was administered in a series of murine IC disease models including collagen-induced arthritis (CIA), collagen antibody-induced arthritis (CAIA), reversed passive anaphylactic reaction (RPA), and passive cutaneous anaphylaxis (PCA). Clinical and pathologic features characteristic of each model were examined following treatment. PCI-32765 was then examined in assays using immune cells relevant to the pathogenesis of arthritis, and where Btk is thought to play a functional role. These included proliferation and calcium mobilization in B cells, cytokine and chemokine production in monocytes/macrophages, degranulation of mast cells and its subsequent cytokine/chemokine production.

Results: PCI-32765 dose-dependently and potently reversed arthritic inflammation in a therapeutic CIA model with an ED(50) of 2.6 mg/kg/day. PCI-32765 also prevented clinical arthritis in CAIA models. In both models, infiltration of monocytes and macrophages into the synovium was completely inhibited and importantly, the bone and cartilage integrity of the joints were preserved. PCI-32765 reduced inflammation in the Arthus and PCA assays. In vitro, PCI-32765 inhibited BCR-activated primary B cell proliferation (IC(50) = 8 nM). Following FcγR stimulation, PCI-32765 inhibited TNFα, IL-1β and IL-6 production in primary monocytes (IC(50) = 2.6, 0.5, 3.9 nM, respectively). Following FcεRI stimulation of cultured human mast cells, PCI-32765 inhibited release of histamine, PGD(2), TNF-α, IL-8 and MCP-1.

Conclusions: PCI-32765 is efficacious in CIA, and in IC models that do not depend upon autoantibody production from B cells. Thus PCI-32765 targets not only B lymphocytes but also monocytes, macrophages and mast cells, which are important Btk-expressing effector cells in arthritis.
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http://dx.doi.org/10.1186/ar3400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3239353PMC
July 2011

JAK3 inhibition significantly attenuates psoriasiform skin inflammation in CD18 mutant PL/J mice.

J Immunol 2009 Aug 13;183(3):2183-92. Epub 2009 Jul 13.

Rigel Pharmaceuticals, Inc, South San Francisco, CA 94080, USA.

JAK3, a member of the Janus kinase family, is predominantly expressed in hemopoietic cells and binds specifically to the common gamma chain of a subfamily of cytokine receptors that includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Previous studies suggest that this tyrosine kinase plays key roles in mediating T cell functions, and inhibition of JAK3 has been shown to prevent graft rejection and decrease the severity of arthritis in rodent models. However, the functions of JAK3 in the development of skin immune responses and diseases such as psoriasis have not been determined. CD18 mutant PL/J mice develop spontaneous T cell-dependent psoriasiform skin disease with several similarities to human psoriasis. In this study, we treated mice with established skin disease with R348, a small molecule inhibitor of JAK3, and observed a marked attenuation of skin lesions following 6 wk of treatment. Histological analyses revealed major reductions of both epidermal and dermal lesion severity scores in R348-treated CD18-deficient PL/J mice compared with vehicle controls, which was associated with decreased CD4(+) T cell infiltration. In addition, systemic levels of IL-17, IL-22, IL-23, and TNF-alpha were significantly lower in mice receiving the compound, and T cells isolated from R348-treated mice also showed reduced phosphorylation of Stat5 after stimulation with IL-2. These findings suggest that small-molecule inhibitors of JAK3 may be useful in the treatment of inflammatory skin diseases such as psoriasis and strongly implicate JAK signaling events as important in the pathogenesis of this disease.
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http://dx.doi.org/10.4049/jimmunol.0804063DOI Listing
August 2009

RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells.

FEBS Lett 2004 Jun;567(2-3):321-6

Department of Medicine, Stanford University, Stanford, CA 94305, USA.

Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.
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http://dx.doi.org/10.1016/j.febslet.2004.03.125DOI Listing
June 2004

The Xenopus laevis cortical granule lectin: cDNA cloning, developmental expression, and identification of the eglectin family of lectins.

Comp Biochem Physiol A Mol Integr Physiol 2004 Jan;137(1):115-29

Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616, USA.

A Xenopus laevis egg cortical granule, calcium-dependent, galactosyl-specific lectin participates in forming the fertilization layer of the egg envelope and functions in establishing a block to polyspermy. We report the cDNA cloning of the lectin, expression of the cortical granule lectin gene during oogenesis and early development, and identification of a new family of lectins. The translated cDNA for the cortical granule lectin had a signal peptide, a structural sequence of 298 amino acids, a molecular weight of 32.7 K, contained consensus sequence sites for N-glycosylation and a fibrinogen domain. The lectin cDNA was expressed during early stages of oogenesis. Lectin glycoprotein levels were constant during development with 2/3 of the lectin associated with the extracellular perivitelline space and the egg/embryo fertilization envelope. Lectin mRNA levels were from 100- to 1000-fold greater in ovary than in other adult tissues. The lectin had no sequence homology to the previously identified lectin families. The lectin had 41-88% amino acid identity with nine translated cDNA sequences from an ascidian, lamprey, frog, mouse, and human. Based on the conserved carbohydrate binding and structural properties of these glycoproteins, we propose a new family of lectins, the eglectin family.
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http://dx.doi.org/10.1016/s1095-6433(03)00269-1DOI Listing
January 2004

Detection of protein kinase-binding partners by the yeast two-hybrid analysis.

Methods Mol Biol 2003 ;233:327-43

Department of Medicine, Stanford University School of Medicine, CA, USA.

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http://dx.doi.org/10.1385/1-59259-397-6:327DOI Listing
February 2004

RACK1: a novel substrate for the Src protein-tyrosine kinase.

Oncogene 2002 Oct;21(50):7619-29

Department of Medicine, Stanford University, Stanford, California, CA 94305, USA.

RACK1 is one of a group of PKC-interacting proteins collectively called RACKs (Receptors for Activated C-Kinases). Previously, we showed that RACK1 also interacts with the Src tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various tyrosine kinases in vitro, and by kinase-active and inactive mutants of Src in vivo. We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor tyrosine kinases and is involved in the regulation of Src function and cell growth.
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http://dx.doi.org/10.1038/sj.onc.1206002DOI Listing
October 2002