Identification of Bruton's tyrosine kinase as a therapeutic target in acute myeloid leukemia

Bruton's tyrosine kinase (BTK) is a cytoplasmic protein found in all hematopoietic cell lineages except for T cells. BTK mediates signalling downstream of a number of receptors. Pharmacological targeting of BTK using ibrutinib (previously PCI-32765) has recently shown encouraging clinical activity in a range of lymphoid malignancies. This study reports for the first time that ibrutinib inhibits blast proliferation from human acute myeloid leukaemia (AML) and that treatment with ibrutinib significantly augmented cytotoxic activities of standard AML chemotherapy cytarabine or daunorubicin. Here we describe that BTK is constitutively phosphorylated in the majority of AML samples tested, with BTK phosphorylation correlating highly with the cell's cytotoxic sensitivity towards ibrutinib. BTK targeted RNAi knock-down reduced colony forming capacity of primary AML blasts and proliferation of AML cell lines. We showed ibrutinib binds at nanomolar range to BTK. Furthermore, we also showed ibrutinib's anti-proliferative effects in AML are mediated via an inhibitory effect on downstream nuclear factor-κB (NF-κB) survival pathways. Moreover, ibrutinib inhibited AML cell adhesion to bone marrow stroma. Furthermore, these effects of ibrutinib in AML were seen at comparable concentrations efficacious in chronic lymphocytic leukemia (CLL). These results provide a biologic rationale for clinical evaluation of BTK inhibition in AML patients

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

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

Bruton's Tyrosine Kinase Regulates the Activation of Gene Rearrangements at the λ Light Chain Locus in Precursor B Cells in the Mouse

Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an ∼50% reduction in the frequency of immunoglobulin (Ig) λ light chain expression, already at the immature B cell stage in the bone marrow. Conversely, transgenic mice expressing the activated mutant BtkE41K showed increased λ usage. As the κ/λ ratio is dependent on (a) the level and kinetics of κ and λ locus activation, (b) the life span of pre-B cells, and (c) the extent of receptor editing, we analyzed the role of Btk in these processes. Enforced expression of the Bcl-2 apoptosis inhibitor did not alter the Btk dependence of λ usage. Crossing 3-83μδ autoantibody transgenic mice into Btk-deficient mice showed that Btk is not essential for receptor editing. Also, Btk-deficient surface Ig+ B cells that were generated in vitro in interleukin 7-driven bone marrow cultures manifested reduced λ usage. An intrinsic defect in λ locus recombination was further supported by the finding in Btk-deficient mice of reduced λ usage in the fraction of pre-B cells that express light chains in their cytoplasm. These results implicate Btk in the regulation of the activation of the λ locus for V(D)J recombination in pre-B cells

Bruton's Tyrosine Kinase Is Required for Activation of Iκb Kinase and Nuclear Factor κb in Response to B Cell Receptor Engagement

Mutations in the gene encoding Bruton's tyrosine kinase (btk) cause the B cell deficiency diseases X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. In vivo and in vitro studies indicate that the BTK protein is essential for B cell survival, cell cycle progression, and proliferation in response to B cell antigen receptor (BCR) stimulation. BCR stimulation leads to the activation of transcription factor nuclear factor (NF)-κB, which in turn regulates genes controlling B cell growth. We now demonstrate that a null mutation in btk known to cause the xid phenotype prevents BCR-induced activation of NF-κB. This defect can be rescued by reconstitution with wild-type BTK. This mutation also interferes with BCR-directed activation of IκB kinase (IKK), which normally targets the NF-κB inhibitor IκBα for degradation. Taken together, these findings indicate that BTK couples IKK and NF-κB to the BCR. Interference with this coupling mechanism may contribute to the B cell deficiencies observed in XLA and xid

Nanoemulsion stability: experimental evaluation of the flocculation rate from turbidity measurements

The coalescence of liquid drops induces a higher level of complexity compared to the classical studies about the aggregation of solid spheres. Yet, it is commonly believed that most findings on solid dispersions are directly applicable to liquid mixtures. Here, the state of the art in the evaluation of the flocculation rate of these two systems is reviewed. Special emphasis is made on the differences between suspensions and emulsions. In the case of suspensions, the stability ratio is commonly evaluated from the initial slope of the absorbance as a function of time under diffusive and reactive conditions. Puertas and de las Nieves (1997) developed a theoretical approach that allows the determination of the flocculation rate from the variation of the turbidity of a sample as a function of time. Here, suitable modifications of the experimental procedure and the referred theoretical approach are implemented in order to calculate the values of the stability ratio and the flocculation rate corresponding to a dodecane-in-water nanoemulsion stabilized with sodium dodecyl sulfate. Four analytical expressions of the turbidity are tested, basically differing in the optical cross section of the aggregates formed. The first two models consider the processes of: a) aggregation (as described by Smoluchowski) and b) the instantaneous coalescence upon flocculation. The other two models account for the simultaneous occurrence of flocculation and coalescence. The latter reproduce the temporal variation of the turbidity in all cases studied (380 \leq [NaCl] \leq 600 mM), providing a method of appraisal of the flocculation rate in nanoemulsions

Hepatitis B virus infected health care workers in the Netherlands, 2000-2008

In response to the confirmed transmission of hepatitis B virus (HBV) from a surgeon to several patients in the Netherlands, a ‘Committee for Prevention of Iatrogenic Hepatitis B’ was established in 2000. During the years 2000–2008, the committee reviewed 99 cases of HBV-infected health care workers. Fifty of them were found to perform exposure prone procedures (EPPs). Because of high levels of HBV DNA (>100,000 copies/ml), a ban on performing EPPs was applied in 11/50 cases; 25/50 low-viremic health care workers were allowed to continue EPPs while their HBV load was being monitored; and 14/50 cases had stopped working or changed profession. In five restricted workers who started oral antiviral treatment, HBV replication was persistently suppressed, enabling the ban on EPPs to be lifted. Throughout the European Union different levels of HBV viremia have been chosen, above which health care workers are not allowed to perform EPPs. It remains unknown how this affects the safety of patients. Application in the Netherlands of a European or a British guideline would have, respectively, doubled or tripled the number of restricted health care workers

Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5′RACE and 3′RACE we identified three IBTKα, IBTKβ and IBTKγ mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk