Dasatinib inhibits CXCR4 signaling in chronic lymphocytic leukaemia cells and impairs migration towards CXCL12

Chemokines and their ligands play a critical role in enabling chronic lymphocytic leukaemia (CLL) cells access to protective microenvironmental niches within tissues, ultimately resulting in chemoresistance and relapse: disruption of these signaling pathways has become a novel therapeutic approach in CLL. The tyrosine kinase inhibitor dasatinib inhibits migration of several cell lines from solid-organ tumours, but effects on CLL cells have not been reported. We studied the effect of clinically achievable concentrations of dasatinib on signaling induced by the chemokine CXCL12 through its' receptor CXCR4, which is highly expressed on CLL cells. Dasatinib pre-treatment inhibited Akt and ERK phosphorylation in CLL cells upon stimulation with CXCL12. Dasatinib also significantly diminished the rapid increase in actin polymerisation observed in CLL cells following CXCL12 stimulation. Moreover, the drug significantly inhibited chemotaxis in a transwell assay, and reduced the percentage of cells able to migrate beneath a CXCL12-expressing murine stromal cell line. Dasatinib also abrogated the anti-apoptotic effect of prolonged CXCL12 stimulation on cultured CLL cells. These data suggest that dasatinib, akin to other small molecule kinase inhibitors targeting the B-cell receptor signaling pathway, may redistribute CLL cells from protective tissue niches to the peripheral blood, and support the investigation of dasatinib in combination strategies

An increased fraction of circulating miR-363 and miR-16 is particle bound in patients with chronic lymphocytic leukaemia as compared to normal subjects.

In vitro culture studies have shown that miR-363 is enriched in extracellular vesicles from chronic lymphocytic leukaemia cells. We wondered whether miR-363 was detectable in plasma, which is an essential precondition for further studies to assess its usefulness as a biomarker. Using samples from two clinical trials: one enrolling patients with advanced disease and the other asymptomatic patients with early stage disease, we determined plasma miR-363 levels and secondly investigated the distribution of this miRNA between plasma and particle bound fractions in patients and normal subjects.Advanced disease (n = 95) was associated with higher levels of miR-363 than early stage disease (n = 45) or normal subjects (n = 11) but there was no association with markers of prognosis. The distribution of specific miRNA between particle bound and plasma protein fractions was investigated using size exclusion chromatography on plasma from patients (n = 4) and normal subjects (n = 3). ~ 20% of total miR-16 and miR-363 is particle bound in patients while there was no detectable particle bound material in normal subjects. Our work demonstrates that miR-363 levels are raised in chronic lymphocytic leukaemia patients and raises the possibility that distribution of circulating miRNA between plasma fractions differs in health and disease

The miR-17/92 cluster: a comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and disease.

The miR-17/92 cluster is among the best-studied microRNA clusters. Interest in the cluster and its members has been increasing steadily and the number of publications has grown exponentially since its discovery with more than 1000 articles published in 2012 alone. Originally found to be involved in tumorigenesis, research work in recent years has uncovered unexpected roles for its members in a wide variety of settings that include normal development, immune diseases, cardiovascular diseases, neurodegenerative diseases and aging. In light of its ever-increasing importance and ever-widening regulatory roles, we review here the latest body of knowledge on the cluster\u27s involvement in health and disease as well as provide a novel perspective on the full spectrum of protein-coding and non-coding transcripts that are likely regulated by its members

Regulation of CD38 in proliferating chronic lymphocytic leukaemia cells stimulated with CD154 and IL-4

Background and Objectives Chronic lymphocytic leukemia (CLL) cells, like normal B-cells, exist in two populations in vivo : quiescent cells in the peripheral circulation and proliferating cells in lymph nodes. The surface marker CD38 has roles in cell adhesion and signaling. Its expres- sion correlates with poor clinical outcome and is associated with expression of the signaling intermediate ZAP-70, which is also a marker of poor prognosis. We investi- gated the regulation of CD38 and ZAP-70 in proliferating CLL cells. Design and Methods W e cultured CLL cells on a stromal cell la yer that maintains viability and also with some stromal cells expressing CD40 ligand (CD154) in order to measure changes in expression of CD38 and ZAP-70. Results W e demonstrated up-regulation of CD38 expression b y CD154. The degree of up-reg - ulation did not cor relate with clinical stage or mutational status. In addition in the majority of cases tested ZAP-70 expression increased in parallel with up-regulation of CD38 although discordant cases were also observed. Interpretation and Conclusions Overall w e demonstrated that regulation of CD38 in CLL is dynamic and dependent on signals from CD154 and a stromal cell la yer . W e speculate that CD38 and ZAP-70 are expressed in lymph node leukemic cells in both good and poor prognosis patients, but, in cases with good clinical outcome, these molecules are down-regulat- ed in the peripheral blood whereas in cases with poor prognosis their expression is maintaine

An in vitro based investigation of the cytotoxic effect of water extracts of the Chinese herbal remedy LD on cancer cells

BACKGROUND: Long Dan Xie Gan Wan (LD), a Chinese herbal remedy formulation, is traditionally used to treat a range of conditions, including gall bladder diseases, hepatitis, hyperthyroidism, migraines but it is not used for the management or treatment of cancer. However some of its herbal constituents, specifically Radix bupleuri, Radix scutellariae and Rhizoma alismatis have been shown to inhibit the growth of cancer cells. Thus, the aim of the study was to investigate the impact of LD on cancer cells in vitro. METHODS: HL60 and HT29 cancer cell lines were exposed to water extracts of LD (1:10, 1:50, 1:100 and/or 1:1000 prepared from a 3 mg/30 ml stock) and for both cell lines growth, apoptotic induction, alterations in cell cycle characteristics and genotoxicity were investigated. The specificity of the action of LD on these cancer cell lines was also investigated by determining its effect on human peripheral blood lymphocytes. Preliminary chemical analysis was carried out to identify cytotoxic constituents of LD using HPLC and LCMS. RESULTS: LD was significantly cytotoxic to, and induced apoptosis in, both cell lines. Apoptotic induction appeared to be cell cycle independent at all concentrations of LD used (1:10, 1:50 and 1:100) for the HL60 cell lines and at 1:10 for the HT29 cell line. At 1:50 and 1:100 apoptotic induction by LD appeared to be cell cycle dependent. LD caused significant genotoxic damage to both cell lines compared to their respective controls. The specificity study showed that LD exerted a moderate cytotoxic action against non-proliferating and proliferating blood lymphocytes but not apoptosis. Chemical analysis showed that a number of fractions were found to exert a significant growth inhibitory effect. However, the molecular weights of compounds within these fractions did not correspond to those from the herbal constituents of LD. CONCLUSION: It is possible that LD may have some chemotherapeutic potential. However, further studies are required to determine its cytotoxic constituents

CD40 and B-cell receptor signalling induce MAPK family members that can either induce or repress Bcl-6 expression

Bcl-6 is essential for germinal centre development and normal antibody responses, and has major roles in controlling B-cell proliferation and differentiation. Bcl-6 expression is tightly controlled, but neither the nature of all the regulatory signals nor their interactions are known. Bcl-6 expression is induced in Bcr-Abl expressing lymphoid cell lines by the tyrosine kinase inhibitor, imatinib. We show that p38 MAPK mediates induction of Bcl-6 following inhibition of Bcr-Abl by imatinib. Next we analyze p38 function in a germinal centre B-cell line, Ramos. p38 is phosphorylated under basal conditions, and studies with p38 inhibitors show that it induces Bcl-6 expression. Membrane bound CD40 ligand activates p38 but also other MAPK pathways that strongly repress Bcl-6 and the overall effect is reduction in Bcl-6 expression. Surprisingly soluble CD40 ligand induces Bcl-6 by activating p38 without activating the repressive pathways. Hence different types of CD40 signalling are associated with varying effects on Bcl-6 expression. Transcription reporter assays demonstrate p38 responsive sequences at about 4.5 kb from the transcription start site. Immunocytochemistry of tonsil sections show phosphorylated p38 in a minor population of germinal centre B-cells. We demonstrate for the first time that p38 induces Bcl-6 transcription, but increased protein expression occurs only when the strong pathways repressing Bcl-6 are not activated.Ana Batlle, Vasiliki Papadopoulou, Ana R. Gomes, Shaun Willimott, Junia V. Melo, Kikkeri Naresh, Eric W.-F. Lamb and Simon D. Wagne

The level of BCR-ABL1 kinase activity before treatment does not identify CML patients who fail to achieve a complete cytogenetic response on Imatinib

Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However, 20–25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown, but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34+ cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway