The effect of the dual Src/Abl kinase inhibitor AZD0530 on Philadelphia positive leukaemia cell lines

Background Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells. Methods Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively. Results AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15. Conclusion Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl

The role of Src family tyrosine kinases in imatinib resistant philadelphia chromosome positive leukaemias

Imatinib (GleevecTM; GlivecTM; formerly STI571), a specific inhibitor of Abl tyrosine kinase, is efficacious in treating Philadelphiachromosomepositive (Ph+) leukaemias such as chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL) (Ottmann, Druker et al. 2002). Within a few years of its introduction to the clinic, Imatinib had dramatically altered the firstline therapy for CML, because it was found that most newly diagnosed CML patients in the chronic phase achieve durable responses when treated with Imatinib (Goldman and Melo 2003). However, a small percentage of these patients, as well as most advancedphase CML and Ph+ ALL patients, relapse on Imatinib therapy (Yokota, Kimura et al. 2006). Several mechanisms of refractoriness and relapse have been reported. These include point mutations within the Abl kinase domain, overexpression of BcrAbl mRNA (Hofmann, Jones et al. 2002), decreased intracellular drug levels mediated by Pglycoprotein (Pgp) (Hegedus, Orfi et al. 2002), and nonBcrAbl dependent mechanisms (activation of the SFKs) (Donato, Wu et al. 2003). In this research work, a possible means of overcoming resistance to Imatinib by the use of the specific dual Src/Abl kinase inhibitor AZD0530 has been investigated. The efficacy of AZD0530 in the treatment of Ph+ leukaemias, sensitive to or resistant to Imatinib, has been tested on cell lines, primary patient material and in vivo in transduction/transplantation mouse model of Imatinib sensitive or resistant BcrAbl dependent CML-like disease. Data with AZD0530 has been compared to cells treated with Imatinib. The potential of inhibiting both Src and Abl kinases while inducing growth arrest and apoptosis has been analysed. AZD0530 specifically inhibited the growth of CML and Ph+ ALL cells in a dosedependent manner, but has shown a marginal effect on Ph- ALL cells. Treatment of p185BcrAbl expressing Ba/F3 cells with AZD0530 has led to apoptosis induction and growth inhibition in these cells, while the untransformed Ba/F3 cells have remained unaffected. Resistance to Imatinib due to mutation in the Ba/F3MutY253F cells has been overcomed by this compound. The growth inhibitory effect of AZD0530 correlates with its induction of apoptosis. Combination of AZD0530 and Imatinib at low concentrations has shown an additive effect on the inhibition of proliferation of BV173 cells. The growth inhibition and apoptosis induction by AZD0530 have shown to be uncoupled to major changes in cell cycle. An exception is the CML blast crisis cell line BV173 which has shown a considerable G0/G1 arrest in the presence of AZD0530 and Imatinib as single agents. Immunoblotting of whole cell lysates from Imatinib or AZD0530 treated BV173, Ba/F3 expressing p185(BcrAbl) MutT253F cells and the WTSupB15 cells, for Src and BcrAbl clearly demonstrates that there is an ongoing transphosphorylation taking place between the SFKs and BcrAbl. This transphosphorylation synergizes and influences the aggressive nature of CML blast crisis and Ph+ ALL. Investigations have been carried out on downstream signaling events to determine how Src family members contribute to BcrAbl signaling. Specifically, Stat, Erk and PI3K/ Akt activation status have been characterised in Imatinib sensitive and resistant Ph+ cells. AZD0530 has significantly downregulated the activation of survival signaling pathways as shown by it’s inhibition of Stat5, Akt and Erk kinases in Ph+ cells, resistant or sensitive to Imatinib. The only exception to this has been the Imatinib resistant cell line RTSupB15, in which activated Akt kinase level has remained unaffected. AZD0530 has shown to be efficient in the treatment of cells isolated from three Ph+ leukaemic patients (resistant or sensitive to Imatinib), and has led to an induction of apoptosis. Equally, in the same patients, growth and survival pathways have been inhibited in vitro in the presence of AZD0530. An overall therapeutic effect of AZD0530 in vivo has been studied in mouse model of Imatinib sensitive and Imatinib resistant, BcrAbldependent desease. Mice with a BcrAbllike disease responded to Imatinib treatment but not to AZD0530. Using the CFU assay, an influence on the differentiation status of primary leukaemic blast stem cells have been tested. The in vivo studies as well as the CFU results have shown discrepancies to the effects of AZD0530 tested so far in this research work. These discrepancies have paralleled with the upregulation of BcrAbl in most AZD0530 treated cells. These are to be further analysed. These data elucidate the role of Src kinases in BcrAbl leukaemogenesis. Results gotten from this research work has shown that AZD0530 targets both Src and BcrAbl kinase activity and reduces the transforming potential of BcrAbl. It also shows that there is an ongoing transphosphorylation between SFKs and BcrAbl kinase. AZD0530 has proven effective in CML cell lines, Ph+ ALL cell lines and patient cells resistant to Imatinib. These have demonstrated that AZD0530 is a potential drug target which can be used to overcome Imatinib resistance.Das Philadelphia-Chromosom (Ph) ist das zytogenetische Korrelat der Translokation t(9;22). 95% der chronisch myeloischen Leukämien (CML) und 2025% der akuten lymphatischen Leukämien (ALL) bei Erwachsenen sind Phpositiv (Ph+). Bei der t(9;22) kommt es zur Fusion des Abllocus auf Chromosom 9 und des Bcrlocus auf Chromosom 22. Dies führt zur Bildung des chimären Bcr-Abl-Gens, welches für das BcrAblFusionsprotein kodiert, das für die Pathogenese der Ph+ Leukämien verantwortlich gemacht wird. Das Abl-Protein ist eine Nicht-Rezeptor-Tyrosin-Kinase, die eine wichtige Rolle in der Signaltransduktion und der Regulation des Zellwachstums spielt. Im Bcr-Abl-Fusionsprotein wird die Kinase-Aktivität von Abl, die im Normalfall streng reguliert ist, durch die Fusion mit Bcr konstitutiv, das heißt andauernd, aktiviert. Dadurch kommt es zur Deregulierung vielfältiger intrazellulärer Signalwege, was die maligne Transformation hämatopoetischer Zellen zur Folge hat. Mit dem spezifischen Abl-Kinaseinhibitor Imatinib steht seit wenigen Jahren ein tumorzellspezifischer Wirkstoff für die Therapie der Ph+ Leukämien zur Verfügung, der bei der Mehrzahl der Patienten zur hämatologischen Vollremission führt. Die Begeisterung über den neuen Wirkstoff wird jedoch in letzter Zeit durch die Aufdeckung multifaktorieller Mechanismen der Resistenz der leukämischen Zellen gegen den spezifischen KinaseInhibitor getrübt. Insbesondere bei Patienten mit CML-Blastenkrise und Ph+ ALL kommt es durch klonale Expansion Imatinibresistenter Zellen zu einem frühen Therapierefraktären Rezidiv der Krankheit. Als Resistenzmechanismen werden diskutiert: i.) Punktmutationen, die die Bindung von Imatinib an die ATP-Bindungsstelle von Bcr-Abl inhibieren (Hofmann, Komor et al. 2003); ii.) die Hochregulation der Expression von Bcr-Abl (Hofmann, Jones et al. 2002); iii.) die Selektion von zusätzlichen molekularen Aberrationen unter Imatinib, welche die Abhängigkeit der Leukämiezellen von der deregulierten Bcr-Abl-Kinaseaktivität aufhebt (Donato, Wu et al. 2003); iv.) die Hochregulation der Expression zellmembranständiger Effluxpumpen (‘multi drug resistance gene product’) (Hegedus, Orfi et al. 2002); v.) verminderte Bioverfügbarkeit z.B. durch Bindung an inaktivierende Proteine im Serum (GambacortiPasserini, Barni et al. 2000). Aus diesen Erkenntnissen geht hervor, dass Imatinib mit anderen Therapiemöglichkeit kombiniert werden muss. Attraktive Kandidaten sind dabei verschiedene Substanzklassen, die gleichfalls als Signaltransduktionsinhibitoren fungieren. Bcr-Abl kann Src-Tyrosin-Kinasen (Src family tyrosine kinase (SFK) aktivieren (Klejman, Schreiner et al. 2002; Stanglmaier, Warmuth et al. 2003). Es konnte gezeigt werden, dass Bcr-Abl in myeloischen Zellen mit der Src-Kinase Hck (‚haematopoietic cell kinase’) interagiert, diese phosphoryliert, und dadurch aktiviert. Phophorylierte Hck aktiviert ihrerseits Stat5 (‚signal transducer and activator of transcription’) durch Phosphorylierung. Es ist bekannt, dass die Aktivierung von Stat5 von grundlegender Bedeutung für das transformierende Potential von Bcr-Abl ist. Desweiteren konnte gezeigt werden, dass Bcr-Abl direkt Src-Tyrosin-Kinasen aktiviert. Außerdem wird ihnen eine potentielle Rolle in der Ausbildung der Resistenz gegenüber Imatinib zugeschrieben (Donato, Wu et al. 2003). Ziel dieser Arbeit ist die Aufklärung der Rolle von Src-Tyrosin-Kinasen in Imatinibsensitiven und resistenten Ph+ Zellen. Es wurden die Mechanismen der Wirkung des dualen Src/Abl-Tyrosin-KinaseInhibitors AZD0530 analysiert. Die klinische Implikation besteht in der Vermeidung oder Überwindung der Resistenz der CML-Blastenkrise und Ph+ ALL gegenüber Imatinib. ..