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

Molecular targeting in Philadelphia-chromosome positive leukemias

Acute lymphoblastic leukemia (ALL) is an aggressive, complex disease with a several subtypes. The presence of the Philadelphia chromosome (Ph) - the cytogenetic correlate of the t(9;22) - or the t(4;11) define high risk ALL patients with a poor prognosis (Hoelzer and Gokbuget 2000; Hoelzer et al. 2002; Hoelzer et al. 2002). Ph chromosome lead to expression of p185(BCR-ABL) or p210(BCR-ABL) chimeric proteins depending on translocation breakpoints. The fusion of Abl-tyrosine kinase on Bcr leads to constitutive activation of the Abl kinase which causes the transformation of cells. With the currently applied chemotherapy regimens survival ranges between 0-10%, even though initial complete remission rates of 80% are comparable to those achieved in Ph negative (Ph-) patients. Imatinib (GleevecTM; GlivecTM; formerly STI571), a specific inhibitor of ABL tyrosine kinase, is efficacious in treating Ph+ ALL. However, despite the persistence of BCR-ABL expression throughout all phases of this disease, patients frequently relapse and progress on therapy (Donato et al. 2004). The major problems of Ph+ ALL patients are low long-term survival and development of the Imatinib resistance. Therefore, in this study, mechanisms of Imatinib resistance and alternative therapy approaches have been investigated. One approach was to identify genes with a possible role in Imatinib resistance with the aim of preventing or overcoming resistance in Ph+ ALLs. A second approach was to investigate a new therapy approaches by triggering impaired transcription, induced by BCRABL activated signaling that leads to uncontrolled proliferation, defects in apoptosis and an impaired differentiation potential of leukemic blasts. The aim was to inhibit histone deacetylation which is responsible for aberrant chromatin remodeling. By comparative genetic expression analysis of Imatinib sensitive wild type Ph+ ALL cells to Imatinib resistant derived cells a small number of differentially expressed genes connected with malignant transformation and potential functional meaning for the development of resistance were identified. After the validation of the overexpressed genes in Imatinib resistant cells, Egr-1 was selected for further investigation. In this study it is shown that Imatinib resistant cells have a different gene expression profile than non resistant cells although on protein level these findings could not be confirmed. Egr-1 protein expression in Ph+ human and Ph+ mouse cells showed that the role of Egr-1 in resistance can not be excluded though the dual function of Egr-1 as an oncogene and tumor suppressor gene most probably plays a more dominant role in leukemogenesis. The function of Egr-1 in Ph+ cell lines as well as in mHSC is cell type specific and its role in differentiation and proliferation varies, depending on the hematopoietic cell type. Nevertheless, overexpression of Egr-1 appears to have the ability to enhance proliferation and replating efficiency of mHSC but has no influence on differentiation. Taken together, further studies should be performed to understand functional interactions between Egr-1 and other genes involved in leukemias for a rational drug design. HDAC inhibitors (HDI) are members of a new class of agents able to regulate gene expression by modulating chromatin structure (Struhl 1998; Kouzarides 1999). In the second part of my study, in vitro effects of the novel HDI LAQ824 on ALL-derived cell lines were examined. LAQ824 inhibits intracellular HDAC activity, inducing an accumulation of acetylated histone species. In addition LAQ824 significantly inhibited proliferation and induced apoptosis in B-lineage Ph+ as well as Ph- ALL cells. Apoptotic signal induced by LAQ824 includes G0/G1 cell cycle arrest, up-regulation of p21WAF1/Cip1, cleavage of PARP and concomitant activation of caspases-2,-3 and -9. Considering these data together with down-regulation of Bcl-2, activation of BID and disruption of Dym observed in tested cell lines, it can be concluded that the program cell death induced by LAQ824 goes through intrinsic (mitochondrial) pathway. In cell lines harboring translocations, activation of caspases-3 and -9 is dispensable for LAQ824-induced apoptosis what indicates that the presence of the t(4;11) or t(9;22) translocation may interfere with apoptosis induction. In summary, presented data demonstrated that LAQ824 is a novel HDI with significant antileukemia activity in vitro. The anti-tumor activity correlates well with its ability to arrest the cell cycle and to induce apoptosis even in cells derived from high risk ALL. Taken together this work showed that Imatinib resistant cells have a different gene expression profile than non resistant cells. By used experimental approaches it was not possible to clearly evidence a significant role for Egr-1. To overcome Imatinib resistance new drugs were involved and tested. LAQ824 has been found to be potent inhibitor of tumor cell growth. Collectively, these findings generate the rationale to investigate the clinical efficacy of LAQ824 in the treatment of ALL.Die akute lymphatische Leukämie (ALL) ist eine aggressive Krankheit des hämatopoetischen Systems. Die Gegenwart des Philadelphia - Chromosoms (Ph), das die Translokation t(9;22) kodiert, oder die t(4,11) definieren Hochrisiko - ALL - Patienten mit einer besonders schlechten Prognose (Hoelzer and Gokbuget 2000; Hoelzer et al. 2002; Hoelzer et al. 2002)]. Das Ph Chromosom führt je nach Bruchpunkt der Translokation zur Expression von p185(BCR-ABL) oder p210(BCR-ABL). Die Fusion der Abl-Tyrosin Kinase an Bcr führt zur konstitutiven Aktivierung der Abl-Kinase, die hauptsächlich für die Transformation der Zellen verantwortlich ist. Die Überlebensrate durch aktuelle zytostatische Therapien der Ph+ ALL liegt bei 0-10%, trotz initialer Komplettremission (CR) von 80%, die ähnlich hoch wie die von Ph– Patienten ist. Imatinib (GleevecTM, GlivecTM, früher STI571) ist ein spezifischer Inhibitor der Abl – Kinase mit hoher Effizienz für die Behandlung der Ph+ ALL. Trotz der effektiven Hemmung von BCR-ABL durch Imatinib kommt es beinahe immer zum Rezidiv. Dies verschlechtert weiter die Prognose (Donato et al. 2004). Die Entstehung von Mutationen ist die häufigste Ursache für Resistenz gegen Imatinib, Nilotinib und/oder Dasatinib - Therapie. In dieser Arbeit wurden alternative Therapiestrategien für die Behandlung der ALL untersucht. Dazu wurden zwei Ansätze gewählt, wobei 1.) Gene identifiziert wurden, die zur Imatinib – Resistenz führen, um der Resistenzentwicklung vorzubeugen oder die Resistenz zu überwinden. Weiterhin wurde 2.) versucht die aberrante Transkription leukämischer Zellen zu verhindern. Dazu wurde die Inhibition der Histon – Deazetylierung, die für die aberrante Remodellierung des Chromatins verantwortlich ist, untersucht. ..

Use of a novel histone deacetylase inhibitor to induce apoptosis in cell lines of acute lymphoblastic leukemia.

Background and objectives: Chromatin structure and thereby transcription is controlled by the level of acetylation of histones, which is determined by the balance between histone acetyl transferase (HAT) activity and histone deacetylase (HDAC) activity. HDAC inhibitors are a class of compounds able to regulate gene expression by modulating chromatin structure. There are two major classes of HDAC inhibitors: the hydroxamic acid derivatives such as trichostatin A (TSA) or SAHA, and the butyrates such as phenyl-butyrate. HDAC inhibitors interfere with differentiation, proliferation and apoptosis in tumor cells. Here, we investigated the activity of a new hydroxamic acid derivative, LAQ824, on lymphoblastic cells. Design and methods: Four different pre-B lymphoblastic cell lines: Sup-B15 and TMD-5, both t(9;22) positive, SEM, t(4;11) positive, and NALM-6 cells were exposed to the hydroxamic acid derivatives, LAQ824 and TSA. Histone hyperacetylation, apoptosis, cell cycle and related pathways were assessed by flow cytometry and Western blotting. Results: LAQ824 significantly inhibited the proliferation of leukemic lymphoblastic cell lines. The effect of LAQ824 was due to increased apoptosis accompanied by activation of caspase-3 and caspase-9, cleavage of poly(ADP-ribose)-polymerase (PARP) as well as by down-regulation of Bcl-2 and disruption of the mitochondrial membrane potential. Surprisingly, LAQ824-induced apoptosis was at least partially independent of caspase activation as indicated by the fact that LAQ824-induced apoptosis was inhibited only partially in both t(9;22) positive Sup-B15 and TMD-5 cells, whereas no inhibition was observed in t(4;11) positive SEM cells upon exposure to the polycaspase inhibitor zVAD-fmk. Interpretation and conclusions: Our study establishes that LAQ824 is a promising agent for the therapy of acute lymphoblastic leukemia

Cohort profile: the ESC EURObservational Research Programme Non-ST-segment elevation myocardial infraction (NSTEMI) Registry

Aims The European Society of Cardiology (ESC) EURObservational Research Programme (EORP) Non-ST-segment elevation myocardial infarction (NSTEMI) Registry aims to identify international patterns in NSTEMI management in clinical practice and outcomes against the 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without ST-segment-elevation. Methods and results Consecutively hospitalised adult NSTEMI patients (n = 3620) were enrolled between 11 March 2019 and 6 March 2021, and individual patient data prospectively collected at 287 centres in 59 participating countries during a two-week enrolment period per centre. The registry collected data relating to baseline characteristics, major outcomes (inhospital death, acute heart failure, cardiogenic shock, bleeding, stroke/transient ischaemic attack, and 30-day mortality) and guideline-recommended NSTEMI care interventions: electrocardiogram pre- or in-hospital, prehospitalization receipt of aspirin, echocardiography, coronary angiography, referral to cardiac rehabilitation, smoking cessation advice, dietary advice, and prescription on discharge of aspirin, P2Y12 inhibition, angiotensin converting enzyme inhibitor (ACEi)/angiotensin receptor blocker (ARB), beta-blocker, and statin. Conclusion The EORP NSTEMI Registry is an international, prospective registry of care and outcomes of patients treated for NSTEMI, which will provide unique insights into the contemporary management of hospitalised NSTEMI patients, compliance with ESC 2015 NSTEMI Guidelines, and identify potential barriers to optimal management of this common clinical presentation associated with significant morbidity and mortality