Open Access

Biserka Bacic Toplek

• 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. ...
• 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.