Entwicklung der Vaskularisierung und des Fasergehaltes im Knochenmark von CML-Patienten während der Behandlung mit Imatinib Gesteigerte Gefäßneubildung und retikuläre Fibrose sind zwei morphologische Veränderungen im Knochenmark von CML-Patienten, welche mit einem schlechteren Krankheitsverlauf assoziiert sind. Imatinib, ein selektiver Tyrosinkinaseinhibitor, hemmt die angiogenen und fibrogenen Substanzen VEGF und PDGF. Diese Dissertation untersuchte den Einfluss von Imatinib auf die Vaskularisierung und den retikulären Fasergehalt im Knochenmark von Patienten mit chronischer myeloischer Leukämie (CML). Es wurden 67 repräsentative Knochenmarkbiopsien von insgesamt 19 Patienten, behandelt in Multicenter-Studien an der Universitätsklinik Leipzig, eingeschlossen. Davon waren zehn Patienten bereits mit IFN-α + Cytarabin vorbehandelt worden. Neun Patienten mit neudiagnostizierter CML erhielten Imatinib als Ersttherapie. Knochenmarkbiopsien zu den Zeitpunkten t0 (vor Therapiebeginn), t1 (6-15 Monate) und t2 (21-36 Monate) nach Behandlungsstart mit Imatinib wurden untersucht. Weiter wurde eine Kontrollgruppe aus 19 KM-Biopsien gebildet, welche ohne pathologischen Befund waren. Während der Behandlung mit Imatinib kam es bei der Mehrzahl der Patienten zu einer Normalisierung der Gefäßdichte im Knochenmark. Der Fasergehalt im Knochenmark der CML-Patienten sank bei allen Patienten auf Normwerte ab. Dies zeigt den positiven Einfluss von Imatinib auf die morphologischen Veränderungen Vaskularisierung und Faserdichte im Knochenmark von CML-Patienten. Ein Einfluss der Vorbehandlung mit IFN-α + Cytarabin auf die Dynamik der morphologischen Parameter während der Behandlung mit Imatinib war nicht nachweisbar.

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

Understanding the factors that regulate the growth and differentiation of haemopoietic stem cells (HSC) remains a major challenge. In this study, the proliferation and differentiation of CD34+ cells from normal donors and chronic myeloid leukaemia (CML) patients was compared. The proliferation and entry of CML cells into the cell cycle was decreased relative to cells from normal donors, and greater heterogeneity in the phenotype of CML cells at the initiation of culture was observed. Analysis of phenotype concomitant with cell division also demonstrated that the differentiation of normal CD34+ cells was consistent between donors, while marked variability was observed in the differentiation of CD34+ cells from CML patients. This included expression of CD13, CD33, CD38 and HLA-DR, which were linked to cell division in normal but not CML cells. The tyrosine kinase inhibitor, imatinib, is a novel drug displaying promising results in the treatment of CML by specifically inhibiting the growth of leukaemic cells. To examine whether myelosuppression observed in patients treated with imatinib may arise from inhibition of normal haemopoiesis, imatinib was added to colony assays established using cells from normal bone marrow. Suppression of monocyte/macrophage growth, but not that of eosinophils or neutrophils, was observed at therapeutic concentrations of imatinib. Inhibition of monocytic differentiation to macrophages was also observed and was associated with decreased functional capacity such as altered antigen uptake, production of proinflammatory cytokines and stimulation of responder cells. The specific suppression of monocyte/macrophage differentiation and function was not due to blockade of tyrosine kinases known to be inhibited by imatinib and was consistent with an inhibition of the M-CSF/c-fms signalling pathway. This hypothesis was tested using a cell line that was dependent on M-CSF for growth and survival. Cell proliferation and phosphorylation of c-fms were inhibited at an IC50 of 1.9μM and 1.4μM imatinib respectively and this was not attributable to decreased c-fms expression. These important findings therefore identify c-fms as a further target of imatinib, and suggest that imatinib should be considered for treatment of diseases where c-fms is implicated. This includes breast and ovarian cancer and inflammatory conditions such as rheumatoid arthritis. Potential side effects resulting from imatinib treatment must also be considered. / Thesis (Ph.D.)--School of Medicine, 2004.

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.

Class of 2004 Abstract / Objective: To develop and populate a decision-analytic model comparing the cost and efficacy of imatinib versus allogenic bone marrow transplantation (BMT) with a matched unrelated donor in the treatment of newly-diagnosed, Philadelphia positive (Ph (+)), chronic phase, chronic myelogenous leukemia (CML). Design: Markov cohort analysis and Monte Carlo microsimulation. Measurements and Main Results: Direct medical costs were measured from the perspective of a third-party payer. Efficacy data and probabilities were obtained from survivability findings emanating primarily from randomized controlled trials (RCTs). A two-year time horizon was employed with three month treatment cycles. BMT was established as the baseline comparator and the base case was defined as a 35 year old, Ph(+) male patient with newly-diagnosed CML. Results from the Monte Carlo trial found that the incremental cost-efficacy ratio was −$5,000 for imatinib (95th % Confidence Interval: −$70,000, $84,000). Analysis of the cost-efficacy plane indicated that imatinib dominated BMT in 84.69 percent of cases, while BMT was dominant in 0.76 percent of cases. Sensitivity analyses of costs and discount rates found results to be robust. Conclusion: Imatinib was observed in a majority of cases to be both less costly and more efficacious relative to BMT in the treatment of CML, suggesting that this pharmaceutical agent is a dominant therapeutic strategy. When available, the incorporation of long-term clinical data are required to assess cost-efficacy beyond the two-year time horizon of this study.

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

Imatinib mesylate (imatinib) represents a potent molecularly targeted therapy against the oncogenic tyrosine kinase, BCR-ABL. Although imatinib has shown considerable efficacy against chronic myeloid leukemia (CML), displaying high rates of complete hematological and complete cytogenetic responses, treatment with imatinib is not curative and overtime advanced-stage CML patients often become refractory to further treatment. Acquired resistance to imatinib has been associated with mutations within the kinase domain of BCR-ABL, BCR-ABL gene amplification, leukemic stem cell quiescence as well as over-expression of the multidrug resistance (MDR1) gene. However, in vitro resistance models often fail to consider the role of the tumor microenvironment in the emergence of the imatinib-resistant phenotype. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrixes, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib sensitivity, we utilized an in vitro co-culture bone marrow stroma model. Using a transwell system, we demonstrated that soluble factors secreted by the human bone marrow stroma cell line, HS-5, were sufficient to cause resistance to apoptosis induced by imatinib in CML cell lines. We subsequently determined that culturing CML cells in HS-5-derived conditioned media (CM) inhibits apoptosis induced by imatinib and other second generation BCR-ABL inhibitors. These data suggest that more potent BCR-ABL inhibitors will not overcome resistance associated with the bone marrow microenvironment. Additionally, we determined that CM increases the clonogenic survival of CML cells following treatment with imatinib. HS-5 cells are reported to express several cytokines and growth factors known to activate signal transducer and activator of transcription 3 (STAT3). Given its crucial role in the survival of hematopoietic cells, we asked whether, 1) CM derived from HS-5 cells can activate STAT3 in CML cells and 2) does activation of STAT3 confer resistance to BCR-ABL inhibitors. We demonstrated that exposure of the CML cell lines, K562 and KU812, to CM caused an increase in phospho-Tyr STAT3, while no increases in phospho-Tyr STAT5 were noted. Moreover, resistance was associated with increased levels of the STAT3 target genes, Bcl-xl, Mcl-1 and survivin. Furthermore, reducing STAT3 levels with siRNA sensitized K562 cells cultured in CM to imatinib-induced cell death (p<0.05, Student’s t-test). Importantly, STAT3 dependency was specific for cells grown in CM, as reducing STAT3 levels in regular growth conditions had no effect on imatinib sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib resistance and provide preclinical rationale for using STAT3 inhibitors to increase the efficacy of imatinib within the context of the bone marrow microenvironment.

HINTERGRUND: Der selektive Tyrosinkinase-Inhibitor Imatinib inhibiert das Wachstum von bcr/abl positiven Zellen und stellt somit eine neue Therapieoption in der Behandlung der CML dar. Mittlerweile sind verschiedene Mutationen innerhalb der abl Sequenz beschrieben worden, die eine adäquate Imatinibbindung verhindern und zu einer zellulären Resistenz der CML Zellen führen. METHODE: Wir untersuchten 69 Patienten unter Behandlung mit Imatinib im Rahmen von laufenden klinischen Studien. Bei Studieneinschluss waren 37 Patienten in chronischer Phase, 21 Patienten in akzelerierter Phase und 11 Patienten in Blastenkrise. Mittels Real-time PCR wurden bcr/abl, WT1, MDR1 und AGP RNA Traskripte aus peripheren Leukozyten quantifiziert. AGP Proteinlevel wurden turbidimetrisch bestimmt. Ausserdem entwickelten wir eine neue, hochsensitive Technik zur Detektion bekannter Mutationen innerhalb der bcr/abl Domäne, indem wir das DNA-Clamping mittels Peptidnukleinsäure (PNA) mit einem Hybridisationssonden-Assay kombinierten. ERGEBNISSE: 1. Unsere Resultate bestätigen die hämatologische Effizienz von Imatinib in Übereinstimmung mit bereits publizierten Daten. 2. Eine komplette molekulare Remission konnte bei einem Patienten erzielt werden. 3. Wir konnten die bcr/abl Mutationen Thr315Ile, Glu255Lys and Tyr253His mit hoher Sensivität nachweisen. In einem Falle konnte die Gly255Lys Mutation vor der Behandlung detektiert werden. SCHLUSSFOLGERUNG: Bcr/abl und WT1 mRNA stellen in der Behandlung der CML Verlaufs- und Prognosemarker dar. Ein Zusammenhang zwischen MDR1 mRNA bzw. AGP mRNA und einer Resistenzentwicklung gegenüber Imatinib konnte nicht bestätigt werden. Durch den PNA-Clamping Assay konnten präexistente und sich entwickelnde bcr/abl Mutationen mit ungünstiger Prognose sicher und frühzeitig detektiert werden. Diese Tatsache ermöglicht eventuell eine Risikostratifizierung der CML und könnte als Model zum individuellen molekularen Monitoring und zur therapeutischen Strategie in anderen malignen Erkrankungen dienen. / BACKROUND: The selective tyrosine kinase inhibitor imatinib inhibits growth of bcr/abl positive cells and, thus, has become a novel therapeutic option for the treatment of Ph+ leukaemic patients. Various mutations within the abl sequence have been described that prevent adequate imatinib binding to bcr/abl resulting in cellular resistance of CML cells. METHODS: We investigated 69 CML patients under treatment with imatinib as part of an ongoing clinical trial. At recruitment 37 patients were in chronic phase, 21 patients in accelerated phase and 11 patients in blast crisis. Bcr/abl, WT1, MDR1and AGP RNA transcripts were quantified in peripheral leucocytes by real time PCR. AGP protein levels in plasma were measured by turbidimetric analysis. By combining peptide nucleic acid (PNA) based DNA clamping with a fluorescence hybridisation probe assay we developed a new and highly sensitive technique for the detection of known mutations within the bcr/abl kinase domain. RESULTS: 1. Our results demonstrate efficacy of imatinib on the haematological level in accordance with previously published data. 2. Complete molecular remission could be achieved in one patient. 3. We could effectively enhance the detection sensitivity (0,2%) for the BCR/ABL mutations Thr315Ile, Glu255Lys and Tyr253His. In one case the Gly255Lys mutation was detectable before treatment. CONCLUSION: Bcr/abl and WT1 mRNA are predictive marker in the treatment of CML. Our investigation could not confirm any relation between MDR1 mRNA nor AGP mRNA and a resistance to imatinib. By the PNA clamping assay pre-existing and evolving bcr/abl mutations associated with an unfavorable prognosis could be safely detected. This may facilitate risk stratification in CML and may serve as a model for individualized molecular monitoring and therapeutic strategies in other malignant diseases.