Fibrosis in the bone marrow is usually denominated myelofibrosis and may contribute to impaired hematopoiesis. Myelofibrosis is seen both in malignant and non-malignant diseases. The normal microenvironment in the bone marrow consists of a heterogenous population of hematopoietic and non-hematopoietic stromal cells, their extracellular products and hematopoietic cytokines. The stromal cells produce a complex array of molecules, among others collagens and glycosaminoglycans (GAGs) of which hyaluronan (HYA) is the most abundant. Marrow fibrosis results from an increased deposition of collagens, which are polypeptides. Staining for reticulin, mostly composed of collagen type III, is the common way of visualizing myelofibrosis. HYA, like the collagens, is widely distributed in connective tissues. Little is known about the distribution of HYA in bone marrow. The aims of this thesis have been to determine how HYA is distributed in normal and malignant bone marrow, compared to reticulin staining, and to follow patients with chronic myeloproliferative diseases (CMPD) during two years treatment with anagrelide considering development of cellularity and fibrosis. In bone marrow biopsies from healthy volunteers, the controls, HYA was found in a pattern that was concordant with the reticulin staining. Comparing patients with different malignant diseases with and without bone marrow involvemen, HYA staining was found to be significantly stronger in both groups compared to the controls. The HYA scores were also significantly higher in the bone marrow of patients with de novo acute myeloid leukemia (AML), compared to the controls. There was a correlation between HYA and reticulin in the patients with de novo AML, and in the patients with different malignant diseases with and without bone marrow involvement as in the controls. Increase of HYA, reticulin and cellularity in the bone marrow of patients with CMPD after two years of treatment with anagrelide indicated progression of fibrosis. Anagrelide is a valuable drug for reduction of platelets but seems unable to stop progression of fibrosis and hypercellularity. HYA is an interesting molecule with properties not only contributing to the structure of extracellular matrix but also to cell signaling and behaviour, although the understanding of the detailed mechanisms is still incomplete.

Acute myeloid leukemia (AML) is a genetic disease whereby sequential genetic aberrations alter essential white blood cell development leading to differentiation arrest and hyperproliferation. Pertinent animal models serve as essential intermediaries between in vitro molecular studies and the use of new agents in clinical trials. We previously generated a transgenic zebrafish model expressing human NUP98-HOXA9 (NHA9), a fusion oncogene found in high-risk AML. This expression yields a pre-leukemic state in both embryos and adults. Using this model, we have identified the overexpression of dnmt1 and the Wnt/β-catenin pathway as downstream contributors to the myeloproliferative phenotype. Targeted dnmt1 morpholino knockdown and pharmacological inhibition with methyltransferase inhibitors rescues NHA9 embryos. Similarly, inhibition of β-catenin with COX inhibitors partially restores normal hematopoiesis. Interestingly, concurrent treatment with a histone deacetylase inhibitor and either a methyltransferase inhibitor or a COX inhibitor, synergistically inhibits the effects of NHA9 on embryonic hematopoiesis. Thus, we have identified potential pharmacological targets in NHA9-induced myeloid disease that may offer a highly efficient therapy with limited toxicity – addressing a major long-term goal of AML research.

Acute myeloid leukemia (AML) is a highly heterogeneous disorder that results from a block in the differentiation of hematopoietic progenitor cells along with uncontrolled proliferation. In approximately 60% of cases, specific recurrent chromosomal aberrations can be identified by modern cytogenetic techniques, and is an important indicator to classify patients into three prognostic categories: favorable, intermediate, and poor risk. Currently, favorable risk patients are usually treated with chemotherapy while poor risk patients receive allogeneic stem cell transplantation. However, the largest subgroup of AML patients (approximately 40%) has no identifiable cytogenetic abnormalities and is classified as intermediate risk. In this special subgroup of patients, a number of studies have demonstrated the relationship between different translocations involving the mixed lineage leukemia (MLL) gene and patient prognosis. The heterogeneity of MLL-rearranged AML is reflected by the identification of more than 70 different fusion partners of this gene and the panel is continuously increasing. The aim of this study is to develop a sensitive molecular profiling test for relevant risk stratification that can help in the decision of treatment and/or follow-up strategy.

Gain- and loss-of-function studies of the BCL-2 family of proteins have shown that they can impact chemotherapeutic sensitivity. However, cells contain myriad anti-apoptotic and pro-apoptotic BCL-2 family members making it difficult to predict cell fate decisions based on the initial conditions of these proteins. BH3 profiling is a tool that measures mitochondrial priming, the readiness of a cell to die through the intrinsic (or mitochondrial) apoptotic pathway. Priming is due to the cumulative effect of the BCL-2 family of proteins that act as the gate keepers of the mitochondrial apoptotic pathway. Priming is measured by determining the sensitivity of mitochondria to perturbation by peptides derived from the BH3 domains of pro-apoptotic proteins. Using BH3 profiling, we now have a functional readout that can quantify priming and assess its contribution to drug sensitivity. Here we show that priming affects the sensitivity of acute myeloid leukemia (AML) cell lines to various standard chemotherapeutics, especially topoisomerase II inhibitors. Priming predicts clinical response to conventional induction chemotherapy as well as the long term maintenance of remission in AML patients. Interestingly, the priming of normal hematopoietic stem cells (HSCs) sits at the boundary line between the priming of cured and refractory patient AML. This HSC priming likely defines the therapeutic index since AML that are lower primed than HSCs are often refractory and cannot be cured without transplantation. Additionally, our BH3 profiles revealed that AML cells are more sensitive to BCL-2 antagonism than normal HSCs, which are primarily dependent on MCL-1. Indeed, we were able to kill primary refractory AML cells in vitro with the BCL-2 antagonist ABT-737 at doses that left HSCs unharmed. Cumulatively, these findings show that priming is a major mechanistic determinant of AML response in vitro and in the clinic to standard induction chemotherapy. With the ability to predict outcome, BH3 profiling may offer physicians and patients a promising tool for treatment decision-making.

Hematopoietic stem and progenitor cells proliferate and differentiate to reconstitute all lineages of functional blood cells. They are regulated by intricate cellular and molecular signals, on both genetic and epigenetic levels. Alterations in these regulatory signaling networks can lead to hematopoietic dysfunction, as well as transformation of hematopoietic cells and induction of leukemogenesis. This thesis focuses on uncovering molecular modulators that are crucial for the proper regulation of hematopoietic stem/progenitor cells. In Chapter II, I describe studies investigating functional roles of the histone demethylase UTX in normal and malignant hematopoiesis, using a short hairpin RNA-mediated knockdown approach. My data revealed that UTX is required for proliferation, self-renewal and differentiation of hematopoietic progenitor cells ex vivo through transcriptional regulation of hematopoiesis- specific transcriptional factors. I also discovered that UTX is critical for the proliferation of leukemia cells, implicating UTX as a possible target for clinical therapy. In Chapter III, I focus on understanding the process of leukemogenesis by generating and characterizing a novel model of myeloid sarcoma and acute myeloid leukemia in mice. This model induces these hematopoietic malignancies by introduction of multiple oncogenetic lesions (specifically, p16/p19-/-;Kras(G12V)) into bone marrow cells, and subsequent transplantation of these gene-modified cells into immunodeficient NOD.SCID mice. This model is very rapid and reproducible, and represents the first transplantable myeloid sarcoma model reported. Moreover, the disease induced in mice recapitulates the pathological progression of myeloid sarcoma in patients, providing a powerful model for dissection of critical leukemogenic events and discovery of new candidate therapeutic targets. Together, these studies help to reveal novel molecular modulators required for normal hematopoiesis, and offer potential animal model and drug target for therapeutic applications.

Voriconazole is a broad spectrum antifungal agent. Patients with acute myeloid leukemia that are susceptible to fungal infections receive simultaneously voriconazole and antitumor regimens. Drug-drug interactions between voriconazole and cytarabine involving the CYP3A4 enzyme are thought to affect the pharmacokinetic profile of the drugs and as a result their toxicological or pharmacological outcome. Population pharmacokinetic models were used to characterize these interactions and optimize the dose schemes after coadministration. Simulations and estimations were conducted by using NONMEM. The optimal dose for one hour intravenous infusion of voriconazole was estimated to 5mg/h. The proposed time points based on the pharmacokinetic profile of voriconazole for validated the model by using a small cohort of patients are 2h, 26h, 27h, 50h, 51h, 120h, 335h, 336h after the first administration of the antifungal agent. / Η βορικοναζόλη αποτελεί έναν ευρέως φάσματος αντιμυκητιασικό παράγοντα. Ασθενείς με οξεία μυελογενή λευχαιμία (ΟΜΛ) που θεωρούνται ευπαθείς στην ανάπτυξη μυκητιάσεων λαμβάνουν βορικοναζόλη σε συγχορήγηση με τα αντικαρκινικά φάρμακα. Αλληλεπιδράσεις φαρμάκων που λαμβάνουν χώρα κατά την διάρκεια του μεταβολισμού των χρησιμοποιούμενων φαρμάκων από κοινά ισοένζυμα του κυττοχρώματος P450 πιθανότατα να επιρεάζουν το φαρμακοκινητικό προφίλ τών φαρμάκων. Πληθυσμιακά φαρμακοκινητικά μοντέλα χρησιμοποιήθηκαν για τον χαρακτηρισμό αυτών των αλληλεπιδράσεων και την βελτιστοποίηση του δοσολογικού σχήματος. Προσομοιώσεις και υπολογισμοί πραγματοποιήθηκαν μέσω του προγράμματος NONMEM. Η βέλτιστη δόση μετά μια ώρα έγχυσης της βορικοναζόλης είναι 5mg/L. Τα προτεινόμενα χρονικά σημεία βασισμένα στο φαρμακοκινητικό προφίλ της βορικοναζόλης για την διεξάγωγή μικρού μήκους κλινικής μελέτης που θα επιτρέψει την αξιολόγηση του μοντέλου είναι 2h, 26h, 27h, 50h, 51h, 120h, 335h, 336h μετά την χορήγηση του αντιμυκητιασικού φαρμάκου.

The value of the combination of gemtuzumab ozogamicin (GO) and chemotherapy for the treatment of acute myeloid leukemia (AML) is currently analyzed within clinical trials. GO (6 mg/m2) and standard-dose cytarabine (100 mg/m2) is evaluated for the treatment of newly diagnosed AML in elderly patients in the SAL phase II trial. Preliminary results of the MRC AML15 trial support the application of GO 3 mg/m2 with standard- and high-dose cytarabine and anthracyclines for the treatment of de novo AML. Within this trial the addition of GO seems especially of value for favorable and intermediate cytogenetic risk groups. The combination of GO (3 mg/m2) and high-dose cytarabine (3 g/m2) is safe and more effective for the treatment of refractory AML than previous combinations from the AMLSG study group. First results prove the possibility of allogeneic stem cell transplantation after GO therapy. Initial data of a phase II trial document the safety and efficacy profile of GO within a reduced-intensity conditioning protocol applying fludarabine and total body irradiation. / Der Stellenwert von Gemtuzumab Ozogamicin (GO) in der Kombination mit Chemotherapie für die Behandlung der akuten myeloischen Leukämie (AML) wird derzeit auch in Europa untersucht. Der Einsatz von GO (6 mg/m2) in Kombination mit Cytarabin (100 mg/m2) bei der Primärbehandlung älterer Patienten mit AML wird in der SAL-Phase-II-Studie geprüft. Das in der MRC-AML15-Studie nachgewiesene verbesserte krankheitsfreie Überleben belegt den Stellenwert von GO (3 mg/m2) in Kombination mit Standard- und hoch dosiertem Cytarabin und einem Anthrazyklin für die Induktion und Konsolidierung bei neu diagnostizierter AML. Insbesondere Patienten mit einem günstigen und intermediären zytogenetischen Risikoprofil scheinen von der Gabe von GO zu profitieren. In der Behandlung von AML-Rezidiven oder refraktärer Erkrankung erwies sich GO (3 mg/m2) als sicher mit hoch dosiertem Cytarabin (3 g/m2) kombinierbar und war in der Wirksamkeit historischen Vergleichskollektiven der AMLSG-Studiengruppe überlegen. Erste Ergebnisse dokumentieren die Möglichkeit einer allogenen Stammzelltransplantation nach GO-Therapie. Erste Daten einer laufenden Studie belegen auch die Einsatzmöglichkeit und das Sicherheitsprofil von GO als Bestandteil einer Konditionierungstherapie von reduzierter Intensität mit Fludarabin und Ganzkörperbestrahlung. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

Objective: CSL360 is a chimeric IgG1 mAb recognizing CD123+/CD131- LSCs responsible for acute myeloid leukemia (AML). The in vitro targeting properties of 111In-labeled CSL360 modified with nuclear localization sequence (NLS) were evaluated in AML cells. Methods: 111In-NLS-CSL360 was constructed and its binding affinity, cellular uptake and nuclear importation were analyzed on CD123+ cells. Cytotoxicity was evaluated by clonogenic assays on AML cells (CD123+/CD131-). Results: 111In-NLS-CSL360 exhibited preserved binding to CD123. High cellular and nuclear uptake was observed at 266 nM after 24 hour of incubation. Nuclear uptake of 111In-NLS-CSL360 (266 nM) was 2.0-fold higher than 111In-CSL360 (266 nM) after 24 hour of incubation. Clonogenic survival (CS) of AML cells was reduced to 27.5 ± 4.1%. The nuclear uptake and cytotoxicity were reduced when pre-exposed to unlabeled CSL360, indicating 111In-NLS-CSL360 was CD123-specific. Conclusion: 111In-NLS-CSL360 could be a promising radioimmunotherapeutic agent specific for LSCs.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.