京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第21305号 / 人健博第61号 / 新制||人健||5(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 足立 壯一, 教授 藤井 康友, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Myelodysplastic syndrome (MDS) is a group of clonal bone marrow disorders characterized by bone marrow dysplasia with myeloblasts <20%, typically seen in older patients. MDS has a significant risk of transformation to Acute Myeloid Leukemia (AML). We report a case of MDS transformed to AML, with sustained Complete Remission and incomplete count recovery (CRi) with treatment. A 78-year-old male with a 2-year history of leukopenia had a workup including bone marrow biopsy (BMBX) revealing intermediate- risk MDS with 13% blasts (Refractory Anemia Excess Blasts II), deletion 20 on cytogenetics and normal MDS FISH panel. He was categorized as revised IPSS score 4.5 on risk stratification. Patient initiated treatment with hypomethylating agent Azacytidine with subsequent improved BMBX with 7% blasts. He continued Azacytidine with dose reductions due to cytopenia only to develop 14% blasts on another follow up BMBX. He continued successful treatment for over 3 years before developing with 40-50% CD 34+/CD117+ blasts in the bone marrow consistent with transformation to AML. He commenced salvage treatment with Venetoclax and full dose Azacytidine as advanced age and performance status precluded transplant options. Repeat BMBX 4 weeks following Venetoclax showed hypocellular marrow, blasts percentage less than 2% indicating a CRi. Two other subsequent marrow exams have demonstrated sustained CRi twelve months after transformation with continued Venetoclax and Azacytidine administration. Around 30% of MDS patients eventually transform to secondary AML. Azacytidine therapy has significantly improved survival and time to AML transformation in intermediate-2 and high-risk MDS patients. Venetoclax, a BCL-2 inhibitor, in treating AML. Based on the results of the VIALE-A trial, the incidence of CR (complete remission) was higher around 36.7% with Azacytidine-Venetoclax (A-V) compared to 17.9% with Azacytidine. The composite CR (CR+ Cri) was higher in the A-V group, 66.4% compared to 28.3% with Azacytidine group. The median overall survival was 14.7 months in the A-V group compared to 9.6 months in the Azacytidine group. Our patient achieved a CRi with A-V treatment and has demonstrated a durable response beyond 16 months in secondary AML which has a bleak prognosis indicating the promise of this new combination treatment.

Patients with myelodysplastic syndromes (MDS) frequently receive red blood cell (RBC) transfusions to alleviate symptoms of anemia and improve health-related quality of life (HR-QoL). Patients can sometimes continue to feel unwell after transfusion and the age of the transfused RBCs could contribute to this observation. Three pilot studies were conducted to inform the design of a randomized crossover trial to determine if fresh blood to MDS patients could improve HR-QoL post-transfusion. A systematic review was performed to inform the background and rationale for the trial. The results showed a dearth of literature addressing the research question. Only two clinical trials have been conducted to date where fatigue and HR-QoL were the primary outcomes of interest. Although results of the trials were negative, several limitations and generalizability issues warrant additional research in this area. Crossover designs necessitate patients have a stable prognosis while being observed; hence, a chart review of adult MDS patients was conducted to assess clinical stability using the following criteria: interval of days between transfusions; pre-transfusion Hb; number of hospital admissions; and severe infections. Results indicated that the majority of patients who had received greater than 3 transfusions within the 6-month observation period had stable disease and were appropriate for a crossover trial. The criteria defining stability will be useful for identifying eligible patients. Finally, an applied qualitative study in adult MDS patients in Hamilton was conducted to inform the selection of an appropriate outcome measure (i.e. HR- QoL tool). Short semi-structured interviews were conducted with participants to elicit information about anemia related symptoms and changes in well-being in response to transfusion. The results of the study support clinical observations that suggest patients do not immediately recover post-transfusion. Findings indicate that an appropriate HR-QoL tool should be short, be disease specific, and have a short recall period. Currently, the Quality of Life-E tool, validated in patients with MDS, fits most of these criteria. In conclusion, data from the systematic review and the two pilot studies suggest that it may be feasible to conduct an age of blood crossover trial in MDS patients where the primary outcome of interest is HR-QoL. / Thesis / Master of Science (MSc)

Patients with myelodysplastic syndromes (MDS) often experience chronic anemia and long-term red blood cell transfusion dependence associated with significant burden on clinical and health-related quality of life (HRQoL) outcomes. In the MEDALIST trial (NCT02631070), luspatercept significantly reduced transfusion burden in patients with lower-risk MDS who had ring sideroblasts and were refractory to, intolerant to, or ineligible for prior treatment with erythropoiesis-stimulating agents. We evaluated the effect of luspatercept on HRQoL in patients enrolled in MEDALIST using the EORTC QLQ-C30 and the QOL-E questionnaire. Change in HRQoL was assessed every 6 weeks in patients receiving luspatercept with best supportive care (+ BSC) and placebo + BSC from baseline through week 25. No clinically meaningful within-group changes and between-group differences across all domains of the EORTC QLQ-C30 and QOL-E were observed. On one item of the QOL-E MDS-specific disturbances domain, patients treated with luspatercept reported marked improvements in their daily life owing to the reduced transfusion burden, relative to placebo. Taken together with previous reports of luspatercept + BSC reducing transfusion burden in patients from baseline through week 25 in MEDALIST, these results suggest luspatercept may offer a treatment option for patients that reduces transfusion burden while providing stability in HRQoL.

No description available.

Myelodysplastic neoplasms (MDS) are a group of heterogeneous, clonal disorders characterised by ineffective haematopoiesis and peripheral blood cytopenia. MDS is highly progressive, difficult to treat, and is one of the most common blood cancers, affecting 4-5/100.000 people below the age of 70 and many more thereafter. Single or multiple driver gene mutations and chromosomal abnormalities in the haematopoietic compartment lead to MDS. These somatic gene mutations account for the dysregulation of epigenetic, DNA repair, cohesion complex, and spliceosome pathways. The International prognostic scoring system (IPSS) that was developed in 1997, revised (IPSS-R) in 2016 and updated in 2022 (IPSS-M) classifies MDS into low risk (LR-), intermediate (Int-), and high risk (HR-) groups. The haematopoietic disorder is accompanied by changes in the bone marrow microenvironment (BMME) and especially in mesenchymal cells (MSCs). BMME provides a supportive milieu for haematopoiesis and can be targeted by clinically available drugs such as AZA. The non cellular component of the BMME, the extracellular matrix (ECM), is a framework providing structural and biochemical support via cell-ECM interactions and the maintenance of growth factor gradients. To date, studies of bone marrow interactions in homeostasis and disease have focused largely on soluble and membrane-associated factors, while the involvement of the ECM in MDS and its response to therapy is underexplored. Therefore, this study aimed to characterise the MDS MSC derived ECM of both LR- and HR-MDS in comparison to that from healthy age matched donors in terms of composition, biophysical properties and functional haematopoietic support. This study also aimed to evaluate the impact of in vivo and in vitro AZA treatment on MDS MSC derived ECM. To investigate this, in vitro ECMs were generated by culturing of MSC monolayers on chemically prepared coverslips followed by decellularization using NH4OH and DNase-1 solution. The biophysical properties of the ECM were analysed using atomic force microscopy (AFM). Using targeted approaches, a selection of biochemical ECM components including glycoprotein (fibronectin), collagens and glycosaminoglycans (GAGs) were analysed in the various ECMs generated from the different MSC samples. AFM analysis revealed that MDS MSCs producer a softer ECM than the healthy donor MSCs, and that this difference becomes more prominent as the disorder progresses from LR-to HR- MDS. An increase in overall collagen content and a specific increase in collagens I and IV was observed in the ECM deposited by both LR- and HR-MDS MSCs when compared to healthy donor MSCs. Lectin staining revealed disease stage-specific differences in GAG composition: The levels of GAGs carrying N acetyl glucosamine and those carrying N-acetyl galactosamine sugars were both increased in ECM from LR-MDS, while ECM from HR-MDS retained high levels of N acetyl glucosamine but contained only low levels of N-acetyl galactosamine GAGs. The changes in N acetyl galactosamine and N acetyl glucosamine GAGs were further confirmed by chondroitin sulphate (CS) immunostaining, and hyaluronic acid (HA) ELISA respectively. Electrophoretic analysis revealed the presence of low molecular weight (LMW)-HA in one of the LR-MDS MSC derived ECM. Furthermore, the stimulation of MNCs with LMW-HA showed an increase in gene expression of pro-inflammatory cytokines like IL6 suggesting the possible involvement of LMW-HA in the inflammatory bone marrow state of LR-MDS. ECM derived from both LR- and HR-MDS MSCs had a reduced ability to support HSPC, as revealed by a loss of both polar morphology and subsequent colony-forming potential. The decreased rigidity of the ECM produced by MSCs from MDS patients was reversed in MSCs isolated from the patients post-AZA therapy. Similarly, direct exposure of cultured MDS MSCs to AZA also resulted in a corresponding increase in the rigidity of the ECM, although this remained lower than that observed from MDS MSCs isolated post-AZA therapy. A reduction in the collagen content of the ECM was only observed when using MSC from AZA-treated patients, but not following in vitro AZA treatment of MSCs from untreated patients. This indicated that the AZA-mediated restoration of ECM rigidity is an indirect result of effects in the context of the BMME and not on the MSCs alone. Interestingly, a few ECMs derived from MDS patients after AZA therapy had an improved ability to maintain functional HSPCs, as assessed by subsequent colony formation assay. Moreover, a polarized morphology of HSPCs cultured on the ECM derived from both in vivo and in vitro AZA-treated MDS MSCs, suggests a partial restoration of the HSPC behaviour on the AZA-treated MDS ECM. In conclusion, this study has demonstrated changes in the structure, collagen content, and GAG composition of ECM derived from MSCs from MDS patients compared to healthy donors. This study is one of the first to demonstrate an impact of MDS-derived ECM on both the morphology and function of HSPCs, supporting the relevance of the bone marrow ECM in haematological malignancies. The partial revision of the MDS ECM phenotype following in vivo AZA treatment suggests that the ECM itself may be a potential therapeutic target. An improved, in-depth understanding of the contribution of ECM to disease processes is therefore likely to enable us to find novel therapeutic targets to improve drug response in MDS in the future. / Myelodysplastische Neoplasien (MDS) sind eine Gruppe heterogener, klonaler Erkrankungen, die durch ineffektive Hämatopoese und Zytopenie des peripheren Blutes gekennzeichnet sind. MDS sind hochgradig progressiv, schwer zu behandeln und gehören zu den häufigsten Blutkrebserkrankungen, von denen 4-5/100.000 Menschen unter 70 Jahren betroffen sind. Die Inzidenz steigt mit zunehmendem Alter deutlich an. MDS wird durch einzelne oder mehrfache Mutationen von Treibergenen und Chromosomenanomalien im hämatopoetischen Kompartiment verursacht. Diese somatischen Genmutationen sind für die Dysregulation von epigenetischen, DNA-Reparatur-, Kohäsionskomplex- und Spleißosomen-Signalwegen verantwortlich. Das Internationale Prognosesystem (IPSS) wurde 1997 entwickelt, 2016 überarbeitet (IPSS-R) und 2022 aktualisiert (IPSS M), um MDS in Gruppen mit niedrigem Risiko (LR-), mittlerem (Int ) und hohem Risiko (HR-) einzuteilen. Die hämatopoetische Erkrankung geht mit Veränderungen in der Mikroumgebung des Knochenmarks (BMME) einher, insbesondere bei mesenchymalen Zellen (MSCs). Das BMME bietet ein unterstützendes Milieu für die Hämatopoese und kann durch klinisch verfügbare Medikamente wie AZA beeinflusst werden. Die nichtzelluläre Komponente der BMME, die extrazelluläre Matrix (ECM), ist ein Gerüst, das durch Zell-ECM-Interaktionen und die Aufrechterhaltung von Wachstumsfaktorgradienten strukturelle und biochemische Unterstützung bietet. Bislang haben sich Studien über die Interaktionen im Knochenmark bei Homöostase und Krankheit hauptsächlich auf lösliche und membranassoziierte Faktoren konzentriert, während die Beteiligung der ECM an MDS und ihre Reaktion auf die Therapie noch nicht ausreichend erforscht ist. Daher zielte diese Studie darauf ab, die aus MDS-MSCs abgeleitete ECM sowohl bei LR- als auch bei HR-MDS im Vergleich zu der von gesunden, altersgleichen Spendern zu charakterisieren, und zwar hinsichtlich der Zusammensetzung, der biophysikalischen Eigenschaften und der funktionellen hämatopoetischen Unterstützung. Ziel dieser Studie war es auch, die Auswirkungen einer in vivo und in vitro AZA-Therapie auf die aus MDS-MSCs stammende ECM zu untersuchen. Hierfür wurden in vitro ECMs durch Kultivierung von MSC-Monolayern auf chemisch-präparierten-Deckgläsern und anschließender Dezellularisierung mit NH4OH und DNase-1-Lösung erzeugt. Die biophysikalischen Eigenschaften der ECM wurden mittels Rasterkraftmikroskopie (AFM) analysiert. Mit gezielten Ansätzen wurde eine Auswahl biochemischer ECM-Komponenten, darunter Glykoproteine (Fibronektin), Kollagene und Glykosaminoglykane (GAGs), in den ECMs analysiert. Die AFM-Analyse ergab eine weichere ECM, die von MDS-MSCs im Vergleich zu gesunden Spender-MSCs gebildet wurde, was mit dem Fortschreiten der Erkrankung von LR- zu HR-MDS noch deutlicher wurde. Sowohl in LR-MDS- als auch in HR-MDS-ECMs wurde im Vergleich zu gesunden Spender-ECMs ein Anstieg des Gesamtkollagengehalts und eine spezifische Zunahme der Kollagene I und IV beobachtet. Darüber hinaus zeigte die Lektinfärbung krankheitsspezifische Unterschiede in der GAG-Zusammensetzung: Der Gehalt an N-Acetylglucosamin-tragenden GAGs und an N-Acetylgalactosamin-tragenden GAGs war in der ECM von LR-MDS erhöht, während die ECM von HR-MDS einen hohen Gehalt an N-Acetylglucosamin, aber nur einen geringen Gehalt an N-Acetylgalactosamin-GAGs aufwies. Die Veränderungen bei den N-Acetyl-Galactosamin- und N-Acetyl-Glucosamin-GAGs wurden durch Chondroitinsulfat (CS)-Immunfärbung bzw. Hyaluronsäure (HA) ELISA weiter bestätigt. Eine Elektrophoretische Analyse zeigte das Vorhandensein von niedermolekularem (LMW)-HA in einer der von LR-MDS-MSCs stammenden ECM. Darüber hinaus zeigte die Stimulierung von mononuklearen Zellen mit LMW-HA einen Anstieg der Genexpression von pro-inflammatorischen Zytokinen wie IL6, was auf eine Rolle von LMW-HA im entzündlichen Zustand des Knochenmarks von LR-MDS hindeutet. Darüber hinaus wies die ECM von LR- und von HR-MDS, eine verminderte Fähigkeit, hämatopoetische Stammvorläuferzellen (HSPCs) zu unterstützen, auf. Dies zeigte sich in einem Verlust sowohl der polaren Morphologie von HSPCs als auch des anschließenden koloniebildenden Potenzials selbiger. Darüber hinaus wurde die verringerte Steifigkeit der ECM von MDS-MSCs, die nach der AZA-Therapie aus den Patienten isoliert wurden, umgekehrt. In ähnlicher Weise führte die direkte Exposition von kultivierten MDS-MSCs mit AZA zu einer entsprechenden Erhöhung der Steifigkeit der ECM. Diese war jedoch geringer als bei den nach der AZA-Therapie isolierten MDS-MSCs. Die Verringerung des Kollagengehalts der ECM wurde nur in der in vivo mit AZA behandelten MSC-ECM beobachtet, nicht aber in den in vitro mit AZA behandelten Proben. Dies deutet darauf hin, dass die AZA-vermittelte Wiederherstellung der ECM-Steifigkeit ein Ergebnis der indirekten Wirkung von AZA im Knochenmark ist und eventuell vom MDS-Klon ausgeht. Interessanterweise wurde bei einigen ECMs von MDS-Patienten nach der AZA-Therapie eine Verbesserung der Koloniebildung hierauf- kultivierter HSPCs beobachtet. Darüber hinaus deutet eine polarisierte Morphologie von HSPCs, die auf der ECM von in vivo und in vitro AZA-behandelten MDS-MSCs vorkultiviert wurden, auf eine teilweise Wiederherstellung des Verhaltens von HSPCs auf der AZA-behandelten MDS-ECM hin. Zusammenfassend lässt sich sagen, dass diese Studie Veränderungen in der Struktur, im Kollagengehalt und in der GAG-Zusammensetzung zwischen der ECM von MDS-MSCs und der ECM von gesunden MSCs nachgewiesen hat. Dies ist auch eine der ersten Studien, die einen Einfluss der aus MDS-MSCs stammenden ECM auf die Morphologie und Funktion von HSPCs zeigt. Dies weist auf die Rolle der ECM bei der Entstehung hämatologischer Malignome hin. Darüber hinaus deutet die teilweise Korrektur des MDS-ECM-Phänotyps nach einer in vivo AZA-Behandlung darauf hin, dass die ECM selbst ein potenzielles therapeutisches Ziel sein könnte. Ein besseres und tieferes Verständnis des Beitrags der ECM zu MDS-Krankheitsprozessen wird es uns daher ermöglichen, neue therapeutische Ziele zu finden, um das Ansprechen auf Medikamente verbessern zu können

Mutations and deletions in ribosomal proteins are associated with a group of diseases termed ribosomopathies. Collectively, these diseases are characterized by ineffective hematopoiesis, bone marrow failure, and an increased risk of developing myelodysplastic syndrome (MDS) and subsequently acute myeloid leukemia (AML). This observation highlights the role of dysregulation of this class of proteins in the development and progression of myeloid neoplasms. Analysis of gene expression in CD34+ hematopoietic stem cells (HSC) from 183 MDS patients demonstrated that ribosomal protein L22 (Rpl22) expression exhibited a greater reduction than any other ribosomal protein gene in MDS. Interestingly, we observed that AML patients with lower expression of Rpl22 had a significant reduction in their survival (TCGA cohort, N=200, Log Rank P value&lt;0.05). To assess the mechanism of reduced expression, we developed a FISH probe complementary to the RPL22 locus and assessed for deletion of this locus in an independent set of 104 MDS/AML bone marrow samples. Strikingly, we found that RPL22 deletion was enriched in high-risk MDS and secondary AML cases. We, therefore, sought to investigate whether reduced Rpl22 expression played a causal role in leukemogenesis. Using Rpl22-/- mice, we found that Rpl22-deficiency resulted in a constellation of phenotypes resembling MDS. Indeed, Rpl22-deficiency caused a macrocytic reduction in red blood cells, dysplasia in the bone marrow, and an expansion of the early hematopoietic stem and progenitor compartment (HSPC). Since MDS has been described as a disease originating from the stem cell compartment, we next sought to determine if the hematopoietic defects were cell autonomous and resident in Rpl22-/- HSC. Competitive transplantation revealed that Rpl22-/- HSC exhibited pre-leukemic characteristics including effective engraftment, but a failure to give rise to downstream mature blood cell lineages. Importantly, there was a strong myeloid bias in those downstream progeny derived form Rpl22-/- HSC. To determine how Rpl22-deficiency increased the causes these deficiencies in HSC, we performed whole transcriptome analysis on Rpl22-/- HSC. Interestingly, alterations in genes associated with both ribosomal proteins and mitochondrial components were observed. We found that protein synthesis was unchanged in Rpl22-deficient HSCs, sharply contrasting the reductions in global protein synthesis that usually accompany ribosomal protein insufficiency. Consequently, we shifted our focus to the dysregulated mitochondrial genes, which were linked to the processes of oxidative phosphorylation and fatty acid metabolism. We observed that oxidative phosphorylation was decreased in Rpl22-deficient HSCs while fatty acid oxidation was increased. Increased fatty acid oxidation is associated with maintenance of the hematopoietic stem cells. Interestingly, inhibiting fatty acid oxidation mitigated this attribute in Rpl22 deficient HSCs. Because Rpl22 is an RNA-binding protein, we asked if Rpl22 was regulating fatty acid oxidation by directly binding mRNAs encoding regulators of fatty acid oxidation. We found that Rpl22 is able to directly bind the coding region of an upstream regulator of fatty acid oxidation, Alox12. Thus, we hypothesized that Rpl22-deficiency increased fatty acid oxidation through increased expression of Alox12. Consistent with this hypothesis, knockdown of Alox12 impaired the function of Rpl22 deficient HSC. Because the increased fatty acid oxidation promotes self-renewal of Rpl22-deficient HSC and blocks their differentiation, we also hypothesized that this would predispose them to leukemogenesis. We examined the potential for Rpl22-deficient HSPC to be transformed upon ectopic expression of the MLL-AF9 oncogenic fusion. Indeed, Rpl22 deficiency increased predisposition to transformation both in vitro and in vivo, in MLL-AF9 knockin mice. Furthermore, Rpl22 deficient leukemias were preferentially sensitive to pharmacologic inhibition of fatty acid oxidation or Alox12 knockdown, indicating that leukemia cell survival was also dependent upon fatty acid oxidation. Taken together, these findings indicate that Rpl22-insufficiency predisposes HSPC to leukemic transformation and aggressive growth by regulating mitochondrial function, providing an explanation for the reduced survival observed in Rpl22-low AML patients. We also sought to determine how Rpl22 may be contributing to another subset of AML known as Therapy-related AML. Most commonly, these patients develop AML after previously being treated with an alkylating chemotherapeutic drug. Interestingly, we found that Rpl22-deficient HSPC are resistant to treatment with these agents, despite having evidence of DNA damage. The ultimate consequence of the insensitivity of Rpl22-deficient HSPC to alkylating agents was that mice given serial doses of cyclophosphamide exhibited an increased incidence of leukemic-like changes. This chemo-resistant phenotype in Rpl22-/- cells was related to increased expression of the DNA repair protein MGMT. Inhibition of this protein abrogated the ability of these cells to survive following treatment with cyclophosphamide. Ultimately, this study implicates Rpl22 as a regulator of alkylating DNA damage repair and suggests that both patients with hematologic or solid cancers that express reduced levels of Rpl22 are at increased risk for development of therapy related AML is they are treated with alkylating agents. / Cancer Biology & Genetics

Au sein des hémopathies myéloïdes malignes, les syndromes myélodysplasiques(SMD) et les leucémies aiguës myéloïdes (LAM) représentent des pathologies complexes ethétérogènes résultant d’anomalies clonales des cellules souches médullaires. Elles sontcaractérisées par une hématopoïèse inefficace provoquant des cytopénies sanguines graves.Les connaissances sur les anomalies moléculaires des SMD et des LAM, notammentà caryotype normal, sont globalement pauvres et leur physiopathologie encore mal connue.Une meilleure définition moléculaire est nécessaire pour une évaluation pronostique plusprécise de ces hémopathies et pour optimiser secondairement les stratégies thérapeutiques.Cette thèse présente un panorama des classifications cytogénétiques et moléculairesactuelles des SMD et LAM ainsi que l’étude de certaines altérations moléculairesrencontrées dans ces maladies.Grâce à l’apport des techniques d’analyse génomique à grande échelle, notamment laCGH-array, notre laboratoire a identifié de nouvelles altérations génétiques, parmi lesquellesles mutations du gène ASXL1, ainsi que des altérations des gènes codant les protéines de laCohésine et des régulateurs de la protéine CBL. Nous avons analysé une combinaison demutations de gène et émis l’hypothèse d’un modèle de leucémogenèse à 4 classes demutations, afin d’apporter des pistes dans la compréhension de la physiopathologie des SMDet LAM. / Among myeloid malignancies, myelodysplastic syndromes (MDSs) represent a groupof complex diseases characterized by clonal abnormalities of bone marrow hematopoieticprecursor cells. They are defined by an ineffective hematopoiesis leading to peripheralcytopenias. About 40% of MDSs secondarily evolve to acute myeloid leukemia (AML).This risk of transformation is evaluated by several international prognostic scoringsystems like IPSS and WPSS. The WHO classification recognizes several classes of MDSsessentially based on morphology and cytogenetics features, some with a high progressionrisk, like refractory anemia with excess of blasts type 2, others with a low risk, likerefractory anemia with ringed sideroblasts. However, the classification of MDSs is stillunsatisfactory and relevant prognostic markers allowing earlier treatments for patients with ahigh risk of transformation are still lacking. The physiopathology of SMDs and AMLs withnormal karyotype remains unclear. Currently, the only potentially curative treatment isallogenic stem cell transplant, which is feasible for a restricted number of patients and candisplay side effects and failures.A better knowledge of the molecular biology of MDSs and AMLs is necessary for abetter understanding of these diseases and may provide new early prognosis indicators andbetter strategies of treatments.

Τα μυελοδυσπλαστικά σύνδρομα αποτελούν ετερογενή ομάδα νοσημάτων, με δυσμενή πρόγνωση και δυσκολία θεραπευτικής προσέγγισης. Η κατανόηση των παθογενετικών μηχανισμών που διέπουν την παθολογική αιμοποίηση που παρατηρείται στα σύνδρομα αυτά in vitro και in vivo, μπορεί να βοηθήσει στην αποτελεσματικότερη αντιμετώπισή τους. Σκοπός αυτής της διατριβής ήταν η μελέτη της αιμοποίησης των ΜΔΣ με τη μέθοδο των καλλιεργειών αιμοποιητικών κυττάρων μακράς διάρκειας και η επίδραση διαφόρων παραγόντων στην in vitro αιμοποίηση, με στόχο την αναγωγή των ευρημάτων και στην in vivo διαδικασία. Οι καλλιέργειες βραχείας διάρκειας ανέδειξαν την αδυναμία των προγονικών κυττάρων των ασθενών να δημιουργήσουν φυσιολογικές αποικίες. Η προσθήκη μίγματος αυξητικών παραγόντων στο καλλιεργητικό υλικό είχε θετική επίδραση στην πλειοψηφία των περιπτώσεων. Με μακράς διάρκειας καλλιέργειες συγκρίθηκαν τα αποτελέσματα των ΜΔΣ με αυτά φυσιολογικών μαρτύρων και των υποκατηγοριών ΜΔΣ μεταξύ τους. Αξιολογήθηκαν η έκταση του στρώματος, η διάρκεια ζωής, η εβδομαδιαία και η συνολική κυτταρική απόδοση. Σε όλες τις περιπτώσεις η ανάπτυξη υπολειπόταν ποιοτικά και ποσοτικά στους ασθενείς, σε σχέση με τους μάρτυρες. Η προσθήκη παραγόντων στο καλλιεργητικό υλικό αποσκοπούσε στη βελτίωση των παραμέτρων που προαναφέρθηκαν. Η IFN-α, η βιταμίνη D3, η Ara-c και ο συνδυασμός της με IFN-α δεν βελτίωσαν τα αποτελέσματα. Η προσθήκη IL-3 είχε ευοδωτική επίδραση κυρίως στις κυτταρικές αποδόσεις. Η IL-6 είχε επίσης ευοδωτική δράση, κυρίως στον σχηματισμό στρώματος. Ο συνδυασμός IL-3+IL-6 απέβη ο πιο σημαντικός τροποποιητής της συμπεριφοράς των καλλιεργειών των ασθενών με ΜΔΣ ευοδώνοντας όλες τις παραμέτρους και προκάλεσε διαφορές πολύ σημαντικές σε σχέση με την control καλλιέργεια. Αναδείχθησαν λοιπόν ευρήματα συνέργειας των δύο ιντερλευκινών, σε όλες τις άλλες παραμέτρους αξιολόγησης των καλλιεργειών μακράς διάρκειας. Με βάση τον τρόπο ανάπτυξης και την συμπεριφορά των κυττάρων των ασθενών με ΜΔΣ στην προσθήκη των κυτταροκινών, διακρίθηκαν δύο μοντέλα in vitro ανάπτυξης της καλλιεργειών: Το δυσπλαστικό και το λευχαιμικό. Συμπερασματικά οι μακράς διάρκειας καλλιέργειες στους ασθενείς με ΜΔΣ αποτελούν χρήσιμη προγνωστική μέθοδο και μπορούν να διακρίνουν τους ασθενείς που θα εξελιχθούν ταχέως, από εκείνους που θα έχουν χρονιότερη και ηπιότερη πορεία. Συνδυασμός κυτταροκινών και άλλων παραγόντων μπορεί να βελτιώσει την προβληματική-παθολογική in vitro αιμοποίηση των προγονικών κυττάρων των ασθενών με ΜΔΣ. / Myelodysplastic syndromes comprise a heterogeneous group of hematopoietic stem-cell disorders, with dismal prognosis and difficulty in their therapeutic approach. The revealing of the underlying pathogenetic mechanisms, implicated in the impaired hematopoiesis of these syndromes, is crucial for the development a more comprehensive and effective treatment approaches. The aim of this thesis was the study of hematopoiesis of MDS, by using long term cultures of hemopoietic cells and the investigation of the influence of various exogenous modulating factors-drugs in vitro, in an effort to obtain results, which could direct their use in vivo. Short term cultures revealed the disability of the progenitor cells of patients to form normal colonies. The addition of a mixture of growth factors in the conditioned medium had a positive influence in the majority of cases. By using long term cultures we compared the results obtained from patients with MDS, with those from normal controls, and between the different MDS subgroups For this comparison we used: the extent of the area of the stroma-layer formed, the longevity of the culture, the weekly cell production and the total cell yield of each culture. In all cases the development of cultures derived from patients was inferior to those of controls. The addition of modulating factors to the culture medium was aimed to improve the above parameters. IFN-α, vitamin D3, Ara-c and the combination of IFN-α and Ara-c did not improve any of the culture’s parameter. The addition of IL-3 had a clearly favorable effect mainly to the weekly and the total cell yield. Interleukin-6 had similarly a favourable effect, particularly promi-nent in the stroma-cell formation. The combination of IL-3 plus IL-6 was proved as the most important favourable modulator of the MDS cultures. It improved all culture parameters and produced statistically significant differences in comparison to the control cultures. According to the developmental model obtained by the long-term culture the dysplastic and the leukemic pattern of in vitro growth could be distinguished. In conclusion, long term cultures of hematopoietic cells of MDS patients represent a useful prognostic tool and can distinguish patients who will more rapidly evolve to leukemia from those who will have a more prolonged and stable clinical course. The use of a combination of cytokines and might have a favourable effect on in vitro hematopoiesis of the progenitor cells of MDS patients.

<p>Hematopoiesis is the continual process of blood cell generation that primarily occurs in the bone marrow of adult animals. Hematologic neoplasms can also occur in the bone marrow and often result from dysregulation of signal transduction pathways. One example is the activation of the Ras oncogene, which has been linked to a variety of different cancers, including hematologic neoplasms. Ras is located proximal to the cell membrane and can activate many downstream effector pathways, thus it is difficult to determine which downstream pathway is mediating oncogenic Ras function. My thesis work focused on the effect of inappropriate activation of MEK/ERK, a downstream Ras effector pathway, in the hematopoietic system.</p><p>Using a retroviral transduction system, we expressed a constitutively active form of MEK1 in hematopoietic stem cells (HSCs). Mice transplanted with HSCs expressing active MEK developed a lethal myelodysplastic syndrome/myeloproliferative disease (MDS/MPN) characterized by the expansion of granulocytes/macrophages (GM) at the expense of lymphoid cell development. Transplantation of active MEK-induced MDS/MPNs into naïve mice did not result in further disease, suggesting that the MDS/MPN is not a frank leukemia.</p><p>Bcl-2 is an anti-apoptotic molecule that has been shown to play a role in leukemia development and maintenance. Coupling expression of active MEK and Bcl-2 resulted in MDS/MPNs that were phenotypically identical and had very similar disease onset compared to active MEK-induced MDS/MPNs. However, transplantation of Bcl-2/active MEK-induced MDS/MPNs did not result in a myeloid disease; rather, it resulted in the development of T-acute lymphoblastic leukemia (T-ALL) that was marked by activated Notch signaling. </p><p>These results led us to conclude that activation of MEK/ERK was sufficient to cause a pre-leukemic myeloid disease; however, additional oncogenic factors, such as Bcl-2 and Notch, were necessary for frank leukemia development. Moreover, additional oncogenic factors can alter the disease phenotype and disease course. Future analysis of the interplay between oncogenic factors will help shed light on disease development and aid in the development of more effective cancer treatments.</p> / Dissertation