The diagnosis and monitoring of AML relies predominantly on the identification of blast cells in the bone marrow and peripheral blood. While at the time of diagnosis the identification of leukemic cells is relatively easy, during remission the identification of small numbers of blasts is problematic. This is most evident by the fact that patients who achieve complete remission frequently relapse, despite pathologic examination indicating a marked reduction in leukemic cell burden. In this thesis I have explored the potential of using serum proteins secreted by leukemic cells as a means of monitoring disease in patients. To identify proteins that might be useful for monitoring, I took advantage of published gene expression arrays and looked into online bioinformatics databases. Using specific characteristics, I was able to identify approximately 107 candidate proteins secreted by AML cells. RT-PCR analysis and ELISA assays were performed to evaluate the variability of expressions and serum level differences of twelve different proteins in the list.

La leucémie myéloïde aigue (LMA), cancer du sang causé par une prolifération excessive des précurseurs myéloïdes à un stade précoce de maturation, est associée à une survie variant entre 20 et 30% à cinq ans en dépit des traitements de chimiothérapie les plus intensifs. L’antigène CD33 est exprimé chez les cellules malignes dans 90% des LMA ce qui en fait une cible de choix pour le développement d’immunoconjugué (IC). Trois IC composés d’un anticorps monoclonal anti-CD33 couplé à la maytansine, une toxine s’attaquant aux fuseaux mitotiques, ont été créés. Nous avons étudié l’effet de ces IC sur des cellules primaires et des lignées cellulaires LMA et étudier les mécanismes pouvant expliquer différents niveaux de sensibilité. Les études effectuées ont permis de déterminer que le niveau d’expression du CD33 n’explique pas la variation de sensibilité face aux IC. Il a été démontré que les IC anti-CD33 sont internalisés rapidement par la cellule et que le conjugué est retrouvé au niveau de l’endosome en premier lieu. Il a été confirmé que le lysosome est essentiel à l’effet anti-mitotique induit par le conjugué. Aussi, il est proposé que la protéine SOCS3 pourrait jouer un rôle dans la résistance aux IC anti-CD33 en dirigeant le complexe IC-CD33-SOCS3 vers le protéasome et ainsi empêcher la libération du composé toxique par le lysosome. Nous avons aussi conclu que les variations d’agent de liaison et l’augmentation du nombre de molécules toxiques entre les 3 IC n’ont pas été suffisantes pour augmenter leur efficacité à éliminer les cellules LMA. L’évaluation de ces IC ainsi que l’identification des mécanismes de résistance permettra de cibler les patients les plus susceptibles de bénéficier de ce type de traitement et potentiellement d’identifier de nouvelles voies pour améliorer l’efficacité des traitements. / Acute myeloid leukemia (AML), a cancer where hematopoietic precursors are arrested in an early stage of development, is associated with a poor survival rate of 20 to 30% over five years, despite intensive chemotherapy treatments. In approximately 90% of AML cases the malignant cells express CD33 antigen, which makes it a target of choice for development of immunotoxin based therapy. Three immunoconjugates (IC) composed of anti-CD33 monoclonal antibody coupled with maytansine derivative, which prevent tubulin polymerization and thus formation of mitotic spindle, were designed. These three IC were tested for their activity against several AML cell lines and primary AML patient cells and we investigate mechanisms responsible for variation in sensitivity to IC treatment. In this report, we show that differences in number of CD33 molecules on AML cell surface does not explain the observed differences in IC sensitivity. We demonstrate that binding of huMy9-6 antibody to CD33 induces rapid internalization and that it is first process through endosome. We confirm that lysosomal processing is essential for the antimitotic effect induced by IC treatment. Also, we provide evidence that SOCS3 protein may play a role in resistance of AML cells to anti-CD33 therapy by directing IC-CD33-SOCS3 complex to the proteasome and therefore affecting lysosomal decoupling of IC-CD33 and intracellular release of maytansine derivatives. Finally, the linkers and maytansine derivative modifications were not sufficient to increase sufficiently the efficacy of conjugates to eliminate higher numbers of AML cells. The identification of mechanisms responsible for increased resistance of AML cell lines and primary AMLs may allow us to identify IC responsive AML cells and also identify strategies to improve the efficacy of IC treatment.

Η Οξεία λευχαιμία (ΟΛ) αποτελεί νεοπλασματικό, αιμοποιητικό νόσημα, που οφείλεται στον πολλαπλασιασμό και την επέκταση κυττάρων, που προέρχονται από τους λευχαιμικούς βλάστες. Η φυσική εξέλιξη του νοσήματος είναι η αντικατάσταση των φυσιολογικών κυττάρων του αιμοποιητικού ιστού από τους απόγονους των λευχαιμικών βλαστών και θάνατος, λόγω των επιπλοκών της έλλειψης των ώριμων αιμοποιητικών κυττάρων, όπως λοιμώξεις, αναιμία και αιμορραγία. Θεραπευτικά για την αντιμετώπιση της ΟΛ χρησιμοποιούνται σχήματα χημειοθεραπείας και ακτινοθεραπείας, που έχουν σαν σκοπό την καταστροφή των λευχαιμικών βλαστών και την αποκατάσταση της φυσιολογικής αιμοποίησης. Συχνά η ΟΛ, ιδιαίτερα σε ασθενείς μεγάλης ηλικίας, εμφανίζεται ταυτόχρονα με δυσπλαστικές διαταραχές των αιμοποιητικών κυττάρων που ωριμάζουν ή εξελίσσεται σε μυελοδυσπλαστικό σύνδρομο μετά από χημειοθεραπεία. Από την βιβλιογραφία είναι γνωστό ότι, τόσο η ΟΛ μπορεί να είναι στάδιο εξέλιξης των μυελοδυσπλαστικών συνδρόμων και κατά συνέπεια μετά τη θεραπεία της ΟΛ επανέρχεται η δυσπλαστική κατάσταση της αιμοποίησης, όσο ότι η χημειοθεραπεία καθαυτή μπορεί να προκαλέσει μυελοδυσπλασία. Είναι σημαντική η διάγνωση των πρωτοπαθών μυελοδυσπλαστικών συνδρόμων που εξελίσσονται σε ΟΛ, ως προς την πρόγνωση των ασθενών, αλλά και τη θεραπευτική τους αντιμετώπιση. Επίσης, είναι σημαντική η διάκριση ομάδων ασθενών που θα αναπτύξουν δυσπλασία μετά από χημειοθεραπεία, σε σχέση με αυτούς που δεν θα αναπτύξουν και ως προς την πρόγνωση και ως προς τη θεραπευτική αντιμετώπιση. Μέχρι σήμερα, κατά την εμφάνιση της ΟΛ η διάγνωση υποκείμενης μυελοδυσπλασίας είναι δύσκολη και στηρίζεται σε μορφολογικά χαρακτηριστικά των ώριμων κυττάρων και στην παρουσία ορισμένων κυτταρογενετικών διαταραχών. Η διάκριση ομάδων που θα αναπτύξουν δυσπλασία μετά από χημειοθεραπεία είναι αδύνατη. Σκοπός της μελέτης ήταν να συμβάλλει στην ανάπτυξη νέων πρωτοκόλλων στο λογισμικό της κυτταρομετρίας ροής, που θα διευκολύνουν τη διάγνωση πρωτοπαθών δυσπλαστικών συνδρόμων κατά την εμφάνιση της ΟΛ, αλλά και θα διακρίνουν τις ομάδες που μπορούν να αναπτύξουν δυσπλασία μετά από θεραπεία. Για τον σκοπό αυτό, παρακολουθήσαμε την έκφραση χαρακτηριστικών αντιγονικών συνδυασμών, που εκφράζονται σε διαφορετικά στάδια ωρίμανσης των φυσιολογικών κυττάρων, παράλληλα με την έκφραση των αντιγόνων των βλαστών. Τα δεδομένα αυτά μελετήθηκαν, τόσο κατά την εμφάνιση της ΟΛ, όσο και κατά τη παρακολούθηση της. Τα δεδομένα αναλύθηκαν με συστήματα ταυτόχρονης ανάλυσης και συσχέτισης 15-20 παραμέτρων, με σκοπό τον καθορισμό συσχετισμών που θα έχουν διαγνωστική και προγνωστική σημασία για τους ασθενείς με ΟΛ. Τα αποτελέσματα του ανοσοφαινοτύπου αναλύθηκαν, επιπλέον, με το λογισμικό πακέτο στατιστικής ανάλυσης SPSS 16.0. Για τους σκοπούς της μελέτης αναλύθηκαν αναδρομικά τα αποτελέσματα της κυτταρομετρίας ροής στο μυελό των οστών 148 ασθενών με ΟΜΛ κατά την εμφάνιση της νόσου, κατά την διάρκεια και μετά από θεραπεία. Αναλύθηκε η έκφραση των αντιγόνων CD11b/CD16/CD13 σε όλα τα στάδια ωρίμανσης της μυελικής σειράς, ενώ η έκφραση των αντιγόνων CD34/CD117 μόνο στα άωρα κύτταρα. Τα ευρήματα του ανοσοφαινοτύπου συγκρίθηκαν με τα μορφολογικά χαρακτηριστικά των αντίστοιχων μυελών των οστών. Συμπερασματικά, τα αποτελέσματα έδειξαν ότι, η έκφραση των αντιγόνων CD11b και CD13 στα μεταμυελοκύτταρα και ουδετερόφιλα των ασθενών διακρίνει αποτελεσματικά τους ασθενείς με de novo ΟΜΛ σε σχέση με αυτούς που εμφάνισαν ΟΜΛ μετά από ΜΔΣ. Στους ασθενείς με de novo ΟΜΛ η έκφραση των αντιγόνων CD11b, CD13, CD16 δεν διέφερε κατά την εμφάνιση της ΟΛ στους υποπληθυσμούς των προμυελοκυττάρων, μυελοκυττάρων, μεταμυελοκυττάρων και ουδετερόφιλων μεταξύ των ασθενών που κατά ή μετά την θεραπεία εμφάνισαν μυελοδυσπλασία, σε σχέση με αυτούς που δεν εμφάνισαν. Επίσης, η θετική συν- έκφραση των αντιγόνων CD34/CD117 στους λευχαιμικούς βλάστες κατά την εμφάνιση δεν συσχετίζονταν με την εμφάνιση ΜΔΣ μετά την θεραπεία. Αντίθετα, η υψηλή έκφραση του λευχαιμικού φαινοτύπου CD34+/CD117- στα άωρα κύτταρα της μυελικής σειράς κατά την εμφάνιση της ΟΜΛ, έδειξε ότι σχετίζεται με την εμφάνιση μυελοδυσπλαστικών χαρακτηριστικών μετά τη θεραπεία. Η ανάλυση των ανοσοφαινοτύπων του μυελού των οστών κατά ή μετά την θεραπεία έδειξε παθολογική έκφραση CD11b, CD13, CD16 στα μεταμυελοκύτταρα και ουδετερόφιλα των ασθενών που εμφάνισαν ΜΔΣ μετά θεραπεία για de novo ΟΜΛ και ασθενών (5/17) που δεν εμφάνισαν ΜΔΣ. Συμπερασματικά, η έκφραση των αντιγόνων CD11b, CD16 και CD13 στα ώριμα κύτταρα της μυελικής σειράς κατά την εμφάνιση της ΟΜΛ διαχωρίζει αποτελεσματικά την de novo ΟΜΛ από την δευτεροπαθή μετά ΜΔΣ. Η έκφραση αυτών των αντιγόνων κατά την εμφάνιση της ΟΜΛ δεν μπορεί, όμως, να προβλέψει την εξέλιξη της de novo ΟΜΛ και την εμφάνιση δυσπλαστικών χαρακτηριστικών μετά τη θεραπεία. Αντιθέτως, η μελέτη των λευχαιμικών φαινοτύπων CD34+/CD117- και CD34+/CD117+ μπορεί να παίξει καθοριστικό ρόλο στην πρόγνωση της εμφάνισης μυελοδυσπλαστικών χαρακτηριστικών κατά τη διάρκεια ή μετά τη θεραπεία του ασθενούς. / --

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa, resultante da expansão clonal da célula-tronco hematopoética pluripotente. A fisiopatologia da LMC está associada a uma translocação entre os braços longos dos cromossomos 9 e 22, o que promove o aparecimento do neogene bcr-abl, cujo gene codifica uma proteína denominada Bcr-Abl. A oncoproteína Bcr-Abl possui atividade tirosina-quinase constitutiva que é a responsável pelo fenótipo maligno da célula, incluindo resistência à apoptose. O tratamento da LMC pode ser realizado com hidroxiuréia, IFN- associado à citarabina, inibidores de TK (mesilato de imatinibe e dasatinibe) e transplante de medula óssea. O tratamento de escolha para pacientes com LMC na fase crônica é o inibidor de tirosina-quinase mesilato de imatinibe e para os refratários utiliza-se o dasatinibe. Apesar do conhecimento acerca do mecanismo de ação dos inibidores de TK, pouco se sabe sobre seu efeito na maquinaria apoptótica. Sendo assim, no presente trabalho foi detectada a expressão dos genes e proteínas anti- (A1, Bcl-2, Bcl-Xl, Bcl-W, C-Flip, Ciap-1, Ciap-2 e Mcl-1) e pró-apoptóticos (Bad, Bak, Bax, Bcl-Xs, Bid, Bik, Bimel, Bmf, Bok, Fas, Fasl, Noxa e Puma) em células mononucleares de 32 indivíduos saudáveis e 26 pacientes com LMC antes e após 12 meses da terapia com mesilato de imatinibe e dasatinibe. Dentre os 26 pacientes avaliados, 13 eram do sexo feminino e 13 do sexo masculino, três eram negros, um amarelo e 22 brancos, com idade média de 48 anos (faixa etária de 25 a 77 anos). O grupo controle foi composto por 32 indivíduos, 16 do sexo feminino e 16 do sexo masculino, 26 eram brancos, quatro negros e dois amarelos, com idade média de 45 anos (idade de 23 a 77 anos). O isolamento das células mononucleares foi realizado pelo método de Ficoll-Hypaque, a determinação da expressão gênica por PCR em tempo real e a protéica por western-blot. Os resultados foram expressos em unidade relativa de expressão (U.R.E.), comparados entre os diferentes grupos (controle e pacientes pré- e pós-tratamento), associados à resposta aos medicamentos e correlacionados ao índice de prognóstico de SOKAL. Na comparação dos dados de expressão gênica entre pacientes e controles, verificou-se que os pacientes apresentaram maior expressão dos genes bcl-xL, c-flip, mcl-1 e fas e níveis reduzidos de bik. O mesilato de imatinibe modulou significativamente a transcrição dos genes bcl-xL, bok, mcl-1 e noxa, enquanto que o dasatinibe agiu sobre a expressão dos genes a1, bmf, c-flip, ciap-1, ciap-2 e mcl-1. Os pacientes refratários ao mesilato de imatinibe apresentaram níveis elevados de expressão de a1 e c-flip e reduzida expressão de bcl-2, ciap-2, bak, bax, bid e fasl em relação aos pacientes em remissão. A expressão protéica refletiu os dados da quantificação do RNAm dos genes. Os dados da presente investigação indicam que as células mononucleares dos pacientes com LMC apresentam desregulação do processo de apoptose celular. Essa alteração pode ser parcialmente associada ao fenótipo de resistência das células leucêmicas Bcr-Abl+ à apoptose e ausência de resposta aos inibidores de TK. Os dados revelam ainda que os inibidores de tirosina-quinase interferem na transcrição e tradução das moléculas envolvidas na regulação do processo de apoptose. / Chronic myeloid leukemia (CML) is a myeloproliferative disease resultant of a clonal expansion of pluripotent hematopoietic stem cells. The CML physiopathology is associated with a translocation between chromosomes 9 and 22 long arms, promoting the formation of a bcr-abl neogene, which codifies the Bcr-Abl protein. The Bcr-Abl oncoprotein presents tyrosine-kinase activity that is responsible for the malign phenotype which includes apoptosis resistance. CML treatment may be performed with hydroxyurea, IFN- plus cytarabine, tyrosine-kinase inhibitors (imatinib mesylate and dasatinib) and bone marrow transplantation. The standard treatment for CML patients in chronic phase is the tyrosine-kinase inhibitor imatinib mesylate (IM) and for IM-refractory patients dasatinibe is employed. Despite of the knowledge related to the mechanism of action of tyrosine-kinase inhibitors, little is known about its effects on the apoptosis machinery. In this work we characterized both the mRNA and protein patterns of expression of the anti- (A1, Bcl-2, Bcl-Xl, Bcl-W, C-Flip, Ciap-1, Ciap-2 e Mcl-1) and pro-apoptotic (Bad, Bak, Bax, Bcl-Xs, Bid, Bik, Bimel, Bmf, Bok, Fas, Fasl, Noxa e Puma) regulators in peripheral blood mononuclear cells (PBMC) from 32 healthy individual and 26 CML patients before and after 12 months of imatinib mesylate and dasatinibe therapy. Thirteen patients were female and thirteen were male, three were black, one was japanese and 22 were white, the mean age was 48 years (varying from 25 to 77 years). The control group was composed of 32 individuals, 16 were female and 16 were male, 26 were white, four black and two japanese, the mean age was 45 years (varying from 23 to 77 years). PBMC isolation was performed by Ficoll-Hypaque, gene expression was assessed by real time PCR and protein expression was carried out by western-blot methodology. Results were given by the relative expression, after comparison between the different groups (control and patients before and after treatment), associated with drug response and related to Sokal index. The comparison of gene expression profiles between patients and controls showed that CML patients present increased levels of bcl-xL, c-flip, mcl-1 and fas expression and reduced levels of bik gene expression. The imatinib mesylate significantly modulated the transcription of bcl-xL, bok, mcl-1 and noxa, whereas dasatinib affected the expression of a1, bmf, c-flip, ciap-1, ciap-2 and mcl-1. MI-refractory patients present higher levels of a1 and c-flip and lower levels of bcl-2, ciap-2, bak, bax, bid and fasl when compared to patients who achieved complete cytogenetic response. Overall, the patterns of protein expression agree with the profiles of mRNA expression. Taken together, the results described in this investigation indicate that PBMC from CML patients present deregulation in cell death pathways. These alterations could partly account for the apoptosis resistance phenotype observed in Bcr-Abl+ leukemic cells and for lack of response to tyrosine kinase inhibitors. Furthermore, our data also reveal that tyrosine kinase inhibitors interfere in the transcription and translation of molecules that regulate the apoptosis process.

Na leucemia mielóide crônica (LMC) a proteína Bcr-Abl possui atividade tirosina-quinase constitutivamente ativada, induzindo a mieloproliferação e a resistência das células à apoptose. A maioria dos pacientes na fase crônica da LMC tratados com inibidores de tirosina-quinase (TKIs), como o mesilato de imatinibe, apresenta remissão citogenética completa da doença, mas uma parcela desses pacientes tem se mostrado resistente à terapia. A fisiopatologia da LMC e os mecanismos celulares e moleculares envolvidos na resistência à terapia com TKIs são diversos e precisam ser melhor estudados. Nesse contexto, o objetivo do presente estudo foi quantificar os níveis de expressão do gene DIDO, incluindo suas diferentes isoformas (DIDO 1, 2 e 3) e promotores (DIDO PP e PD) em controles e em pacientes com LMC nas diferentes fases da doença, tratados ou não com TKIs, bem como em linhagens celulares BCR-ABL1+ sensíveis (S) e resistentes (R) ao mesilato de imatinibe (MI). A literatura relata que DIDO 1 participa do processo de apoptose e que alterações na expressão de DIDO 2 e DIDO 3 podem estar associadas com o desenvolvimento de neoplasias mielóides. Dessa forma, foram estudados 60 pacientes com LMC e 57 controles, assim como as linhagens celulares HL-60, HL-60.Bcr-Abl+, LAMA 84 S, LAMA 84 R, KCL 22 S e KCL 22 R. Foram separadas as células mononucleares de sangue periférico dos pacientes e controles, com posterior extração de RNA e síntese de cDNA, que foi então empregado nas reações de PCR em tempo real (qPCR) para quantificação das expressões gênicas de DIDO 1, 2, 3, PP, PD e ORF. As linhagens celulares foram tratadas por 4h com TKIs e então a expressão das diferentes isoformas de DIDO foi também quantificada por qPCR. A avaliação da expressão proteica de Bcr-Abl, c-Abl e proteínas fosforiladas nas linhagens foi realizada por Western-blotting. A expressão de DIDO 1 e 2 foi maior nos pacientes nas fases avançadas da LMC e nos pacientes na fase crônica da doença tratados com TKIs (mesilato de imatinibe ou dasatinibe) do que nos controles. Os pacientes na fase crônica da LMC tratados com MI expressaram mais DIDO 1 e 3 do que os pacientes na fase crônica sem tratamento. Houve uma correlação positiva entre expressão de BCR-ABL1 e de DIDO 2 e entre índice de Sokal dos pacientes e expressão de DIDO 2. Na linhagem HL60.Bcr-Abl+, a expressão de proteínas fosforiladas reduziu após tratamento de 4h com TKIs, mas não houve alteração na expressão gênica de DIDO. Nas linhagens celulares S e R, houve aumento da expressão de DIDO 1 após o tratamento de 4h com MI. Conclui-se, portanto, que as diferentes isoformas de DIDO parecem exercer funções distintas na leucemia mielóide crônica; que o tratamento de pacientes e linhagens BCR-ABL1 positivas com inibidores de tirosina-quinase aumenta expressão de DIDO 1; e que a expressão de DIDO 2 correlaciona-se positivamente à expressão de BCR-ABL1 e ao índice de Sokal dos pacientes. / In chronic myeloid leukemia (CML) the Bcr-Abl protein has constitutively activated tyrosine kinase activity, that induces to myeloproliferation and apoptosis resistance of the cells. Most patients in chronic phase of CML treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate have a complete cytogenetic remission, but a portion of these patients have been shown to be resistant to therapy. The pathophysiology of CML and the cellular and molecular mechanisms involved in resistance to TKIs therapy are diverse and require further study. In this sense, the aim of this study was to quantify the expression levels of the DIDO gene, including its isoforms (DIDO 1, 2 and 3) and promoters (DIDO PP and PD) in controls and in patients with CML in different phases of the disease treated or not treated with TKIs, as well as in cell lines BCR-ABL1+ sensitive (S) and resistant (R) to imatinib mesylate (IM). The literature reports that DIDO 1 is involved in apoptosis process and that alterations of DIDO 2 and DIDO 3 expression may be associated with the development of myeloid neoplasms. Thus, 60 CML patients, 57 control individuals and the cell lines HL-60, HL-60.Bcr-Abl+, LAMA 84 S, LAMA 84 R, KCL 22 S and KCL 22 R were studied. Peripheral blood mononuclear cells of patients and controls were isolated and RNA extraction and cDNA synthesis were performed. The cDNA samples were used in Real-Time PCR reactions (qPCR) to quantify the DIDO 1, 2, 3, PP, PD and ORF gene expression. The cell lines were treated during 4h with TKIs and then the expression of DIDO different isoforms was also quantified by qPCR. The assessment of protein expression of Bcr-Abl, c-Abl and phosphorylated proteins in this cell lines was performed by Western blotting. The DIDO 1 and 2 expression was higher in advanced phases patients and in chronic phase patients CML treated with TKIs (imatinib mesylate and dasatinib) than in controls. Chronic phase CML Patients treated with IM expressed more DIDO 1 and 3 than chronic phase untreated patients. There was a positive correlation between BCR-ABL1 expression and DIDO 2 expression and between Sokal score prognostic and DIDO 2 expression. In HL60.Bcr-Abl+ cells the expression of phosphorylated proteins was lower after treatment during 4h with TKIs, but there was no change in DIDO gene expression. There were an increase of DIDO 1 expression in all S and R cell lines after treatment during 4h with IM. Therefore we conclude that the DIDO different isoforms may have different functions in chronic myeloid leukemia; the treatment of patients and BCR-ABL1+ cell lines with TKIs increases DIDO 1 expression; and that the DIDO 2 expression is positively correlated to the BCR-ABL1 expression and Sokal score prognostic of CML patients.

Les longs ARN non codants (lncRNAs) sont définis comme des transcrits de plus de 200nt et n'ayant pas de potentiel codant. Des études récentes ont démontré que les lncRNAs pouvaient être impliqués dans la régulation de la transcription, de la traduction, de la différenciation cellulaire, de l'expression génique, du cycle cellulaire et des modifications de la chromatine. De plus, il a été montré un impact fonctionnel de certains lncRNAs dans le processus de cancérogenèse mais nos connaissances actuelles sur ces molécules dans le cancer, et plus particulièrement dans la leucémie, restent extrêmement limitées. Au cours de cette étude, nous avons analysé l'expression des lncRNAs par RNA-sequencing sur 40 patients atteints de leucémie aiguë myéloblastique (LAM) à caryotype normal. Parmi les 11065 lncRNAs exprimés dans nos échantillons, nous avons identifié une signature de lncRNAs associée à la mutation de NPM1. Afin de mettre en évidence les fonctions putatives des lncRNAs sélectionnés, nous avons utilisé un algorithme de prédiction d'interaction protéine/ARN. De manière intéressante, plus de la moitié des lncRNAs présentent des sites d'interactions potentiels à SUZ12, une sous unité du complexe PRC2 (Polycomb repressive complex 2), connu pour être recruté par des lncRNAs pour la régulation épigénétique de gènes cibles. Par RNA immunoprécipitation (RIP) de SUZ12, nous avons pu démontrer que le lncRNA XLOC_087120 interagissait avec SUZ12. De plus, son expression est anti-corrélée avec celle des gènes voisins codants des histones, suggérant un rôle dans la régulation négative des histones par ce lncRNA. L'impact de la dérégulation de XLOC_087120 sur les histones a été confirmé par des expériences de surexpression et d'inhibition de ce lncRNA dans des lignées de LAM. De plus, même si la mutation NPM1 ne semble pas affecter directement l'expression de ce lncRNA, des expériences d'infection de la forme mutée de NPM1 dans une lignée LAM ont montré que NPM1 pourrait réguler la localisation nucléaire/cytoplasmique de XLOC_087120 et moduler sa fonction de répresseur. En conclusion, ces données suggèrent que les lncRNAs sont des facteurs clés dans la pathogenèse des LAMs. / Long noncoding RNAs (lncRNAs) are defined as RNA transcripts that are larger than 200 nt but do not appear to have protein- coding potential. Recent studies have demonstrated that lncRNAs regulate many processes such as transcription, translation, cellular differentiation, gene expression regulation, cell cycle regulation, and chromatin modification. Cumulative evidence points towards an important role of lncRNAs in cancer initiation, development, and progression. However, our overall knowledge of lncRNAs in cancer, including leukemia, remains extremely limited. In this study, we investigated lncRNA expression by RNA-sequencing in 40 acute myeloid leukemia (AML) patients with normal karyotype. Among 11065 lncRNA expressed in our samples, we identified specific lncRNA signature associated with the presence of NPM1 mutation. To go further into the putative function of these lncRNAs, we used catRAPID Omics algorithm to predict potential protein partners. Interestingly, the majority of the selected lncRNAs contains putative SUZ12 binding sites, a PRC2 (Polycomb Repressive Complex 2) component known to be linked to lncRNAs and to epigenetically regulates target genes. By using SUZ12 RNA Immunoprecipitation, we identify one lncRNA named XLOC_087120 linked to SUZ12. XLOC_087120 is located in a region enriched in histone genes. Pearson correlation showed a significative anti-correlation between XLOC_087120 and histone neighboring coding gene expression suggesting a role of this lncRNA in the regulation of histone genes. The impact on histone genes expression was confirmed by overexpression and inhibition of XLOC_087120 in AML cell lines. Overexpression of NPM1 mutant in an AML cell line showed that NPM1 modulates the nuclear/cytoplasmic localization of XLOC_087120 and consequently its repressive function. Altogether, these data suggest that lncRNAs should be considered as key players in the pathogenesis of acute myeloid leukemias.

abstract: The phycologist, M. R. Droop, studied vitamin B12 limitation in the flagellate Monochrysis lutheri and concluded that its specific growth rate depended on the concentration of the vitamin within the cell; i.e. the cell quota of the vitamin B12. The Droop model provides a mathematical expression to link growth rate to the intracellular concentration of a limiting nutrient. Although the Droop model has been an important modeling tool in ecology, it has only recently been applied to study cancer biology. Cancer cells live in an ecological setting, interacting and competing with normal and other cancerous cells for nutrients and space, and evolving and adapting to their environment. Here, the Droop equation is used to model three cancers. First, prostate cancer is modeled, where androgen is considered the limiting nutrient since most tumors depend on androgen for proliferation and survival. The model's accuracy for predicting the biomarker for patients on intermittent androgen deprivation therapy is tested by comparing the simulation results to clinical data as well as to an existing simpler model. The results suggest that a simpler model may be more beneficial for a predictive use, although further research is needed in this field prior to implementing mathematical models as a predictive method in a clinical setting. Next, two chronic myeloid leukemia models are compared that consider Imatinib treatment, a drug that inhibits the constitutively active tyrosine kinase BCR-ABL. Both models describe the competition of leukemic and normal cells, however the first model also describes intracellular dynamics by considering BCR-ABL as the limiting nutrient. Using clinical data, the differences in estimated parameters between the models and the capacity for each model to predict drug resistance are analyzed. Last, a simple model is presented that considers ovarian tumor growth and tumor induced angiogenesis, subject to on and off anti-angiogenesis treatment. In this environment, the cell quota represents the intracellular concentration of necessary nutrients provided through blood supply. Mathematical analysis of the model is presented and model simulation results are compared to pre-clinical data. This simple model is able to fit both on- and off-treatment data using the same biologically relevant parameters. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2015

La duplication interne en tandem au sein du gène du Fms-like tyrosine kinase 3 (FLT3) est l’une des mutations les plus fréquemment observées dans les leucémies aiguës myéloblastiques (LAM). Elle est corrélée à un mauvais pronostic. Des inhibiteurs de tyrosine kinase anti-FLT3 (FLT3-ITK) sont en cours de développement mais les premiers essais cliniques ont été décevants. Les rémissions sont de courte durée, et si une clairance leucémique sanguine est observée, la LAM persiste au sein de la moelle osseuse. Dans ce travail, nous avons démontré que les cytokines activatrices de STAT5, telles que l’interleukine-3 et la thrombopoïétine, et les basses pressions en oxygène, telles que celles observées au sein de la niche hématopoïétique augmentent l’expression et l’activité du récepteur tyrosine kinase AXL qui protège les cellules de LAM FLT3-ITD de l’apoptose induite par le FLT3-ITK quizartinib (AC220). Nous avons démontré dans un modèle murin que les cellules de LAM FLT3-ITD « knock-down » pour AXL sont plus sensibles au quizartinib, et que cette différence se révèle spécifiquement dans un modèle de prise de greffe hématopoïétique. La combinaison de stratégies inhibitrices du FLT3-ITD et d’AXL permettra d’améliorer l’efficacité des FLT3-ITK en atteignant la fraction de cellules responsable des rechutes, nichée dans son microenvironnement. A l’issue, nous avons démontré que le gilteritinib (ASP2215), double FLT3/AXL-ITK est plus efficace que le quizartinib pour atteindre ces cellules leucémiques médullaires. Enfin, nous avons démontré que la combinaison d’un anticorps monoclonal anti-AXL avec un FLT3-ITK ou de la cytarabine était une stratégie thérapeutique prometteuse dans les LAM FLT3-ITD ou sauvage. / Internal tandem duplication in Fms-like tyrosine kinase 3 gene (FLT3-ITD) is the most frequent mutation observed in acute myeloid leukemia (AML), and correlates with poor prognosis. FLT3 tyrosine kinase inhibitors (FLT3-TKI) have been promising for therapeutic strategies but clinical trials have revealed rarely long-lasting remission with persistent leukemic cells present in the bone marrow. In this work, we show that the hematopoietic niche microenvironment protects FLT3-ITD AML cells from FLT3-TKI quizartinib (AC220) through convergent up-regulation of AXL expression and activity. Cytokine-dependent activation of STAT5 enhances AXL gene transcription and expression, while low O2 concentration up-regulates AXL protein levels. Moreover, cytokines such as thrombopoietin or interleukin-3 directly activate AXL. RNA interference-based inhibition of AXL expression in FLT3-ITD AML cells allowed a selective purge of leukemic cells within their microenvironment when combined with FLT3-TKI in immuno-compromised mice. Altogether, our data support a strategy combining FLT3-TKI and anti-AXL therapy to eradicate FLT3-ITD AML cells, including those protected by the hematopoietic niche. In such a setting, we performed a study to test the efficacy of gilteritinib (ASP2215) and we showed in vitro and in vivo that this dual FLT3/AXL-TKI is more efficient to eradicate leukemic cells in their microenvironment than quizartinib which is a more specific FLT3-TKI. Finally, we also studied an anti-AXL monoclonal antibody on primary AML cells and showed that its efficacy could be interesting with FLT3-TKI and cytarabine in both FLT3-wild type and FLT3-ITD AML.

Made available in DSpace on 2014-12-17T14:16:28Z (GMT). No. of bitstreams: 1 RobertoCV_DISSERT.pdf: 2201581 bytes, checksum: a5ed04f6ae5794f328192fe513bdf03f (MD5) Previous issue date: 2010-02-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The acute myeloid leukemia (AML) is a disease in which malignant myeloblasts expand, build up and suppress normal hematopoietic activity would represent a major diagnostic challenge. With the advent of immunophenotyping by flow cytometry, the diagnosis of these tumors have become more faithful, facilitating the treatment and monitoring of patients. The objectives of this study: diagnosis and classification of AML based on immunophenotyping by flow cytometry with a panel of AcMo specific for acute leukemias, set the frequency of AML in samples from patients with acute leukemias sent to the Department of Hematology Blood Center of Rio Grande do Norte - HEMONORTE, establish standards of antigen expression for different subtypes of acute leukemia and its correlation with the newly diagnosed cases refractory to treatment and recurrence of the disease, standardization of methods for detection and labeling of surface antigens by flow cytometry and intracytoplasmic flow, and observe the frequency of acute leukemia with aberrant phenotypes rare. During the study, 351 were diagnosed acute leukemia, and 179 (51%) classified as AML and 172 (49%) and ALL, which were excluded from the present work. Of the 179 AML, 92 (51.4%) were female and 87 (48.6%) were male, with ages ranging from 3 to 95 years of ag, with higher incidence in individuals in the age group of 41 to 65. Splenomegaly was the clinical finding more present, a total of 147 cases (82.1%), followed by hepatomegaly present in 132 cases (73.7%). The hemorrhagic events were observed in 55 cases (30.7%). Lymphadenopathy in turn was detected in 20 of 179 cases (11.2%). In order to classify subtypes of AML, we used a large panel of monoclonal antibodies, obtaining the following results: AML M0, 02 (1.1%) AML M1, 40 (22.3) AML M2, 60 (33.5) AML M3, 22 (12.3%) AML M4, 10 (5.6) AML M5, 13 (7.3%) AML M6 06 (3.4%) and AML M7 01 (0.6%). We observed some cases with aberrant expression of some antigens such as CD7, CD4, CD19, CD3, CD5 and TdT, CD 7 was present in 30 (16.8%), CD4 in 5 (2.8%), the CD 3 in 5 (2.8%), the CD19 in 3 (1.7%), the CD5 in 3 (1.7%) and TDT was in 7 (3.9%) cases of AML .the CD8 and CD79a was present in only a 1 case. / A Leucemia Miel?ide Aguda (LMA) ? uma doen?a maligna em que os mieloblastos expandem-se, acumulam-se e suprimem a atividade hematopo?tica normal, constituindo um grande desafio diagn?stico. Com o advento da imunofenotipagem por citometria de fluxo, o diagn?stico dessas neoplasias se tornaram mais fi?is, facilitando o tratamento e o acompanhamento dos pacientes. Foram objetivos deste estudo: diagnosticar e classificar as LMA com base na imunofenotipagem por citometria de fluxo, com um painel de AcMo espec?fico para leucemias agudas; estabelecer a frequ?ncia de LMA nas amostras de pacientes com leucemias agudas encaminhadas ao Departamento de Hematologia do Hemocentro do Rio Grande do Norte HEMONORTE; estabelecer padr?es de express?o antig?nica para os diversos subtipos de leucemias agudas e a sua correla??o com os casos rec?m diagnosticados, refrat?rios ao tratamento e recorr?ncia da doen?a; padroniza??o dos m?todos de detec??o e marca??o de ant?genos de superf?cie e intracitoplasm?tico por citometria de fluxo; e observar a freq??ncia de leucemias agudas com fen?tipos aberrantes raros. Durante o estudo, foram diagnosticados 351 leucemias agudas, sendo 179 (51%) classificadas como LMA e 172 (49%) como LLA, as quais foram exclu?das do presente trabalho. Das 179 LMA, 92 (51,4%) eram do sexo feminino e 87 (48,6%) do sexo masculino, com faixa et?ria variando de 3 a 95 anos de idade, com maior incid?ncia em indiv?duos na faixa et?ria de 41 a 65 anos. A esplenomegalia foi o achado cl?nico mais presente, perfazendo um total de 147 casos (82,1%), seguida da hepatomegalia presente em 132 casos (73,7%). Os fen?menos hemorragicos foram observado em 55 casos ( 30,7%). A linfoadenopatia por sua vez foi constatada em 20 dos 179 casos (11,2%). Para classifica??o dos subtipos de LMA foi utilizado um painel amplo de anticorpos monoclonais, obtendo os seguintes resultados: LMA M0, 02 (1.1%) LMA M1, 40 (22.3) LMA M2, 60 (33.5) LMA M3, 22 (12.3%) LMA M4, 10 (5.6) LMA M5, 13 (7.3%) LMA M6 06 (3.4%) e LMA M7 01 (0.6%). Foram observados alguns casos com express?o aberrante de alguns ant?genos tais como CD7, CD4, CD19, CD3, CD5 e TdT, O CD7 esteve presente em 30 (16,8%) , o CD4 em 5 (2,8%), o CD 3 em 5 (2,8%), o CD19 em 3 (1,7%), o CD5 em 3 (1,7%) e o TDT esteve em 7 (3,9%) casos de LMA diagnosticados.O CD8 e o CD79a esteve presente em apenas um 1 caso.

Orientador: Célia Regina Nogueira / Resumo: A Leucemia Mielóide Crônica (LMC) é uma doença malígna, clonal, da célula tronco hematopoética. A descoberta do cromossomo filadélfia e subsequente identificação do gene BCR-ABL, levaram a compreensão da biologia da doença que culminou com o desenvolvimento de drogas alvo-específicas, assim como o de métodos moleculares para o monitoramento da doença. O foco atual das pesquisas em LMC está voltado para o maior entendimento dos mecanismos moleculares e epigenéticos que levam a resistência terapêutica e progressão da doença. Estudos recentes demonstram que a expressão de micro-RNAs específicos modula oncogenes e genes supressores envolvidos no desenvolvimento de neoplasias. Ao encontro a esta tendência, propomos um estudo que procurou identificar o perfil de micro-RNAs dos pacientes bons respondedores aos tratamentos de primeira linha para LMC. Avaliamos o perfil de micro-RNAs, de 41 pacientes com LMC que atingiram resposta citogenética completa (ausência do cromossomo Filadélfia) após o tratamento com inibidor de tirosinaquinase e transplante alogênico de células progenitoras hematopoéticas, por meio do sistema de micro-RNA PCR arrays (TaqMan® Human Micro-RNA Array A e B). Identificamos uma assinatura de micro-RNA distinta entre os grupos tratados, apesar de se encontrarem no mesmo patamar de resposta clínica e citogenética. Palavras-chave: Leucemia Mielóide Crônica, micro-RNA, imatinibe, transplante, qPCR array. / Abstract: Chronic Myeloid Leukemia (CML) is a malignant clonal hematopoietic stem cell disease. The discovery of the Philadelphia chromosome and subsequent identification of the gene BCR-ABL have led to understanding the biology of the disease and the development of target-specific drugs, as well as molecular methods for monitoring the disease. The current focus of research in the CML is facing the greatest understanding of the molecular and epigenetic mechanisms that lead to therapy resistance and disease progression. Recent studies show that the expression of specific micro-RNAs modulates oncogenes and tumor suppressor genes involved in cancer development.We are proposing a new study in the literature that aims to identify the profile of micro-RNAs of patients good responders to first-line treatments for CML.Evaluated the profile of micro-RNAs in 41 CML patients who achieved a complete cytogenetic response (absence of Philadelphia chromosome) after treatment with tyrosine kinase inhibitor and allogeneic bone marrow transplantation, through the micro-RNA- PCR arrays (TaqMan® Human Micro-RNA Array A e B). We identified a distinct micro-RNA signature between the treated groups, despite being on the same level of cytogenetic and clinical response.Key Words: Chronic Myeloid LeuKemia, micro-RNA, imatinib, bone marrow transplantation, qPCR array. / Mestre