Longitudinal monitoring of BCR-ABL transcript levels in peripheral blood of CML patients treated with tyrosine kinase inhibitors (TKI) revealed a typical biphasic response. Although second generation TKIs like dasatinib proved more efficient in achieving molecular remission compared to first generation TKI imatinib, it is unclear how individual responses differ between the drugs and whether mechanisms of drug action can be deduced from the dynamic data. We use time courses from the DASISION trial to address statistical differences in the dynamic response between first line imatinib vs. dasatinib treatment cohorts and we analyze differences between the cohorts by fitting an established mathematical model of functional CML treatment to individual time courses. On average, dasatinib-treated patients show a steeper initial response, while the long-term response only marginally differed between the treatments. Supplementing each patient time course with a corresponding confidence region, we illustrate the consequences of the uncertainty estimate for the underlying mechanisms of CML remission. Our model suggests that the observed BCR-ABL dynamics may result from different, underlying stem cell dynamics. These results illustrate that the perception and description of CML treatment response as a dynamic process on the level of individual patients is a prerequisite for reliable patient-specific response predictions and treatment optimizations.

Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive. First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells. Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model. Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients. Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.

Die Verwendung von nukleinsäuretemplatgesteuerten Reaktionen stellt einen innovativen Ansatz zur Therapie von Krankheiten dar, deren Ursprung in einer genetischen Veränderung von Zellen liegt. Im Rahmen der hier vorliegenden Arbeit erfolgte eine Weiterentwicklung der RNA-templatgesteuerten Acyltransferreaktion nach Seitz et al. hinsichtlich des Aufbaus von aromatischen Amidbindungen, wie sie in vielen niedermolekularen Wirkstoffen vorkommen. Als Modellverbindungen dienten die Bcr-Abl-Tyrosinkinaseinhibitoren Nilotinib, Ponatinib und GZD824, die durch zentrale Benzanilidmotive charakterisiert sind und zur Therapie der chronischen myeloischen Leukämie eingesetzt werden. Das konzipierte Reaktionssystem beruht auf dem Mechanismus der nativen chemischen Ligation, weswegen die Bcr-Abl-Inhibitoren durch die Einführung einer Thiolgruppe modifiziert werden mussten. Es wurde gezeigt, dass diese Modifikation in den meisten Fällen nur zu einer geringen Beeinträchtigung der biologischen Aktivität führt. Der RNA-templatgesteuerte Aufbau der Benzanilidstrukturen konnte sowohl mit PNA- als auch mit DNA-verknüpften Inhibitorfragmenten basierend auf Ponatinib und GZD824 erzielt werden. In Gegenwart eines komplementären RNA-Templats erfolgte die benachbarte Hybridisierung der reaktiven Konjugate, infolgedessen ein Benzoyltransfer von einem thioestermodifizierten Donor auf ein ortho-Mercaptoanilinderivat als Akzeptor stattfand. Mit PNA/PNA-Reaktionssystemen wurde auch in Abwesenheit des RNA-Templats eine signifikante Produktbildung beobachtet, was auf hydrophobe Wechselwirkungen der Konjugate zurückgeführt wurde. Unter Verwendung von DNA/DNA- bzw. PNA/DNA-Konjugatpaaren blieb die templatunabhängige Produktbildung hingegen ausreichend gering. Die entsprechenden Reaktionssysteme wurden hinsichtlich der erzielten Produktausbeuten optimiert, wobei sowohl die Linkerlänge bzw. -struktur der reaktiven Konjugate als auch die Templatarchitektur variiert wurde. / The use of nucleic acid-templated reactions represents an innovative approach to the therapy of diseases that originate in genetic alterations of cells. In this work, the RNA-templated acyl transfer reaction invented by Seitz et al. was extended towards the formation of aromatic amide bonds which occur in a variety of small molecule drugs. The Bcr-Abl tyrosine kinase inhibitors nilotinib, ponatinib, and GZD824 were used as model compounds, as they are characterized by central benzanilide motifs and find application in the treatment of chronic myeloid leukemia. The designed reaction system is based on the mechanism of native chemical ligation which required the modification of the Bcr-Abl inhibitors by introducing a thiol group. It was shown that thiolation affected the biological activity only moderately. The RNA-templated formation of the benzanilide structures was achieved with both PNA- and DNA-linked inhibitor fragments of ponatinib and GZD824. The presence of a complementary RNA template enabled adjacent binding of the reactive conjugates, triggering a rapid benzoyl transfer from a thioester-linked donor to an ortho-mercaptoaniline derivative as an acceptor. With PNA/PNA reaction systems, significant product formation was observed in absence of the RNA template, which was attributed to hydrophobic interactions between the conjugates. However, the template-independent product formation remained low when DNA/DNA or PNA/DNA conjugate pairs were used. The product yields of the corresponding reaction systems were optimized by varying the linker length and structure of the conjugates as well as the template architecture.

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Σκοπός της παρούσας εργασίας είναι η εκτίμηση των περιβαλλοντικών και υδρογεωλογικών συνθηκών της ευρύτερης περιοχής του Μεσολογγίου, η εκτίμηση της ποιότητας νερών και πηλών στης περιοχές ενδιαφέροντος όσον αφορά τους ιαματικούς φυσικούς πόρους της περιοχής και τέλος η διερεύνηση των πιθανών πηγών ρύπανσης της περιοχής και ειδικότερα των παραπάνω θέσεων ενδιαφέροντος. Από τα αποτελέσματα της παρούσας εργασίας προκύπτει ότι τα υπόγεια νερά της περιοχής παρουσιάζουν αυξημένη αλατότητα όπως αυτό ερμηνεύεται από τις πολύ υψηλές τιμές της ηλεκτρικής αγωγιμότητας που παρουσιάζουν. Λόγω του ασήμαντου ρυπαντικού τους φορτίου δεν καθιστούν πηγή ρύπανσης των θέσεων ενδιαφέροντος. Από της άλλη μεριά τα νερά και οι πηλοί που συλλέχθησαν από ρέματα της περιοχής που καταλήγουν στην λιμνοθάλασσα, παρουσίασαν αυξημένες τιμές βαρέων μετάλλων που οφείλονται κατά κύριο λόγο στις ανθρωπογενείες δραστηριότητες της περιοχής, ενδέχεται να επιβαρύνουν τους πηλούς κυρίως σε βαρέα μέταλλα. Το μεγαλύτερο μέρος της συγκέντρωσης των βαρέων μετάλλων που ανιχνεύτηκε στους πηλούς της περιοχής δεν συνιστά πηγή δευτερογενούς ρύπανσης. Από τα αποτελέσματα της μεθόδου διαδοχικής εκχύλισης BCR-SEP φάνηκε ότι το μεγαλύτερο μέρος των βαρέων μετάλλων είναι ισχυρά συνδεδεμένο με το ίζημα (συνδεδεμένα με το κρυσταλλικό πλέγμα των ορυκτών των ιζημάτων) και ένα μικρό μέρος μπορεί να διαφύγει και να προκαλέσει ρύπανση της υδάτινης στήλης. Οι πηγές ρύπανσης των θέσεων ενδιαφέροντος είναι ο Βιολογικός Καθαρισμός του Δήμου Μεσολογγίου, οι ανεξέλεγκτες χωματερές που γειτνιάζουν με την λιμνοθάλασσα, η πόλη του Μεσολογγίου (κυρίως στο βόρειο τμήμα της Δυτικής Κλεισοβας) και τέλος οι διάφορες ανθρωπογενείς δραστηριότητες της περιοχής απόβλητα των οποίων ρίχνονται στα ρέματα και μέσω αυτών καταλήγουν στην λιμνοθάλασσα. / The aim of this thesis was the assessment of environmental and hydro geologic conditions of Mesolonghi’s area, the assessment of the quality of water and sediments in the areas in question regarding their medicinal natural resources and finally the estimation of possible sources of pollution. According the results of that thesis all groundwater samples present an increased salinity (increased rates of Electrical conductivity). Due to their of no importance pollution load it is believed that groundwater do not affect areas in question. On the contrary the surface samples of water and sediments collected from flumes shows that this water which ends up in Kleisova lagoon may affect the areas mostly with heavy metals. Great part of heavy metals concentration found in sediments does not threaten areas as ectypal source of pollution. Results from sequential extraction procedure showed that heavy metals are being highly associated with minerals crystalline lattice of sediments. Sources of pollution at areas in question are the wastewater treatment plant of Mesolonghi’s Municipality, many incontrollable refuse dumps which are neighboring on Kleisova lagoon, the Mesolonghi’s city and finally the surface water of flumes which ends up in lagoon and carries an high amount of heavy metals.

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Made available in DSpace on 2016-04-12T12:32:35Z (GMT). No. of bitstreams: 2 596.pdf: 1353914 bytes, checksum: bee2473adf5a37f943c4f715f4043519 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / A Leucemia Mielóide Crônica (LMC) é uma neoplasia mieloproliferativa crônica, cuja incidência é de 1,5 casos por 100.000 habitantes ao ano, com predomínio do gênero masculino. A predisposição hereditária parece não existir e o único fator bem caracterizado de risco é a exposição à radiação ionizante. O geoprocessamento é uma ferramenta informacional de auxílio aos profissionais e estudiosos da área da saúde capaz de mapear as doenças e permitir a análise de riscos sócio-ambientais. O objetivo deste estudo foi caracterizar a distribuição dos casos de LMC e de exposição a tóxicos industriais dos pacientes residentes no estado de Pernambuco com registro no Hospital Hemope nos últimos cinco anos. O estudo tipo série de casos incluiu todos os pacientes com diagnóstico de LMC, idade igual ou superior a 18 anos, de ambos os gêneros, atendidos no período de janeiro de 2004 a dezembro de 2009 e procedentes das diversas regiões de desenvolvimento do Estado. Para coleta e análise dos dados foram utilizados os registros dos prontuários de saúde e os aplicativos Excel 2003 e Terra View. No período do estudo foram diagnosticados 201 casos de LMC, com mediana de idade de 49 anos (extremos 18 a 93) e maior pico de incidência entre os 40 e 50 anos (24 por cento). Houve predomínio do sexo masculino (1,1:1) e de residentes de áreas urbanas (86 por cento). A incidência da LMC para as 12 regiões de desenvolvimento do Estado variou entre 0,5 e 3,8 casos por 100.000 habitantes/ano com média de 1,8 casos. As Regiões do Agreste Meridional e Sertão do Pajéu apresentaram maior incidência de casos de LMC, no entanto, não houve variação significante no número de casos ao longo dos anos e nem foram identificados determinantes sócio-ambientais relevantes associados. Apenas 10 por cento dos prontuários apresentaram registro sobre contato do pacientes com tóxicos industriais. A taxa de incidência da LMC encontrou-se dentro da frequência esperada, porém, com variações entre os municípios e as diferentes regiões de desenvolvimento do Estado. O estudo por geoprocessamento auxiliou na análise da distribuição espacial dos casos para uso dos dados como estudo de linha de base. A interface entre o profissional e o serviço de saúde pública é essencial para reconhecer e responder as preocupações com mudanças no perfil epidemiológico de doenças crônicas, frente aos novos cenários de desenvolvimento econômico

Des lésions de l’ADN sont impliquées dans les mécanismes de l’oncogenèse. De plus, la prolifération incontrôlée des cellules tumorales induit l’accumulation d’aberrations géniques. En réponse à ce stress génotoxique, les cellules en transformation expriment les ligands NKG2D MICA et MICB, molécules du CMH de classe I non conventionnelles qui activent une réponse cytotoxique T et NK contre cette transformation. Dans les syndromes lymphoprolifératifs chroniques, les mécanismes de la leucémogenèse reposent essentiellement sur une stimulation antigénique ou une activation des voies du récepteur à l’antigène (BCR) qui induit la prolifération cellulaire. De plus, les ligands MICA/B ne sont pas retrouvés à la surface de ces cellules. Les objectifs de cette thèse sont (i) rechercher si l’activation de la prolifération lymphocytaire peut induire l’expression de MICA/B et (ii) étudier les mécanismes induisant cette expression et leurs liens avec les voies de lésions/réparations de l’ADN. Pour cela, nous avons mis en place des conditions d’activation du récepteur à l’antigène permettant d’obtenir une prolifération (objectivée après marquage par CFSE) de lymphocytes B sains et de lymphocytes issus de patients porteurs de leucémie lymphoïde chronique (LLC), la plus fréquente des leucémie de l’adulte. L’expression des ligands MICA et MICB a ensuite été évaluée par qPCR, cytométrie en flux, western blots et ELISA. L’implication des différentes voies de signalisation en aval du récepteur à l’antigène a été analysée, ainsi que la cinétique d’apparition des lésions de l’ADN durant ce processus. Mes résultats montrent que MICA/B ne sont pas exprimés à la surface des lymphocytes B issus de donneurs sains ou de patients porteurs de LLC. Cependant, l’activation de la prolifération lymphocytaire induit une activation transcriptionnnelle de MICA ainsi que son expression à la surface de ces cellules. Cette expression est induite par différentes voies du récepteur à l’antigène ainsi que par la voie JAK/STAT et est indépendante des lésions de l’ADN qui surviennent plus tardivement dans la cellule. Au total, l’activation du récepteur à l’antigène qui induit la prolifération lymphocytaire induit également l’expression du ligand MICA (et non MICB) à la surface des lymphocytes sains et cette capacité d’expression est conservée dans les cellules de LLC qui ne l’expriment pas. Ces résultats suggèrent que MICA pourrait jouer un rôle crucial aux stades précoces de l’immunité anti-proliférative, ce qui ouvre la voie à de potentielles applications thérapeutiques. / Tumor cell’s uncontrolled proliferation induces an accumulation of genetic aberrations. In response to this genotoxic stress, most cells in transformation express NKG2D ligands (not expressed on resting cells), including MICA and MICB, which are non-conventional MHC class I molecules that could induce a cytotoxic T and NK response against the transformed cell. In chronic lymphoproliferative conditions, leukemogenic mechanisms rely in part on antigenic stimulations and/or activation of the B cell antigen receptor (BCR) pathways that induce cell proliferation. My thesis aims at studying : (i) the induction of MICA/B expression during lymphocyte proliferation and (ii) the mechanisms inducing this expression and their relationship with the DNA damage/repair pathways.I did generate BCR activation conditions to obtain B cells proliferation from healthy control individuals and from patients suffreing from chronic lymphocytic leukemia (CLL), the most common leukemia in adults. MICA and MICB expression was assessed by quantitative PCR, flow cytometry, Western blotting and ELISA after activation of B-cell proliferation. The different signaling pathways downstream BCR were analyzed, as were the kinetics of the DNA damage during this process. The results show that MICA/B aren’t expressed on cell surface of B cells from healthy control individuals or CLL patients before activation. Lymphoproliferative stimulation however up-regulates both MICA mRNA and surface protein in these same cells. This expression was induced by several BCR and by JAK/STAT pathways and seems to be indpendant of DNA damage. In conclusion, antigen receptor activation that induces lymphocyte proliferation also induces MICA expression (but not MICB) on B cells surface from healthy control individuals and this expression capacity is conserved in B cells from patients suffering from CLL. These results suggest that MICA may play a crucial role in the early stages of anti-proliferative immunity, which opens the avenue for therapeutic interventions.

1 Abstract BCR/ABL is a constitutively active tyrosine kinase that has been shown to be at the heart of the development of chronic myeloid leukemia (CML) and about 30% of acute lymphoblastic leukemia (ALL). With the recent advent of tyrosine kinase inhibitors (TKIs), exemplified by Imatinib, Nilotinib, Dasatinib and Bosutinib, patients with Ph+ CML or ALL are candidates for the therapy with these agents. From the available TKIs, Imatinib is considered as front-line therapy for CML patients in chronic phase, while for Ph+ ALL patients, 2nd generation TKIs (nilotinib, dasatinib, bosutinib) might be considered as more effective therapeutic option. Since the treatment with TKIs is a long-term affair, a substantial proportion of patients acquire some sort of mutation in kinase domain of BCR- ABL, which could be a reason of treatment failure. To date, over ninety BCR/ABL kinase domain mutations have been identified, affecting over 50 amino acids. Recurrent BCR/ABL kinase domain mutations have already been in vitro tested to approximate for their in vivo behavior. Our goal is to invent in vitro technique that would allow testing TKI sensitivity of novel BCR/ABL kinase domain mutations, identified at very low MRD levels. The technique makes use of site-directed mutagenesis to create the novel BCR/ABL kinase domain...

The presence of the translocation t(9;22)(q34;q11), leading to the BCR::ABL1 fusion transcript, is the hallmark of chronic myeloid leukemia (CML). Nevertheless, atypical presentation at diagnosis can be challenging. However, although most patients with CML are diagnosed with the e13a2 or e14a2 BCR:: ABL1 fusion transcripts, about 5% of them carry rare BCR::ABL1 fusion transcripts, such as e19a2, e8a2, e13a3, e14a3, e1a3, and e6a2. In particular, the e6a2 fusion transcript has been associated with clinically aggressive disease frequently presenting in accelerated or blast crisis phases. To date, there is limited evidence on the efficacy of front-line second-generation tyrosine kinase inhibitors for this genotype. Here, we report two patients, in whom the diagnosis of CML was challenging. The use of primers recognizing more distant exons from the common BCR::ABL1 breakpoint region correctly identified the atypical BCR::ABL1 e6a2 fusion transcript. Treatment with the second-generation tyrosine kinase inhibitor nilotinib was effective in our patient expressing the atypical e6a2 BCR::ABL1 fusion transcript.