In chronic myelogenous leukemia (CML), activation of BCR-ABL, the product of the bcr-abl chimeric gene, leads to constitutive activation of pathways that increase genomic instability through endogenous production of reactive oxygen species (ROS) that cause oxidative DNA damage and inactivate the function of repair proteins leading to unfaithful DNA repair. If misrepaired, oxidative DNA damage, such as 8-oxoguanine (8-oxoG), may result in point mutations and/or DNA double-strand breaks (DSBs) leading to drug resistance to the BCR-ABL kinase inhibitor imatinib mesylate (IM) and accumulation of chromosomal aberrations associated with malignant CML progression from a benign chronic phase (CP) to a fatal blast phase (BP). To determine which population of CML-CP cells, leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs), displays elevated levels of ROS and oxidative DNA damage, and whether these elevated levels of ROS and oxidative DNA damage in CML-CP subpopulations result in the accumulation of genomic instability, we employed the tetracycline-inducible SCLtTA/BCR-ABL transgenic mouse model. We showed that LSCs, including the quiescent subpopulation, but not LPCs, displayed elevated levels of ROS and oxidative DNA damage, perhaps due to deregulated expression of genes involved in ROS metabolism, resulting in genomic instability manifested by both point mutations and genetic alterations. We also examined the effect of IM on ROS, oxidative DNA damage and genomic instability displayed by CML-CP subpopulations, and determined that elevated ROS and oxidative DNA damage were not inhibited by IM in quiescent LSCs, nor was genomic instability and deregulated gene expression prevented. To explore underlying mechanisms, i.e. BCR-ABL expression levels, by which CML-CP cells accumulate genomic instability, we examined the effect of low and high BCR-ABL expression on ROS and oxidative DNA damage in BCR-ABL-transduced human CD34+ cells. We detected elevated ROS and oxidative DNA damage in high BCR-ABL-expressing CD34+ cells compared to low BCR-ABL-expressing cells. Furthermore, BCR-ABL exerted a kinase-dependent effect on ROS-dependent DNA damage. These data support the hypothesis that genomic instability may originate from LSCs, but do not exclude the potential role of LPCs, and may have important clinical implications for CML treatment since additional genetic aberrations that encode primary resistance may protect LSCs, including the quiescent subpopulation, from eradication by tyrosine kinase inhibitors (TKIs), and the continuous accumulation of genetic errors may trigger disease relapse and progression. / Microbiology and Immunology

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa e sua patogênese está associada à expressão de um neogene, bcr-abl, que codifica uma proteína tirosina quinase Bcr-Abl. Esse trabalho tem como objetivos o estudo dos mecanismos envolvidos na resistência à morte das células Bcr-Abl positivas e a identificação de alterações gênicas nessas células. Dados de expressão gênica global obtidos por \"microarray\" mostraram uma superexpressão nas células HL-60.Bcr-Abl com relação a HL-60 dos genes faim e nipa, que foi confirmada por qRT-PCR em diferentes linhagens celulares Bcr-Abl positivas. Já os genes de trail e osm, apresentaram uma diminuição significativa em HL-60.Bcr-Abl, que foi confirmada para trail, porém osm não teve seu resultado validado. A avaliação da expressão dos genes em células de pacientes portadores de LMC, em diferentes fases da doença também foi estudada. Com esses resultados, o presente estudo visa a melhor compreensão de como alterações na expressão desses genes contribuem na fisiopatologia da LMC. / Chronic myelogenous leukemia (CML) is a stem cell disease characterized by the presence of the Bcr-Abl oncoprotein, which is the cause of the malignant transformation and the extreme resistance to apoptosis displayed by CML patients. Our aim was to analyze the alteration in global gene expression in Bcr-Abl expressing cells. Data obtained from microarray analysis showed significant up-regulation of nipa and faim in HL60.Bcr-Abl and down-regulation of osm and trail. These results were further confirmed by Real-Time PCR to nipa, faim and trail, but not for osm expression in HL-60.Bcr-Abl cells. To evaluate the potential of some of the modified genes as therapeutic targets or prognostic markers for CML, we also analyzed the expression of these genes in samples from CML patients.

Chronic Myelogenous Leukemia (CML) is a blood cancer that arises when hematopoietic cells acquire an abnormal protein known as BCR-ABL. Current therapies for CML include drugs that inhibit BCR-ABL. However, these drugs only suppress the disease and do not cure it. One reason is that BCR-ABL drugs fail to kill the primitive population of CML cells, referred to as leukemia stem cells (LSCs), which are responsible for initiating and propagating CML. Since LSCs are not killed, the cancer is not cured and many affected patients eventually relapse. Recent studies suggest that LSCs are protected from current therapies by the bone marrow micro-environment where they reside. There, cytokine signaling molecules are present, which mediate processes that protect LSCs from BCR-ABL drugs. The stem cell factor (SCF) is one of these signaling molecules. It activates the receptor c-Kit located on the surface of LSCs, and this activation in turn allows proliferating LSCs to resist BCR-ABL drugs, even without prior exposure to these drugs, i.e., innate resistance is observed. In this thesis, the mechanism of this innate resistance is investigated, so that a suitable treatment strategy can be developed. To this end, a co-agent approach based on synthetic antibodies (sABs) is proposed to inhibit the receptor c-Kit, with the goal of disrupting its activation by the ligand SCF. This disruption should in turn block the SCF-mediated innate resistance, thus potentially restoring BCR-ABL drug apoptotic activity. The method for this disruption involves targeting the c-Kit structural susceptibility. Specifically, the sABs are designed via antibody phage display technology to target the D1–D2–D3 domains representing the SCF binding sites, hence preventing downstream pathway activation. The hypothesis is that, by blocking the SCF-mediated innate resistance, a suitable combination of such an sAB co-agent and a BCR-ABL drug should be conducive to suppressing LSCs, thereby providing a potential means to improve CML treatment. In addition, to assess the performance of the proposed treatment strategy, a set of in vitro tests is conducted, focusing on performance behaviors such as cell binding, cell death, and the progenitor inhibition. The experimental results support the hypothesis that the proposed combinatorial strategy is indeed a promising approach to mitigate the innate resistance, thus restoring BCR-ABL drug apoptotic activity.

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa e sua patogênese está associada à expressão de um neogene, bcr-abl, que codifica uma proteína tirosina quinase Bcr-Abl. Esse trabalho tem como objetivos o estudo dos mecanismos envolvidos na resistência à morte das células Bcr-Abl positivas e a identificação de alterações gênicas nessas células. Dados de expressão gênica global obtidos por \"microarray\" mostraram uma superexpressão nas células HL-60.Bcr-Abl com relação a HL-60 dos genes faim e nipa, que foi confirmada por qRT-PCR em diferentes linhagens celulares Bcr-Abl positivas. Já os genes de trail e osm, apresentaram uma diminuição significativa em HL-60.Bcr-Abl, que foi confirmada para trail, porém osm não teve seu resultado validado. A avaliação da expressão dos genes em células de pacientes portadores de LMC, em diferentes fases da doença também foi estudada. Com esses resultados, o presente estudo visa a melhor compreensão de como alterações na expressão desses genes contribuem na fisiopatologia da LMC. / Chronic myelogenous leukemia (CML) is a stem cell disease characterized by the presence of the Bcr-Abl oncoprotein, which is the cause of the malignant transformation and the extreme resistance to apoptosis displayed by CML patients. Our aim was to analyze the alteration in global gene expression in Bcr-Abl expressing cells. Data obtained from microarray analysis showed significant up-regulation of nipa and faim in HL60.Bcr-Abl and down-regulation of osm and trail. These results were further confirmed by Real-Time PCR to nipa, faim and trail, but not for osm expression in HL-60.Bcr-Abl cells. To evaluate the potential of some of the modified genes as therapeutic targets or prognostic markers for CML, we also analyzed the expression of these genes in samples from CML patients.

L’existance de cellules souches leucémiques (CSL) dans la Leucémie Myéloïde Chronique (LMC) prédit que que seule la destruction des CSL conduirait à une guérison. Une proportion importante de patients atteints de LMC développe une résistance aux drogues, environ ~ 30% des cas, Les mécanismes de résistance aux inhibiteurs de tyrosine kinase (TKI) dans la leucémie myéloïde chronique (LMC) restent souvent obscures. Les cellules souches leucémiques de la LMC pourraient rester viables et en repos, malgré la présence de facteurs de croissance ou de médicaments qui semblent les protéger de l'apoptose. Nous avons montré dans la première parti de cette étude que certains transporteurs telles ABCG2, hOCT-1 pourraient jouer un rôle avec le micro environnement dans la résistance des lignées LMC en adhésion au stoma médullaire. De plus, dans deuxième parti nous avons montré que le gène TWIST-1 (est un acteur clé de l'embryogenèse) est déréglementé dans les cellules de LMC innée des résistants à l'imatinib, et que la sur-expression de l’oncogène TWIST-1 pourrait représenter un nouveau facteur clé de pronostiques potentiellement utiles pour améliorer la guérison de LMC aux TKI. De plus, nous avons pu également montrer que le gène TP73 est impliqué dans la résistance des CSL de LMC. Ce gène pourrait être un facteur prédictif pour identifier une résistance potentielle des patients de LMC au moment du diagnostic. Nous avons montré également que ce gène est régulé par le micro-environnement. Nous avons montrés une sur-expression des isoformes tronquées dans les lignées LMC avec l’adhésion au stroma. Les résultats suggèrent que les molécules intrinsèques comme TWIST-1, les transporteurs et les isoformes de p73 sont dérégulées dans les CSL par des mécanismes extrinsèques qui interviennent avec le micro environnement leucémique par le mécanisme d’adhésion dans le phénomène de résistance aux traitements. Ce mécanisme spécial de résistance due à la conservation de ces CSL dans l’état immature en adhésion avec son micro environnement. / The existence of Leukemia stem cells (CSL) in chronic myelogenous leukemia (CML) predicts that only the destruction of CSL lead to a cure. A significant proportion of CML patients develop resistance to drugs, ~ 30% cases, mechanisms of resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) often remain obscure. Leukemic stem cells of CML could remain viable and quiet, despite the presence of growth factors or drugs that seem to protect them from apoptosis. We have shown in the first part of this study that some carriers such as ABCG2, hOCT could to be play a role with the microenvironment in the resistance among CML adhesion of stoma Bone marrow. Furthermore, in the second part we showed that the gene TWIST-1 (is a key player of the embryogenesis) is deregulated in cells of CML innately resistant to imatinib, and that overexpression of the oncogene TWIST-1 could represent a new prognostic factor key potentially useful for improving the querison CML to TKI. In addition, we also could show that the TP73 gene is involved in the resistance of CSL CML. This gene could be a predictor to identify potential resistance of CML patients at diagnosis. We have also shown that this gene is regulated by the microenvironment. We have shown an overexpression of truncated isoforms in CML cell lines with the accession to the stroma. The results suggest that intrinsic molecules such as TWIST-1 carriers and p73 isoforms are deregulated in CSL by extrinsic mechanisms involved with the leukemia microenvironment by the mechanism for participation in the phenomenon of drug resistance. This mechanism with its microenvironment.

Une des principales causes d’échec dans le traitement des cancers est le développement de résistances aux drogues par les cellules tumorales. Les cellules souches cancéreuses (CSC) sont suspectées d’être responsables de ces rechutes, conduisant à la récurrence de la maladie et bien souvent au décès des patients. En clinique, il est donc nécessaire de développer des stratégies thérapeutiques capables de cibler ces CSC résistantes et aboutir à la guérison des patients. Les CSC sont régulées par un ensemble de signaux aussi bien biologiques que physiques au sein de la niche tumorale. Mon projet a pour objectif de déterminer l’implication du microenvironnement tumoral (voie de signalisation BMP et contraintes mécaniques) dans le maintien et la résistance des cellules souches leucémiques (CSLs) de la leucémie myéloïde chronique (LMC). Pour cela, nous avons combiné tests fonctionnels et moléculaires ainsi que l’analyse de la niche tumorale sur plus de 200 échantillons de patients atteints de LMC. Nous avons ainsi démontré que l’altération de la voie BMP intrinsèque aux cellules immatures de la LMC corrompt et amplifie la réponse à BMP2 et BMP4, présents en quantités anormalement abondantes au sein de la niche tumorale. Ces résultats récemment publiés dans Blood nous ont amenés à évaluer le rôle de la voie BMP dans le maintien des CSLs sous traitement par les ITK. La microscopie à force atomique nous a permis de démontrer que l’expression de BCR-ABL est suffisante pour induire une augmentation de la rigidité des cellules immatures de LMC par rapport à des cellules saines. Enfin, l’utilisation d’un système de confinement cellulaire nous a permis de démontrer que le stress mécanique contrôle la prolifération des cellules leucémiques immatures en régulant l’expression de gènes mécano-sensibles comme Twist-1. Ces résultats pourraient expliquer comment des CSLs tirent profit des contraintes mécaniques issues de leur microenvironnement afin d’acquérir un avantage prolifératif par rapport aux cellules saines. Ultimement, nous espérons que cette approche transdisciplinaire permettra d’identifier les molécules clés de la transduction de signaux mécaniques potentiellement impliqués dans le maintien et la résistance des CSC et ainsi proposer de nouvelles cibles pour contrer ces effets. / One of the main causes of treatment failure in cancers is the development of drug resistance by cancer cells. The persistence of cancer stem cells (CSCs) might explain cancer relapses as they could allow reactivation of cancer cells proliferation following therapy, leading to disease persistence and ultimately to patients’ death. Clinically, it is crucial to develop therapeutic strategies able to target resistant CSCs in order to cure the patients. CSCs are controlled by a variety of biochemical and biomechanical signals from the leukemic niche. My project aims to determine the involvement of the tumor microenvironment (BMP signaling pathway and mechanical stress) in the maintenance and resistance of Leukemic Stem Cells (LSCs) in Chronic Myelogenous Leukemia (CML). For this, we combined functional and molecular assays to the analysis of tumor microenvironment on more than 200 CML patients’ samples. We demonstrated that alterations of intracellular BMP signaling pathway in CP-CML primary samples corrupt and amplify the response to exogenous BMP2 and BMP4, which are abnormally abundant in the tumor microenvironment. These results, recently published in Blood led us to evaluate the role of the BMP pathway in LSC maintenance under TKI treatment. Atomic force microscopy allowed us to demonstrate that BCR-ABL expression alone is sufficient to increases the rigidity of immature CML cells compared to healthy ones. Finally, using a unique cell confining system, we were able to demonstrate that mechanical stress controls the proliferation of immature leukemic cells by regulating the expression of mechano-sensitive genes such as Twist-1. These results could explain how LSCs can benefit from a mechanical stress exerted by their microenvironment to acquire a proliferative advantage over normal cells. Ultimately, we hope that this transdisciplinary approach will help to identify key molecules in the transduction of mechanical signals potentially involved in maintenance and resistance of CSCs and thus offer new targets to counter these effects.