Submitted by admin tede (tede@pucgoias.edu.br) on 2016-11-22T18:16:35Z No. of bitstreams: 1 LUCIANA CARDOSO MARINHO.pdf: 2007688 bytes, checksum: 7b1d1ab427984f805bae729662ab0296 (MD5) / Made available in DSpace on 2016-11-22T18:16:35Z (GMT). No. of bitstreams: 1 LUCIANA CARDOSO MARINHO.pdf: 2007688 bytes, checksum: 7b1d1ab427984f805bae729662ab0296 (MD5) Previous issue date: 2016-08-10 / The latest research has shown that the angiogenic factors play an important role in the modulation of tumor angiogenesis process. Thus, this study aimed to analyze the influence of this process on the development of leukemias. a systematic review based on scientific articles that investigated the interference of angiogenic factors in cases of leukemia early and advanced stages was performed. The major evidence indicates that these factors, in special VEGF, are expressed intensely in malignant cells and may generate, for example, a hematologic malignancy. The regulation of expression of certain genes is also involved in abnormal proliferation, differentiation and apoptosis of these cells. The expression, function and regulation mechanism of angiogenic factors have been gradually elucidated in this review and in conclusion, it is noted that some of them have relation to the development and leukemias prognosis, and provide potential strategies for the treatment of patients with leukemia. / As últimas pesquisas demonstram que os fatores angiogênicos desempenham um importante papel na modulação do processo da angiogênese tumoral. Neste sentido, o presente estudo tem como objetivo a análise da influência deste processo no desenvolvimento de leucemias. Foi realizada uma revisão sistemática com base em artigos científicos que investigaram a interferência de fatores angiogênicos em casos de leucemias em estágios iniciais e avançados. As principais evidências indicam que estes fatores, em especial o VEGF, são expressos de forma intensa em células malignas e podem gerar, por exemplo, uma malignidade hematológica. A regulação de expressão de determinados genes também está envolvida na proliferação anormal, na diferenciação e apoptose destas células. A expressão, função e o mecanismo de regulação dos fatores angiogênicos foram elucidados gradualmente nesta revisão e como conclusão, salienta-se que alguns deles apresentam relação com o desenvolvimento e prognóstico de leucemias, além de fornecer possíveis estratégias para o tratamento de pacientes com leucemia.

Les Leucémies Aigues Myéloïdes (LAM) sont des hémopathies au pronostic sombre. A l’exception des Leucémies Aigues Promyélocytaires (LAP), un sous type minoritaire de LAM, la plupart des patients sont traités par une chimiothérapie d’induction composée de la cytarabine et d’une anthracycline. Cependant, une fraction importante (20-30%) des patients ne répond pas à la chimiothérapie d’induction et les taux de rechutes sont très élevés.Il n’existe actuellement pas d’outils disponibles au diagnostic permettant de prédire la réponse des patients aux chimiothérapies. De tels tests permettraient d’adapter les doses utilisées pour augmenter le taux de réponse et limiter la toxicité des chimiothérapies, très importante chez les personnes âgées.Les protéines de la famille de l’Ubiquitine (UbL), dont l’ubiquitine, SUMO (-1,-2,-3) et Nedd8 sont les membres les plus étudiés, sont des modificateurs post-traductionnels peptidiques conjugués de façon covalente sur des milliers de protéines. Elles sont impliquées dans la plupart des fonctions cellulaires et dérégulées dans de nombreuses pathologies. De nombreux travaux suggèrent que leur dérégulation est associée à la chimiorésistance des LAM.La première partie de nos travaux a consisté à identifier un ensemble de protéines dont le niveau de modification par l’ubiquitine, SUMO et NEDD8 pourrait constituer des biomarqueurs de la réponse des LAM aux drogues chimiothérapeutiques. Nous avons de plus développé un test permettant d’évaluer leur niveau de modification par des extraits de cellules de patients de manière simple et rapide, utilisable en clinique au diagnostic.Le ciblage des voies Ubiquitine, SUMO et Nedd8 est en train d’apparaître comme une stratégie thérapeutique prometteuse dans les cancers et plusieurs molécules ciblant ces voies ont été récemment décrites. Le développement clinique de ces molécules nécessite la mise au point de tests compagnons pour suivre leur efficacité sur leurs cibles moléculaires. Nous avons ainsi développé un test d’activité permettant de mesurer de façon simple et quantitative l’efficacité des molécules ciblant les modificateurs de la famille de l’ubiquitine,Enfin, compte tenu du potentiel thérapeutique du ciblage de la SUMOylation, nous avons débuté le développement d’un inhibiteur de la SUMOylation basé sur l’inhibition de l’interaction entre les enzymes E1 et E2 par des peptides agrafés. / Acute Myeloid Leukemias (AML) are severe haematological malignancies with a poor prognosis. Except Acute Promyelocytic Leukemia (APL), a minor subtype of AML, most patients receive an induction chemotherapy consisting of cytarabine and an anthracycline. However, a large number (20-30%) of patients do not respond to this induction chemotherapy, and relapses are frequent.There are currently no tools available at diagnosis to predict patient response to chemotherapy. Such tests would make it possible to adapt the doses used to increase the response rate and limit the toxicity of chemotherapies, which is significant in older patients.Ubiquitin-like modifiers (UbL), among which ubiquitin, SUMO (-1, -2, -3) and Nedd8 are the most studied members, are post-translational modifiers covalently conjugated to thousands of proteins. They are involved in most cellular pathways, and deregulated in several pathologies. Many studies suggest that their deregulation is associated with LAM chemoresistance.The first part of our work consisted in identifying a set of proteins whose level of modification by ubiquitin, SUMO and NEDD8 could constitute biomarkers of AMLs response to chemotherapeutic drugs. We have also developed a test to evaluate their level of modification by extracts of patient cells in a simple and rapid manner, usable in clinical diagnosis.Targeting of the Ubiquitin, SUMO and Nedd8 pathways is emerging as a promising therapeutic strategy in cancers and several molecules targeting these pathways have recently been described. The clinical development of these molecules requires the development of companion tests to monitor their efficacy on their molecular targets. We have developed an activity test to measure in a simple and quantitative way the efficacy of molecules targeting ubiquitin-like modifiers.Finally, given the therapeutic potential of targeting SUMOylation, we have started the development of a SUMOylation inhibitor based on the inhibition of the interaction between E1 and E2 enzymes by stapled peptides.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-17T19:27:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ShayenneSchefferHomrich.pdf: 1706469 bytes, checksum: 48ab5f162c893018778d1ac573ca031d (MD5) / Made available in DSpace on 2018-08-17T19:27:03Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ShayenneSchefferHomrich.pdf: 1706469 bytes, checksum: 48ab5f162c893018778d1ac573ca031d (MD5) Previous issue date: 2017-03-28 / The Acute Promyelocytic Leukemia (APL), firstly described in 1957, is the most malignant type of acute leukemia. Currently, the knowledge of its pathophysiological mechanism at molecular levels became possible the development of efficient therapies making APL the most curable leukemia type. In this sense, APL is also used as a model on cancer advances, being the molecular treatment performed with all trans-retinoic acid (ATRA) that presents high efficiency at its control. However, several patients develop differentiation syndrome, as a side effect of this drug. In these terms, compared to another drugs, the cytotoxic antineoplastic agents present particular problems, as poor specificity, high toxicity and resistance susceptibility. An alternative strategy to decrease the ATRA cytotoxicity is the incorporation of this drug in polymer nanocapsules with oily core. In this work nanocapsules with lipid core containing ATRA (NA) were evaluated as their potential to inhibit cellular grow, to induce apoptosis, to interfere the cell cycle, and at APL cellular differentiation using NB4 cell line. Results showed that NA was able to overcome the cellular resistance to AL treatment, decreasing cell viability, inducing apoptosis, through BAX/BCL-2 gene expression, cell cycle arresting at G1 phase and cellular differentiation under 1.5 and 2.0 μM. Additionally, theses systems can contribute to increase the efficacy and reduce the toxicity due to the potential accumulation of nanoparticles at the tumor region due to increased vascular permeability of tumor vases. The ATRA incorporation in lipid nanocarriers is a interesting alternative to make possible its intravenous administration. Moreover, these systems present potential to drug accumulation at tumor tissue through a passive targeting called effect of permeability and increased retention. / A Leucemia Promielocítica Aguda (LPA), primeiramente descrita em 1957 é a forma mais maligna de leucemia aguda. Atualmente, o conhecimento dos seus mecanismos fisiopatológicos em nível molecular possibilitou o desenvolvimento de terapias eficazes fazendo com que a LPA seja a forma mais curável de leucemia. Por este motivo a LPA é também utilizada como modelo para os avanços no tratamento do câncer, sendo o tratamento molecular com ácido all-trans-retinoico (ATRA) o que apresenta alta eficiência no seu controle. Entretanto, inúmeros pacientes adquirem a Síndrome de Diferenciação, como efeito adverso a este medicamento. Diante disto, em comparação com outros fármacos, os antineoplásicos citotóxicos apresentam problemas únicos, como pobre especificidade, elevada toxicidade e susceptibilidade para induzir resistência. Uma estratégia para diminuir a citotoxicidade do ATRA é a incorporação do mesmo em nanocápsulas poliméricas com núcleo oleoso. Neste trabalho nanocápsulas de núcleo lipídico contendo ATRA (NA) foram avaliadas quanto ao seu potencial de inibir o crescimento, induzir a apoptose e interferir com o ciclo celular e na diferenciação de células de LPA, linhagem NB4. Os resultados demonstraram que a NA foi capaz de superar a resistência celular ao tratamento com AL, reduzindo a viabilidade celular, induzindo apoptose, pela expressão dos genes BAX/BCL-2, parada do ciclo celular em G1 e diferenciação celular nas concentrações de 1,5 e 2,0 μM. Adicionalmente, estes sistemas podem contribuir para o aumento da eficácia e redução da toxicidade devido ao potencial para acúmulo das nanopartículas na região tumoral graças à permeabilidade vascular aumentada dos vasos tumorais. A incorporação de ATRA em nanocarreadores lipídicos constitui uma alternativa interessante para viabilizar sua administração intravenosa. Além disso, estes sistemas apresentam potencial para acúmulo do fármaco na região tumoral, por meio de um direcionamento passivo chamado de efeito de permeabilidade e retenção aumentada.

Un défi de la transcriptomique est d'explorer l'intégralité du répertoire des transcrits normaux et anormaux. Les analyses de GEP (Gene Expression Profiling) basées sur la technologie des puces à ADN sont largement exploitées en cancérologie depuis plusieurs années. Parallèlement, les nouvelles méthodes basées sur le séquençage à haut débit offrent désormais la possibilité de réaliser des analyses précises et sensibles nécessaires à l'étude des cellules normales et cancéreuses. Quelle que soit la méthode, la caractérisation de l'ensemble des transcrits codants et non-codants représente un réel défi biologique pour la recherche de nouveaux marqueurs de diagnostic et de pronostic, et pour la bonne prise en charge des patients. Dans ce travail, j'ai eu l'occasion de traiter deux aspects différents de la biologie qui convergent vers l'identification de transcrits exprimés de manière aberrante dans les leucémies myéloïdes. Le premier aspect a consisté à proposer une sélection de biomarqueurs moléculaires pour la caractérisation de la leucémie myélomonocytaire chronique (LMMC). A partir de l'expression de ces gènes, nous avons développé un score de pronostic qui a permis de définir deux groupes de patients cliniquement distincts. Nous avons ensuite complété notre étude par une caractérisation phénotypique par cytométrie en flux des sous-populations cellulaires aberrantes constituant les lignages mono- et granulocytaires. Une partie de ce travail a été étendue aux leucémies aiguës myéloïdes (LAM) à caryotype normal (CN). L'autre aspect a consisté à participer à la mise en place d'une approche computationnelle intégrée pour caractériser de nouveaux ARNs non annotés et fort probablement non-codants. En explorant des données de Digital Gene Expression (DGE), nous avons quantifié et caractérisé la fraction de ces transcrits dans les régions intergéniques. Nous avons vérifié l'expression de ces nouveaux transcrits dans les tissus normaux et cancéreux en croisant avec d'autres données d'expression, telles que le RNA-Sequencing, et regarder leur conservation et leur expression dans d'autres espèces. / A challenge of transcriptomics is to explore the full repertoire of normal and abnormal transcripts. Gene expression profiling analyses based on microarray technology are widely used in cancer research since many years. Meanwhile, new methods based on high-throughput sequencing methods offers henceforth the possibility to undergo accurate and sensitive analyses necessary for studying normal and cancer cells. Despite the method, the characterization of all coding and non-coding transcripts is a real biological challenge in identifying novel diagnostic and prognostic markers, and for the proper care of patients. In the present work, I had the opportunity to address two different aspects of biology, both convergent toward the identification of aberrantly expressed transcripts in myeloid leukemia. The first aspect was to provide a selection of molecular biomarkers for the characterization of chronic myelomonocytic leukemia (CMML). We developed a gene expression-based prognostic score which identified two clinically distinct groups of patients. We then completed our study with a phenotypic characterization by flow cytometry of aberrant subpopulations constituting the myeloid and granulocytic lineages. A part of this work has been extended to acute myeloid leukemia (AML) patients with normal karyotype. The other aspect was to participate in the implementation of an integrated computational approach in order to characterize novel non annotated RNAs, more likely non-coding. We quantified and characterized the proportion of these transcripts in intergenic regions by exploring Digital Gene Expression (DGE) data. We checked their expression in normal and cancer tissues by crossing with RNA-Seq data, and their conservation and expression in other species.

The availability of several methods to unambiguously mark individual cells has strongly fostered the understanding of clonal developments in hematopoiesis and other stem cell driven regenerative tissues. While cellular barcoding is the method of choice for experimental studies, patients that underwent gene therapy carry a unique insertional mark within the transplanted cells originating from the integration of the retroviral vector. Close monitoring of such patients allows accessing their clonal dynamics, however, the early detection of events that predict monoclonal conversion and potentially the onset of leukemia are beneficial for treatment. We developed a simple mathematical model of a self-stabilizing hematopoietic stem cell population to generate a wide range of possible clonal developments, reproducing typical, experimentally and clinically observed scenarios. We use the resulting model scenarios to suggest and test a set of statistical measures that should allow for an interpretation and classification of relevant clonal dynamics. Apart from the assessment of several established diversity indices we suggest a measure that quantifies the extension to which the increase in the size of one clone is attributed to the total loss in the size of all other clones. By evaluating the change in relative clone sizes between consecutive measurements, the suggested measure, referred to as maximum relative clonal expansion (mRCE), proves to be highly sensitive in the detection of rapidly expanding cell clones prior to their dominant manifestation. This predictive potential places the mRCE as a suitable means for the early recognition of leukemogenesis especially in gene therapy patients that are closely monitored. Our model based approach illustrates how simulation studies can actively support the design and evaluation of preclinical strategies for the analysis and risk evaluation of clonal developments.

Unsere Untersuchungen belegen, dass nichtmyeloablative Stammzelltransplantationen (NST) bei Patienten mit Hochrisiko-ALL oder -AML eine neue therapeutische Option darstellen. Die NST ermöglichte eine allogene Stammzelltransplantation bei Transplantationskandidaten mit Kontraindikationen gegen eine hochdosierte Strahlen- und Chemotherapie (Standardtransplantation). Nach NST kam es häufig zur Entwicklung von Infektionen und einer spät auftretenden akuten Transplantat-gegen-Wirt Reaktion (GvHD), diese waren aber mit einer geringeren Mortalität verbunden. Das erkrankungsfreie Überleben und das Gesamt-Überleben nach NST und nach Standardtransplantation waren fast gleich. Der höheren Rezidivrate nach NST stand eine höhere transplantationsassoziierte Mortalität nach Standardtransplantation gegenüber. Das Konditionierungsregime selber war ohne Einfluss auf das Überleben der Patienten. Sequenzielle Chimärismusuntersuchungen von Leukozytensubpopulationen erlaubten eine frühe Diagnose eines gemischten Chimärismus, der einen prädiktiven Wert für das Auftreten eines Rezidivs hatte. Ein stabiler gemischter Chimärismus wurde nicht beobachtet. Durch eine adoptive Immuntherapie mit Spenderlymphozyten (DLI) konnte bei der Mehrzahl der transplantierten Patienten mit gemischtem Chimärismus eine Chimärismuskonversion und eine lang anhaltende komplette Remission induziert werden, ein wichtiger Hinweis auf einen Transplantat-gegen-Leukämie Effekt (GvL) der Spenderzellen. Durch die NST rückt der immunologische Effekt der Transplantation gegenüber der Zytoreduktion bei der Standardtransplantation stärker in den Vordergrund. Unsere Ergebnisse sprechen für die Wirksamkeit eines GvL-Effektes bei akuten Leukämien auch nach NST. Die Erfahrungen mit der NST bei akuten Leukämien haben zu einer Anwendung bei refraktären Nierenzellkarzinomen geführt. Eine Tumorregression wurde erst nach Chimärismuskonversion und/oder Entwicklung einer GvHD beobachtet; diese Befunde sind vereinbar mit einem Transplantat-gegen-Tumor Effekt (GvT). Die transplantationsassoziierte Morbidität war allerdings bei diesen meist älteren Patienten erheblich. Die Therapie sollte ausschließlich im Rahmen klinischer Studien erfolgen, da es sich nach wie vor um ein experimentelles Therapieverfahren handelt. Die Analyse minimal residueller Erkrankung und der Einsatz hochauflösender Chimärismusuntersuchungen von Leukozytensubpopulationen sollte die Bedeutung des gemischten Chimärismus weiter klären und zu einem gezielteren Einsatz von DLI führen. Prospektive vergleichende Studien müssen in naher Zukunft den Stellenwert der NST untersuchen. / Our clinical investigations demonstrate, that nonmyeloablative stem cell transplantation (NST) is a novel therapeutic option in patients with high-risk ALL or AML. NST can be administered to patients eligible for allogeneic stem cell transplantation with contraindications against high-dose radio- and chemotherapy (standard SCT). After NST, infections and late onset acute graft-versus-host disease (GvHD) frequently occurred, but transplant-related mortality was low in contrast to standard SCT. Leukemia-free survival and overall survival were similar after NST and standard SCT. A higher relapse rate after NST was balanced by a higher transplant-related mortality after standard SCT. The conditioning regimen itself had no relevant impact on survival. Sequential chimerism analyses of leukocyte subpopulations resulted in early diagnosis of mixed chimerism, which proved to be predictive for later relapses. Stable mixed chimerism was not established in these patients. Adoptive immunotherapy in patients, who were in hematologic remission but had mixed chimerism after transplantation, induced conversion to complete donor chimerism and long-lasting complete remissions in the majority of patients, a strong hint to a graft-versus-leukemia-effect (GvL) of donor T-lymphocyte infusions. A change in the character of stem cell transplantation was achieved by NST with a shift from the predominantly cytoreductive effect of standard SCT towards an emphasis on the immunologic GvL-effect. Our results demonstrated the efficacy of a GvL-effect in acute leukemias after NST. The experiences with NST in acute leukemias have prompted studies in patients with refractory advanced renal cell cancer. Tumor regressions were observed only after chimerism conversion and / or development of GvHD, these results being compatible with a graft-versus-tumor effect (GvT). However, transplant-related morbidity was substantial in these mostly elderly patients. Therapy within clinical studies is mandatory, because allogeneic stem cell transplantation still has to be regarded an experimental procedure in these patients. The analysis of minimal residual disease and application of high-resolution chimerism analysis of leukocyte subpopulations by microarrays could lead to a more profound understanding of mixed chimerism and to a more rational use of DLI in the near future. Prospective randomized trials should be conducted to evaluate the role of NST.

Le traitement des leucémies myéloïdes et lymphoblastiques aiguës a connu des avancées importantes dans la dernière décennie. Malgré ces progrès, le taux de rechutes reste élevé et le besoin médical est réel. Ces leucémies sont caractérisées par une expression aberrante d’antigènes qui peuvent provenir de protéines mutées, mais aussi de séquences rapportées comme non codantes. Les réponses contre ces néoantigènes tumoraux « cryptiques » demeurent non caractérisées. Afin de définir l’existence d’un répertoire varié de récepteurs des lymphocytes T (TCR) qui reconnaitraient ces néoantigènes, des cellules mononuclées du sang périphérique de patients sains sont isolées puis enrichies en cellules T CD8+ naïves. L’expansion et l’activation de ces cellules sont ensuite réalisées avec des cellules dendritiques autologues chargées avec l’antigène d’intérêt puis triées à l’aide de multimères HLA-peptides spécifiques. L’ARN des cellules avec TCR spécifiques aux antigènes spécifiques des tumeurs (TSA) leucémiques est isolé afin de réaliser un séquençage du TCR-bêta. L’expansion cellulaire a été réalisée de façon suffisante pour effectuer le séquençage des cellules identifiées comme positives par le marquage avec dextramères. Une réponse T est obtenue pour 50% des néoantigènes testés avec une réactivité montrée par ELISpot et se traduisant par une sécrétion de cytokines inflammatoires. Des lymphocytes T spécifiques aux TSA d’intérêt sont donc présents dans le sang périphérique de donneurs sains. Le séquençage de ces cellules a permis d’identifier les clonotypes pour lesquels une réponse anti-leucémique forte est possible. Il serait intéressant d’utiliser ces clonotypes spécifiques aux tumeurs cryptiques dans le développement de nouveaux traitements d’immunothérapie adoptive. / The treatment of acute myeloid and lymphoblastic leukemia has seen significant advances in the past decade. Despite this progress, the relapse rate remains high and the medical need is real. These leukemias are characterized by an aberrant expression of antigens, some from mutated proteins but also from sequences of DNA that were reported as non-coding. Responses against these “cryptic” neoantigens remains uncharacterized. In order to verify whether a diverse repertoire of T cell receptors (TCR) does recognize these neoantigens, mononuclear cells from peripheral blood of healthy patients are isolated and enriched with naive CD8+ T cells. The expansion and activation of these cells are then carried out with autologous dendritic cells loaded with the antigen of interest and then sorted using HLA-peptide specific multimers. RNA from cells with TCR specific for leukemic tumor-specific antigens (TSA) is isolated in order to perform TCR-beta sequencing. Cell expansion was sufficient to perform the sequencing of cells identified as positive by staining with dextramers. A T-cell response is obtained for 50% of the neoantigens tested with reactivity shown by ELISpot and resulting in a secretion of inflammatory cytokines. T lymphocytes specific to the TSA of interest are therefore present in the peripheral blood of healthy donors. Sequencing of these cells made it possible to identify clonotypes for which a strong anti-leukemic response can be expected. It would be interesting to use these cryptic tumor-specific clonotypes in the development of new adoptive immunotherapy treatments.

La leucémie aigüe myéloïde est un cancer hautement létal notamment parce que le taux de rechutes est élevé, ce qui traduit l’importance du développement de nouvelles thérapies. Ces dernières peuvent tirer avantage du fait que les cellules leucémiques peuvent exprimer des antigènes qui ne sont pas exprimés par les tissus sains, soit les antigènes spécifiques aux tumeurs (TSA). À cet effet, nos collaborateurs ont découvert une source importante « d'aberrantly expressed TSA » (aeTSA) dans les régions non codantes de l’ADN. Ces aeTSA sont présentés par les molécules de CMH I et plusieurs ont provoqué la réactivité des lymphocytes T (LTs) in vitro. En plus d'être spécifiques aux cellules cancéreuses, ces aeTSA sont partagés entre plusieurs patients ce qui fait d'eux des cibles intéressantes dans le cadre d’immunothérapies. Sachant que c’est le récepteur de cellules T (TCR) qui confère la spécificité aux LTs, le but est d'isoler et de caractériser des TCR anti-aeTSA en vue de leur utilisation comme outils thérapeutiques. Pour ce faire, l’expansion de LTs CD8+ naïfs provenant de donneurs sains a été réalisée grâce à une co-culture avec des cellules dendritiques autologues chargées avec l'aeTSA d’intérêt. Les LTs CD8+ spécifiques du aeTSA ont été triés à l’aide d'un marquage dextramères et l’ARN a été isolé afin de réaliser le séquençage du TCR. Ce dernier a révélé que le répertoire de TCR anti-aeTSA est nettement oligoclonal, facilitant l'identification des séquences des chaînes α et β des clonotypes les plus abondants. En revanche, les répertoires de TCR anti-LMP2 426-434 (antigène viral) et anti-WT1 37-45 (antigène associé aux tumeurs) étaient plus diversifiés. De plus, des tests d'avidité fonctionnelle réalisés à l'aide d'ELISpot en concentrations décroissantes de peptides ont révélé que l'avidité fonctionnelle des LTs qui reconnaissent les aeTSA est similaire à celle du peptide LMP2 426-434, ce qui suggère que les aeTSA stimulent des réponses T de hautes avidités. Ensuite, la délétion du TCR endogène a été réalisée à l'aide de la technique CRISPR-Cas9, montrant plus de 90% d'efficacité. À des fins d'optimisation de protocoles, le TCR 1G4 spécifique de NY-ESO-1 a été introduit dans le locus TRAC et, simultanément, le knock-out de la chaîne α du TCR endogène a été réalisé afin de limiter les mésappariements et la compétition entre ces deux TCR. Les prochaines étapes seront d’introduire le gène codant pour le TCR spécifique d'aeTSA dans des LTs et de vérifier que les cellules éditées sont réactives envers ces aeTSA. Finalement, ce projet pourrait ouvrir la voie au ciblage d'aeTSA à l’aide de l’ingénierie du TCR pour rediriger un grand nombre de LTs envers les cellules leucémiques. / Acute myeloid leukemia is a highly lethal cancer for which effective immunotherapies are actively sought. These immunotherapies can take advantage of the fact that leukemia cells can express antigens that are not expressed by healthy tissues, namely tumor-specific antigens (TSA). In this regard, our collaborator's team has discovered an important source of aberrantly expressed TSA (aeTSA) in the non-coding regions of DNA. These aeTSAs are presented by MHC 1 molecules and can elicit T cells reactivity in vitro. In addition to being specific to cancer cells, these aeTSAs are shared between several patients, which makes them interesting targets in the context of immunotherapies. Knowing that the T cell receptor (TCR) is responsible for T cells specificity, the goal is to isolate and characterize anti-aeTSA TCRs for their use as therapeutic tools. To this end, we expanded aeTSA-specific T cells from naive CD8+ T cells obtained from healthy donors through co-culture with autologous dendritic cells loaded with the relevant aeTSA. The aeTSA-specific CD8+ T cells identified by dextramer staining were sorted for RNA extraction TCR sequencing. Amplicon sequencing reveals that the expanded anti-aeTSA TCR repertoire is markedly oligoclonal, facilitating the identification of dominant TCR α and β chains. In contrast, the anti-LMP2 426-434 (viral antigen) and anti-WT1 37-45 (tumor-associated antigen) TCR repertoires were more diverse. In addition, functional avidity tests, performed using ELISpot in decreasing concentrations of peptides, revealed that the functional avidity of T cells recognizing aeTSA is similar to LMP2 426-434 peptide, suggesting that aeTSAs stimulate high-avidity responses. Then, endogenous TCR knock-out was performed using the CRISPR-Cas9 technique, showing more than 90% efficiency. For protocol optimization purposes, the 1G4 TCR specific for NY-ESO-1 was introduced into the TRAC locus and, simultaneously, the knock-out of the α chain of the endogenous TCR was achieved in order to limit mismatches and competition between these two TCRs. The next steps will be to introduce the gene coding for the aeTSA-specific TCR into T cells and to validate that the edited cells are reactive toward these aeTSAs. Ultimately, this project could pave the way for targeting aeTSAs using TCR engineering to redirect large numbers of T cells toward leukemic cells.

The availability of several methods to unambiguously mark individual cells has strongly fostered the understanding of clonal developments in hematopoiesis and other stem cell driven regenerative tissues. While cellular barcoding is the method of choice for experimental studies, patients that underwent gene therapy carry a unique insertional mark within the transplanted cells originating from the integration of the retroviral vector. Close monitoring of such patients allows accessing their clonal dynamics, however, the early detection of events that predict monoclonal conversion and potentially the onset of leukemia are beneficial for treatment. We developed a simple mathematical model of a self-stabilizing hematopoietic stem cell population to generate a wide range of possible clonal developments, reproducing typical, experimentally and clinically observed scenarios. We use the resulting model scenarios to suggest and test a set of statistical measures that should allow for an interpretation and classification of relevant clonal dynamics. Apart from the assessment of several established diversity indices we suggest a measure that quantifies the extension to which the increase in the size of one clone is attributed to the total loss in the size of all other clones. By evaluating the change in relative clone sizes between consecutive measurements, the suggested measure, referred to as maximum relative clonal expansion (mRCE), proves to be highly sensitive in the detection of rapidly expanding cell clones prior to their dominant manifestation. This predictive potential places the mRCE as a suitable means for the early recognition of leukemogenesis especially in gene therapy patients that are closely monitored. Our model based approach illustrates how simulation studies can actively support the design and evaluation of preclinical strategies for the analysis and risk evaluation of clonal developments.