<p>The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism.</p><p>The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general.</p><p>The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding.</p><p>During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1.</p><p>The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution</p><p>synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.</p>

The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism. The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general. The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding. During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1. The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.

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Environ 30% des cancers humains ont une mutation gain de fonction dans l’oncogène RAS, menant à une prolifération cellulaire accrue et une expansion clonale. Cependant, il est bien établi qu’une hyperactivation soutenue de cette voie mène au phénotype inverse, soit la sénescence cellulaire, définie par un arrêt stable de la prolifération. Ce destin cellulaire caractérise les lésions bénignes et la progression vers une tumeur maligne est associée à son contournement. Toutefois, les mécanismes moléculaires permettant aux cellules de distinguer entre une signalisation normale et oncogénique par RAS afin de les engager vers la sénescence plutôt que la prolifération demeurent inconnus. Ainsi, l’hypothèse à la base de ces travaux est que la décision d’engagement vers la sénescence implique une reprogrammation transcriptionnelle qui précède l’établissement des phénotypes caractéristiques de la sénescence, tel le phénotype sécrétoire (SASP) (Article 1). Nous avons ainsi identifié un point de restriction (SeRP) critique pour l’engagement des cellules vers la sénescence en réponse à l’oncogène HRASG12V. Ce SeRP intègre l'intensité et la durée du stress oncogénique, tout en gardant une mémoire des stress antérieurs, en modulant l’accessibilité à la chromatine via l’induction d’un réseau auto-régulé de facteurs de transcription comprenant notamment ETV4 et RUNX1 (Article 2). Notre modèle actuel nous porte à croire que cette augmentation d’accessibilité à la chromatine impliquerait principalement une décondensation de l’hétérochromatine périnucléolaire. Ceci mènerait à l’induction du SASP et aux défauts de ribogénèse observés dans la sénescence. Nous montrons d’ailleurs via la génération d’un modèle murin transgénique que l’induction de tels défauts de ribogénèse à l’échelle systémique mène à un phénotype de vieillissement prématuré suggérant une sénescence des cellules souches (Article 3). Les cellules souches ayant des niveaux particulièrement élevés de ribogénèse et étant très sensibles à des altérations de leur niche tels que l’inflammation chronique, nous pensons que, de manière fortuite, ce modèle reproduit en quelque sorte les conséquences du SeRP. En somme, l’ensemble des travaux présentés dans cette thèse permettent une meilleure compréhension des mécanismes moléculaires régulant l’engagement vers la sénescence. À termes, ces nouvelles notions permettraient de concevoir des stratégies thérapeutiques permettant de faire pencher la balance vers la sénescence dans un contexte de cancers mutés en RAS. / Around 30% of human cancers have a gain-of-function mutation in the RAS oncogene, resulting in increased cell proliferation and clonal expansion. However, it is well established that a sustained hyperactivation of this same pathway leads instead to the opposite phenotype, namely cellular senescence, which is defined by a stable proliferation arrest. This cell fate characterizes benign lesions and progression to malignancy is associated with its bypass. However, the molecular mechanisms allowing cells to distinguish between normal and oncogenic RAS signaling in order to commit them to senescence rather than proliferation remain unknown. Thus, the hypothesis underlying the present work is that this decision to commit to senescence involves a transcriptional reprogramming that precedes the establishment of the senescence-characteristic phenotypes such as the secretory phenotype (Article 1). We have thus identified a restriction point (SeRP) critical for the commitment of cells towards senescence in response to HRASG12V oncogene. This SeRP integrates both the intensity and duration of oncogenic stress while keeping a memory of previous stresses. This integration is achieved by modulating chromatin accessibility via the induction of a self-regulated network of transcription factors including among others ETV4 and RUNX1 (Article 2). Our current model leads us to believe that this increase in chromatin accessibility during the SeRP would mainly involve decondensation of perinucleolar heterochromatin. This would lead to the induction of the pro-inflammatory secretome of senescent cells (SASP) and the ribogenesis defects observed in senescence. Besides, we show via the generation of a transgenic mouse model that the induction of such ribogenesis defects at the systemic scale leads to a premature aging phenotype suggesting stem cells senescence (Article 3). Stem cells having particularly high levels of ribogenesis and being very sensitive to alterations of their niche such as chronic inflammation, we believe that serendipitously, this model somehow reproduces the consequences of the SeRP. In short, all the work presented in this thesis allows for a better understanding of the molecular mechanisms regulating the commitment to senescence. Ultimately, these new notions would allow to design therapeutic strategies to tip the balance towards senescence in the context of RAS-mutated cancers.

RUNX1 est un facteur de transcription essentiel pour l’hématopoïèse et joue un rôle important dans la fonction immunitaire. Des mutations surviennent dans ce gène chez 5 à 13% des patients atteints de leucémie myéloïde aiguë (LMA) (RUNX1mut) et définissent un sous-groupe particulier de LMA associé à un pronostic défavorable. En conséquence, il est nécessaire de procéder à une meilleure caractérisation génétique et de concevoir des stratégies thérapeutiques plus efficaces pour ce sousgroupe particulier de LMA. Bien que la plupart des mutations trouvées dans le gène RUNX1 dans la LMA soient supposément acquises, des mutations germinales dans RUNX1 sont observées chez les patients atteints du syndrome plaquettaire familial avec prédisposition aux hémopathies malignes (RUNX1-FPD, FPD/AML). En outre, 44 % des individus atteints évoluent vers le développement d’une LMA. Suite au séquençage du transcriptome (RNA-Seq) d’échantillons de la cohorte Leucégène, nous avons montré que le dosage allélique de RUNX1 influence l’association avec des mutations coopérantes, le profil d’expression génique et la sensibilité aux médicaments dans les échantillons primaires de LMA RUNX1mut. Aussi, la validation des mutations trouvées chez RUNX1 a mené à la découverte que 30% des mutations identifiées dans notre cohorte de LMA étaient d’origine germinale, révélant une proportion plus élevée qu’attendue de cas de mutations RUNX1 familiales. Un crible chimique a, quant à lui, révélé que la plupart des échantillons RUNX1mut sont sensibles aux glucocorticoïdes (GCs) et nous avons confirmé que les GCs inhibent la prolifération des cellules de LMA et ce, via l’interaction avec le récepteur des glucocorticoïdes (Glucocorticoid Receptor, GR). De plus, nous avons observé que les échantillons contenant des mutations RUNX1 censées entraîner une faible activité résiduelle étaient plus sensibles aux GCs. Nous avons aussi observé que la co-association de certaines mutations, SRSF2mut par exemple, et les niveaux de GR contribuaient à la sensibilité aux GCs. Suite à cela, la sensibilité acquise aux GCs a été obtenue en régulant négativement l’expression de RUNX1 dans des cellules LMA humaines, ce qui a été accompagné par une régulation positive de GR. L’analyse de transcriptome induit par GC a révélé que la différenciation des cellules de LMA induite par GCs pourrait être un mécanisme en jeu dans la réponse antiproliférative associée à ces médicaments. Plus important encore, un criblage génomique fonctionnel a identifié le répresseur transcriptionnel PLZF (ZBTB16) comme un modulateur spécifique de la réponse aux GCs dans les cellules LMA sensibles et résistantes. Ces observations fournissent une caractérisation supplémentaire de la LMA RUNX1mut, soulignant l’importance de procéder à des tests germinaux pour les patients porteurs de mutations RUNX1 délétères. Nos résultats ont également identifié un nouveau rôle pour RUNX1 dans le réseau de signalisation de GR et montrent l’importance d’investiguer le repositionnement des GCs pour traiter la LMA RUNX1mut dans des modèles précliniques. Enfin, nous avons fourni des indications sur le mécanisme d’action des GCs, en montrant que PLZF s’avère un facteur important favorisant la résistance aux GCs dans la LMA. / RUNX1 is an essential transcription factor for definite hematopoiesis and plays important roles in immune function. Mutations in RUNX1 occur in 5-13% of Acute Myeloid Leukemia (AML) patients (RUNX1mut ) and are associated with adverse outcome, thus highlighting the need for better genetic characterization and for the design of efficient therapeutic strategies for this particular AML subgroup. Although most RUNX1 mutations in AML are believed to be acquired, germline RUNX1 mutations are observed in the familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD, FPD/AML) in which about 44% of affected individuals progress to AML. By performing RNA-sequencing of the Leucegene collection, we revealed that RUNX1 allele dosage influences the association with cooperating mutations, gene expression profile, and drug sensitivity in RUNX1mut primary AML specimens. Validation of RUNX1 mutations led to the discovery that 30% of RUNX1 mutations in our AML cohort are of germline origin, indicating a greater than expected proportion of cases with familial RUNX1 mutations. Chemical screening showed that most RUNX1mut specimens are sensitive to glucocorticoids (GC) and we confirmed that GCs inhibit AML cell proliferation via interaction with the Glucocorticoid Receptor (GR). We observed that specimens harboring RUNX1 mutations expected to result in low residual RUNX1 activity were most sensitive to GCs, and that co-associating mutations, such as SRSF2mut, as well as GR levels contribute to GC-sensitivity. Accordingly, acquired GC-sensitivity was achieved by negatively regulating RUNX1 expression in human AML cells, which was accompanied by upregulation of the GR. GC-induced transcriptome analysis revealed that GC-induced differentiation of AML cells might be a mechanism at play in the antiproliferative response to these drugs. Most critically, functional genomic screening identified the transcriptional repressor PLZF (ZBTB16) as a specific modulator of the GC response in sensitive and resistant AML cells. These findings provide additional characterization of RUNX1mut AML, further stressing the importance of germline testing for patients carrying deleterious RUNX1 mutations. Our results also identified a novel role for RUNX1 in the GR signaling network and support the rationale of investigating GC repurposing for RUNX1mut AML in preclinical models. Finally, we provided insights into the mechanism of action of GCs, which positions PLZF as an important factor promoting resistance to glucocorticoids in AML.