Statistical and network-based methods for the analysis of chromatin accessibility maps in single cells

In questo lavoro, metodi provenienti dalla Fisica, dalla Statistica e dalla Teoria dei Grafi sono stati impiegati per caratterizzare ed analizzare profili di apertura e accessibilità della cromatina ottenuti con la tecnica ATAC-seq in singole cellule, nella fattispecie linfociti B provenienti da tre pazienti affetti da Leucemia Linfocitica Cronica. Una pipeline bioinformatica è stata sviluppata per processare i dati di sequencing ed ottenere le posizioni accessibili del genoma per ciascuna cellula. La quantità di regioni aperte e la loro distribuzione spaziale lungo il DNA sono state caratterizzate. Infine, l’apertura simultanea nelle stesse singole cellule di regioni regolatrici è stata impiegata come metrica per valutare relazioni funzionali, e in questo modo grafi tra enhancer e promoter sono stati costruiti e le loro proprietà sono state analizzate. La distribuzione spaziale lungo il genoma di regioni aperte consecutive ricapitola proprietà strutturali come gli array di nucleosomi e le strutture a loop della cromatina. Inoltre, i profili di accessibilità delle regioni regolatrici sono significativamente conservati nelle singole cellule. I network tra enhancer e promoter forniscono un modo per caratterizzare la rilevanza di ciascuna regione regolatrice in termini di centralità. Le statistiche sulla connettività tra enhancer e promoter confermano il modello di relazione uno-a-uno come il più frequente, in cui un promoter è regolato dall'enhancer ad esso più vicino. Infine, anche il funzionamento dei superenhancer è stato indagato. In conclusione, ATAC-seq si rivela un'efficace tecnica per indagare l'apertura della cromatina in singole cellule, i cui profili di accessibilità ricapitolano caratteristiche strutturali e funzionali della cromatina. Al fine di indagare i meccanismi della malattia, il panorama di accessibilità dei lifociti tumorali può essere confrontato con quello di cellule sane e cellule trattate con farmaci epigenetici

Non-genetic mechanisms of tumor progression in B-cell lymphoma and lung adenocarcinoma

During its progression, a tumor acquires genetic and epigenetic alterations that sustain its growth and invasiveness. Recently, somatic mutations affecting cancer genomes have been thoroughly characterized. In addition, the relevance of alterations in the tumor epigenetic landscape, chromatin structure, and transcriptional regulation has increasingly been recognized. In this thesis, I investigated structural and epigenetic features of B-cell lymphoma, and non-genetic characteristics driving histologic heterogeneity in lung adenocarcinoma. In the first chapter of the thesis, I describe the three-dimensional organization of the genome and how its interplay with widespread histone modifications promotes B-cell lymphoma development. As part of a collaborative effort, we initially evaluated a set of computational approaches to detect genomic regions of increased physical interactions known as topological domains. Then, we analyzed the genome architecture in B-cell lymphoma cell lines with mutation in the EZH2 gene, which leads to increased levels of the repressive histone mark H3K27me3 genome-wide. We found that the transcription of entire topological domains was repressed as a result of the EZH2 mutation, which also led to a rewiring of specific physical interactions among gene promoters. These domains included known tumor suppressor genes, whose silencing had a synergistic effect in fostering tumor growth in vivo. Pharmacological inhibition of EZH2 reactivated transcription in topological domains, restoring the expression of tumor suppressors. In the second chapter of the thesis, I investigated the molecular features underlying histologic heterogeneity in lung adenocarcinoma. In this tumor type, disease progression and prognosis are associated with the appearance of morphologically diverse tumor regions, named histologic patterns. In collaboration with the Pathology department of the Lausanne University Hospital, we generated multi-omics and spatially resolved molecular profiles of multiple histologic patterns of primary lung adenocarcinomas. We found that epigenetic and transcriptional alterations drove the transition from indolent to aggressive patterns. With this result, we developed a prognostic signature that predicted survival in multiple human cohorts. Finally, we proved that diverse tumor microenvironments were associated with histologic patterns, and the most aggressive one showed features of immune exclusion. In conclusion, with this thesis I report novel non-genetic mechanisms of B-cell lymphoma and lung adenocarcinoma progression, which might inform future therapeutic approaches. Résumé Pendant son développement, une tumeur acquiert des altérations génétiques et épigénétiques qui soutiennent sa croissance et son invasivité. Récemment, les mutations somatiques affectant le génome des cancers ont été dûment caractérisés. En outre, l’importance des altérations dans l’épigénétique, la structure de la chromatine et la régulation transcriptionelle du cancer est de plus en plus reconnue. Dans cette thèse, j’ai étudié les propriétés structurales et épigénétiques du lymphome à cellules B, et les caractéristiques non génétique qui cause l’hétérogénéité histologique de l’adénocarcinome pulmonaire. Dans le premier chapitre de ma thèse, je décris l'organisation tridimensionnelle du génome et comment son interaction avec les modifications d'histones favorise le développement du lymphome à cellules B. Dans le cadre d'un effort de collaboration, nous avons initialement évalué un ensemble d'approches informatiques pour détecter les régions génomiques d'interactions physiques accrues, appelées domaines topologiques. Ensuite, nous avons analysé l'architecture du génome dans les lignées cellulaires de lymphomes à cellules B porteurs d’une mutation dans le gène EZH2, cette dernière conduisant à une augmentation des niveaux de la modification d’histone répressive H3K27me3. Nous avons constaté que la transcription de domaines topologiques entiers était réprimée à cause de la mutation de EZH2, qui a également conduit à une reconfiguration des interactions physiques entre les promoteurs de gènes. Ces domaines comprenaient des gènes suppresseurs de tumeurs connus dont l’inactivation avait un effet synergique en favorisant la croissance tumorale in vivo. L’inhibition pharmacologique d’EZH2 a réactivé la transcription dans les domaines topologiques, restaurant l'expression des suppresseurs de tumeurs. Dans le deuxième chapitre de ma thèse, j'ai étudié les caractéristiques moléculaires sous-jacentes à l'hétérogénéité histologique de l'adénocarcinome pulmonaire. Dans ce type de tumeur, la progression de la maladie et le pronostic sont associés à l'apparition de régions tumorales morphologiquement diverses, appelées ‘patterns’. En collaboration avec le service de pathologie du Centre Hospitalier Universitaire Vaudois, nous avons généré des profils moléculaires multi-omiques et spatialement résolus de multiples patterns d'adénocarcinomes pulmonaires primaires. Nous avons constaté que les altérations épigénétiques et transcriptionnelles conduisaient à la transition de patterns indolents à agressifs. Avec ce résultat, nous avons développé une signature pronostique qui prédisait la survie dans plusieurs cohortes humaines. Enfin, nous avons prouvé que différents micro-environnements tumoraux étaient associés aux patterns, et le plus agressif présentait des caractéristiques d'exclusion immunitaire. En conclusion, avec cette thèse, je rapporte de nouveaux mécanismes non génétiques de la progression du lymphome à cellules B et de l'adénocarcinome pulmonaire qui pourraient éclairer les futures approches thérapeutiques

Sustained androgen receptor signaling is a determinant of melanoma cell growth potential and tumorigenesis.

Melanoma susceptibility differs significantly in male versus female populations. Low levels of androgen receptor (AR) in melanocytes of the two sexes are accompanied by heterogeneous expression at various stages of the disease. Irrespective of expression levels, genetic and pharmacological suppression of AR activity in melanoma cells blunts proliferation and induces senescence, while increased AR expression or activation exert opposite effects. AR down-modulation elicits a shared gene expression signature associated with better patient survival, related to interferon and cytokine signaling and DNA damage/repair. AR loss leads to dsDNA breakage, cytoplasmic leakage, and STING activation, with AR anchoring the DNA repair proteins Ku70/Ku80 to RNA Pol II and preventing RNA Pol II-associated DNA damage. AR down-modulation or pharmacological inhibition suppresses melanomagenesis, with increased intratumoral infiltration of macrophages and, in an immune-competent mouse model, cytotoxic T cells. AR provides an attractive target for improved management of melanoma independent of patient sex

Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients