Tesis sobre el tema "Therapeutic target identification"

Tamoxifen has made a significant contribution in decreasing breast cancer related deaths for over 30 years and until recently was the gold standard for treatment of ER positive breast cancer (Fisher et al, 1998). Resistance to tamoxifen is however a considerable issue with cells utilising a number of molecular mechanisms to bypass the growth inhibition caused by blocking ER activity. This move towards an anti-hormone resistant state from an antihormone responsive state is associated with the transition to a much more aggressive phenotype including increased proliferation and also invasiveness. Thus unfortunately, acquisition of tamoxifen resistance is not only associated with a recurrence of breast cancer, but this cancer is also much more aggressive in nature with fewer treatment options available than the initial cancer. This study has identified Lyn kinase as increased in tamoxifen resistant breast cancer cells compared to oestrogen-responsive breast cancer cells. Subsequent removal of Lyn kinase from tamoxifen resistant breast cancer cell lines using RNAi technology led to a significant decrease in cell proliferation, increased apoptosis and also a decrease in migration and invasion. A mechanism has been suggested whereby Lyn kinase is involved in a calcium dependent zinc wave which ultimately leads to the activation of tyrosine kinases. Metastasis to other sites in the body is ultimately responsible for fatalities due to breast cancer and so being able to block its action is key to treating breast cancer in the clinic. Therefore identifying Lyn kinase as a gene target that leads to the advancement of breast cancer to a more aggressive state provides a powerful tool for treating breast cancer in the clinic.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 96-104).
Identification of effective drug targets to intervene, either as single agent therapy or in combination, is a critical question in drug development. As complexity of disease like cancer is revealed, it has become clear that a holistic network approach is needed to identify drug targets that are specially positioned to provide desired leverage on disease phenotypes. In this thesis we develop a computational framework to exhaustively evaluate target behaviors in biochemical network, either as single agent or combination therapies. We present our single target therapy work as a problem of identifying good places to intervene in a network. We quantify a relationship between how interventions at different places in network affect an output of interest. We use this quantitative relationship between target inhibited and output of interest as a metric to compare targets. In network analyzed here, most targets show a sub-linear behavior where a large percentage of targeted molecule needs to be inhibited to see a small change on output. The other key observation is that targets at the top of the network exerted relatively small control compared to the targets at the bottom of the network. In the combination therapy work we study how combination of drug concentrations affect network output of interest compared to when one of the drugs was given alone at equivalent concentrations. By adapting the definitions of additive, synergistic, and antagonistic combination behaviors developed by Ting Chao-Chou (Chou TC, Talalay P (1984), Advances in enzyme regulation 22: 27-55) for our system and systematically perturbing biochemical pathway, we explore the range of combination behaviors for all plausible combination targets. This holistic approach reveals that most target combinations show additive behaviors. Synergistic, and antagonistic behaviors are rare. Even when combinations are classified as synergistic or antagonistic, they show this behavior only in a small range of the inhibitor concentrations. This work is developed in a particular variant of the epidermal growth factor (EGF) receptor pathway for which a detailed mathematical model was first proposed by Schoeberl et al. Computational framework developed in this work is applicable to any biochemical network.
by Nirmala Paudel.
Ph. D.

Friedreich’s ataxia (FRDA) is a rare debilitating, life-shortening, autosomal recessive inherited disease that leads to progressive damage to the nervous system. Onset is usually around the puberty and patients develop a progressive loss of motor coordination, inability to walk, slurred speech, and a cardiac hypertrophy that often leads to premature death. The particular genetic mutation – expansion of an intronic GAA triplet repeat in the FXN gene – leads to reduced expression of the mitochondrial protein frataxin involved in iron-sulfur cluster biogenesis. The subsequent frataxin insufficiency causes mitochondrial dysfunction and oxidative damage with ultimately cell death, particularly in peripheral sensory ganglia. No therapy to prevent or slow down the progression of the disease has been found yet. Since there is an inverse correlation between the amount of residual frataxin and the severity of disease progression, therapeutic approaches aiming at increasing frataxin levels are expected to improve patients’ conditions. We have recently proven the therapeutic relevance of increasing frataxin levels by preventing its degradation. Indeed, we have recently shown that a significant amount of frataxin precursor is degraded by the ubiquitin-proteasome system before its functional mitochondrial maturation and we have described the therapeutic potential of small molecules that promote frataxin accumulation by docking on the frataxin ubiquitination site, thus interfering with its ubiquitination and degradation. In light of these data, inhibition of frataxin E3 ubiquitin ligase, the enzyme responsible for frataxin ubiquitination, could represent another attractive strategy to prevent frataxin degradation. We therefore pursued the identification of such enzyme by performing a functional screening of an E3 ubiquitin ligase small interfering RNA library. HIT2 was identified and validated as a candidate from this screening. Consistently, knockdown of HIT2 promotes frataxin accumulation in cells. Most importantly, silencing of this candidate gene results in frataxin accumulation also in cells derived from FRDA patients, suggesting the therapeutic potential of strategies aimed at inhibiting this E3 enzyme. Additionally, we demonstrated that HIT2 directly interacts with frataxin and its overexpression increased frataxin protein ubiquitination in a catalytic activity-dependent manner, both in cells and in in vitro assay, indicating that this enzyme may actually represent the frataxin E3 ubiquitin ligase. These findings suggest that HIT2 could be a novel important therapeutic target for Friedreich’s ataxia.

Il Difetto di Mevalonato Chinasi (MKD) è una malattia rara autoinfiammatoria autosomica recessiva, causata da mutazioni nel gene MVK che codifica per mevalonato chinasi (MK), enzima chiave della via del mevalonato. Questa via è importante per la produzione di colesterolo, ed anche geranilgeranil pirofosfato e farnesil pirofosfato essenziali per la prenilazione delle proteine. MKD ha fenotipi clinici eterogenei, infatti, si va da una forma lieve, la sindrome iper-IgD (HIDS), ad una forma più grave, la Mevalonica Aciduria (MA). HIDS è caratterizzata da sintomi eterogenei che comprendono febbri ricorrenti, eruzioni cutanee, afte, artralgia, dolori addominali, diarrea e vomito; mentre MA oltre a tutto questo, mostra un fenotipo più grave con coinvolgimento neurologico, ritardo psicomotorio, epatopatia e atassia cerebellare. Più del 50% dei pazienti MA muore durante l'infanzia o nella prima infanzia. Non è tuttora chiara la correlazione tra mutazioni di MVK ed il fenotipo clinico di MKD; infatti a causa della grande eterogeneità genetica e clinica, la correlazione genotipo-fenotipo risulta essere problematica. L’MKD ad oggi è ancora una malattia orfana di trattamento eziologico specifico, sono ancora poco conosciuti i meccanismi patogenetici ed i principali attori coinvolti nella malattia; in particolar modo, non è ancora stata chiarita la patogenesi collegata alle gravi manifestazioni cliniche di MA così come i meccanismi neuro-infiammatori e le interazioni che avvengono tra i diversi tipi cellulari nel sistema nervoso centrale. L'ipotesi patogenetica di MKD ad oggi più accreditata collega il fenotipo infiammatorio con la diminuzione di composti isoprenoidi e del livello di proteine prenilate, causato dal blocco della via del mevalonato. Questa diminuzione di proteine determina attivazione dell’inflammosoma NALP-3, che a sua volta induce l'attivazione ed rilascio di IL-1β. Attualmente, vi è una mancanza di modelli per lo studio dell’MKD. Infatti, il modello biochimico ottenuto in vivo e in vitro mediante somministrazione di inibitori della via del mevalonato (aminobifosfonati o statine) è l'unico modello in grado di mimare le caratteristiche patologiche. L'obiettivo di questo progetto di dottorato è indagare il meccanismo patogenetico del Difetto di Mevalonato Chinasi, ponendo particolare attenzione alla forma più grave, la Mevalonico Aciduria, valutando i meccanismi neuro-apoptotici e neuroinfiammatori tipici di questa sindrome. Per tutti questi motivi, abbiamo eseguito l’analisi dell’esoma di pazienti MKD, per valutare la presenza di eventuali altri geni implicati nelle variazioni fenotipiche; studiato, in modelli biochimici in vitro (ottenuti in cellule neuronali, microgliali e monocitiche), i meccanismi patogenetici di MKD, tra cui l'apoptosi, il danno mitocondriale, lo stress ossidativo e l'infiammazione; abbiamo inoltre valutato l'infiammazione sistemica e la neuro-infiammazione nel modello biochimico in vivo, ottenuto in due diversi ceppi di topi (BALB/C e C57BL/6); infine, abbiamo sviluppato un modello genetico in vitro, utilizzando trasfezione transitoria di due differenti mutazioni tipiche di MKD (I268T associato ad HIDS, e N301T tipico di MA), valutando le basi molecolari della malattia e il meccanismo patologico legato al processo autofagico.
Mevalonate Kinase Deficiency (MKD) is a rare autoinflammatory autosomal recessive inborn disease, caused by mutations in MVK gene that encodes for Mevalonate Kinase (MK) an important enzyme of the mevalonate pathway. Mevalonate pathway is important for the production of cholesterol, geranylgeranyl pyrophosphate and farnesyl pyrophosphate essential for protein prenylation. MKD has heterogeneous clinical phenotypes, with a mild form, Hyper-IgD Syndrome (HIDS), and a severe one, Mevalonic Aciduria (MA). Heterogeneous symptoms including recurrent fevers, cutaneous rash, aphtae, arthralgia, abdominal pain with diarrhoea and vomiting characterize HIDS, while MA shows a more critical neurologic phenotype with psychomotor retardation, hepatopathy and cerebellar ataxia. More than 50% of MA patients die in infancy or early childhood. The correlation between MVK mutations and MKD clinical phenotype is still to be elucidated. Genotype-phenotype correlation is sometimes problematic due to the great genetic and clinical heterogeneity. MKD is also an orphan drug disease and the pathogenic mechanisms as well as the main actors involved in disease’s aetiology are still unknown; especially the pathogenesis of MA clinical manifestations has not been established. Indeed, the neuro-inflammatory mechanisms and the interactions that occur between the different cellular types in the brain have not yet been explained. The most accredited MKD pathogenetic hypothesis is based on the evidence that the mevalonate pathway block induces a decrease in isoprenoid compounds and prenylated proteins, leading to inflammatory phenotypes, caused by the activation of NALP-3 inflammasome that consequently determines IL-1β activation. Currently there is a lack of models for MKD studies. Indeed, the only model able to mimic pathologic features is a biochemical model obtained in vivo and in vitro by administration of mevalonate pathway inhibitors such as aminobisphosphonate or statin. The aim of this PhD project is to investigate the pathogenic mechanism of MKD. Special attention is given to MA, in order to evaluate the neuro-apoptotic and neuro-inflammatoy mechanisms leading to this syndrome. For all these reasons, we performed exome analyse of MKD patients in order to evaluate the presence of eventual other modifiers gene, able to modulate MKD phenotype; we investigated pathogenic mechanisms of MKD, including apoptosis, mitochondrial damage, oxidative stress and inflammation using an in vitro biochemical models (i.e., neuronal, microglia and monocytic cells); we also evaluated systemic inflammation and neuro-inflammation employing an in vivo biochemical model obtained in two different mice strains (BALB/c and C57BL/6); finally, we developed an in vitro genetic model using transient transfection of two different MKD mutations (I268T associated with HIDS, and N301T typical of MA), evaluating the molecular basis of MKD and the pathology mechanism linked to autophagy. The main specific results emerging from this PhD thesis work are: - GRID2 could be a modifier gene of MKD; - biochemical block of mevalonate pathway in neuronal cells caused a balance between apoptosis follows mitochondrial pathway (caspase-9 and caspase-3 dependent) and pyroptosis (caspase-1 dependent); - microglial activation is a direct consequence of mevalonate pathway block, which induces an additional increase of neuronal cell death; - systemic and neuronal inflammations are observed in biochemical in vivo model obtained in two different mice strains; - mevalonate pathway block induced mitochondrial damage, leading to oxidative stress and pro-inflammatory cytokines’ release, which leaded cells to final apoptosis; - MVK mutations cause an alteration in autophagic flux that leads cells to final apoptosis, in in vitro genetic model of MKD in neuronal cells. The findings obtained during the PhD enabled to formulate a new MKD pathogenic hypothesis, based on mitophagy impairment.

En offrant une image sans précédent du génome humain, les études d'association pangénomiques (GWAS) expliqueraient pleinement le contexte génétique des maladies complexes. A ce jour, les résultats ont été pour le moins mitigés. Cela peut être partiellement attribué à la méthodologie statistique adoptée, qui ne prend pas souvent en compte l'interaction entre les variants génétiques, ou l'épistasie. La détection d'épistasie à travers des modèles statistiques présente plusieurs défis pour lesquels nous développons dans cette thèse une paire d'outils adéquats. Le premier outil, epiGWAS, utilise l'inférence causale pour détecter les interactions épistatiques entre un SNP cible et le reste du génome. Le deuxième outil, kernelPSI, utilise à la place des méthodes à noyaux pour modéliser l'épistasie entre plusieurs polymorphismes mononucléotidiques (SNPs) voisins. Il tire également partie de l'inférence post-sélection pour effectuer conjointement une sélection au niveau des SNPs et des tests de signification au niveau des gènes. Les outils développés sont - au meilleur de nos connaissances - les premiers à étendre au domains des GWAS des outils puissants d'apprentissage statistique tels que l'inférence causale et l'inférence post-sélection nonlinéaire. En plus des contributions méthodologiques, un accent particulier a été mis sur l'interprétation biologique pour valider nos résultats dans la sclérose en plaques et les variations d'indice de masse corporelle
By offering an unprecedented picture of the human genome, genome-wide association studies (GWAS) have been expected to fully explain the genetic background of complex diseases. So far, the results have been mitigated to say the least. This, among other things, can be partially attributed to the adopted statistical methodology, which does not often take into account interaction between genetic variants, or epistasis. The detection of epistasis through statistical models presents several challenges for which we develop in this thesis a pair of adequate tools. The first tool, epiGWAS, uses causal inference to detect epistatic interactions between a target SNP and the rest of the genome. The second tool, kernelPSI, instead uses kernel methods to model epistasis between nearby single-nucleotide polymorphisms (SNPs). It also leverages post-selection inference to jointly perform SNP-level selection and gene-level significance testing. The developed tools are -- to the best of our knowledge -- the first to extend powerful statistical learning frameworks such as causal inference and nonlinear post-selection inference to GWAS. In addition to the methodological contributions, a special emphasis was placed on biological interpretation to validate our findings in multiple sclerosis and body-mass index variations

Glioblastoma multiforme (GBM) is the most devastating tumor of the brain, characterized by an almost inevitable tendency to recur after intensive treatments and a fatal prognosis. Indeed, despite recent technical improvements in GBM surgery, the complete eradication of cancer cell disseminated outside the tumor mass still remains a crucial issue for glioma patients management. In my PhD project, we identified Annexin 2A (ANXA2) as an important intracellular cytoskeletal protein expressed also on the surface of various types of cancer cells. Initially, we show that ANXA2 is over-expressed in IV grade GBM at various levels when compared to lower stage tumors. More importantly, we demonstrated that low/absent expression of ANXA2 identifies a subgroup of GBM patients endowed with better prognosis, suggesting that ANXA2 expression can be considered as an independent prognostic factor in glioma. We then analyzed the transcriptional changes associated to different levels of ANXA2 expression. In particular, we generated a series of ANXA2 dependent transcriptional signatures based on the comparison between ANXA2hi versus ANXA2lo expressing GBM patients from the TCGA and GSE13041 datasets (719 differentially expressed genes in common between the two cohorts), modulated transcripts after ANXA2 neutralization by specific antibody (855 differentially expressed genes) and the expression profiles of ANXA2 silenced cells respect to relative controls (3592 differentially expressed genes) in our primary GBM cell cultures. Interestingly, Gene Set Enrichment Analysis (GSEA) on the three different signatures obtained, revealed a negative enrichment of cell migration and mesenchymal transition related genes. These data strongly suggested the important role played by ANXA2 in GBM cell behavior and aggressiveness, allowing us to further setup strategies to specifically modulate its functions and dependent intracellular signaling. For this reason, we further analyzed ANXA2 functional activity in vitro in primary GBM cell cultures, demonstrating as ANXA2 is a major sustainer of GBM cell aggressiveness by regulating cellular invasion and motility together with cancer cell proliferation and differentiation status. Moreover, based on gene expression data of ANXA2 neutralized cells, we were able to test the prognostic potential of an ANXA2down signature in multiple cancer datasets, demonstrating that expression of genes regulated by ANXA2 fluctuations predict cancer patients outcome by themselves. Finally, we then functionally mapped an ANXA2-dependent gene signature (TCGA and GSE13041 datasets analysis) by exploiting the Connectivity Map bioinformatic tool in order to identify compounds and approved drugs able to revert this signature of GBM aggressiveness. The compounds, significantly predicted to be able to counteract the ANXA2-dependent transcriptional signature, were analyzed for their ability to inhibit GBM cell invasion in vitro in primary GBM cultures. Finally, we applied ANXA2 dependent transcriptional signatures, previously generated from our primary GBM cells, to the QUADrATiC tool, which was allowed the exploration of a larger database of reference cell lines and perturbagens.
Il Glioblastoma Multiforme (GBM) è il tumore cerebrale più aggressivo, caratterizzato da una prognosi infausta e dall’inevitabile tendenza a ricadere anche in seguito ad un trattamento intensivo. Nonostante i recenti miglioramenti tecnici nella chirurgia del GBM, la sua completa rimozione rimane ad oggi uno dei maggiori problemi legati all’insuccesso terapeutico di questi pazienti. Questo studio si focalizza sulla caratterizzazione di annessina 2A (ANXA2), proteina presente in diversi compartimenti delle cellule normali e ritrovata anche sulla superficie di diversi tipi di cellule tumorali. Con lo sviluppo di questo progetto, abbiamo dimostrato che ANXA2 è espressa ad alti livelli nei gliomi di IV grado rispetto ai gliomi di grado minore e che una bassa/nulla espressione di ANXA2 identifica un sottogruppo di pazienti caratterizzati da una prognosi migliore, suggerendo come l’espressione di ANXA2 possa essere considerata un fattore prognostico indipendente nei gliomi. Successivamente, con lo scopo di analizzare i cambiamenti trascrizionali associati ai differenti livelli di espressione di ANXA2, abbiamo generato una signature trascrizionale ANXA2-dipendente utilizzando i dati provenienti dai dataset pubblici TCGA e GSE13041 e basata sul confronto tra pazienti esprimenti alti livelli di ANXA2 e pazienti esprimenti bassi livelli di questa proteina (719 geni differenzialmente espressi in comune tra le due coorti). Sono state quindi generate due signature ANXA2-dipendenti basate rispettivamente sui trascritti modulati in seguito alla neutralizzazione di ANXA2 con anticorpo specifico (855 geni differenzialmente espressi) e tramite silenziamento (3592 geni differenzialmente espressi), in colture primarie di GBM. L’analisi di gene set enrichment (GSEA) condotta sulle tre signature, ha rivelato un arricchimento negativo di geni legati ai processi di migrazione cellulare e transizione epitelio-mesenchimale. Questi dati hanno fortemente suggerito l’importante ruolo svolto da ANXA2 nel comportamento e nell’aggressività delle cellule di GBM, portandoci pertanto a programmare differenti strategie per modulare le sue funzioni e le vie di segnale intracellulare ad essa correlate. Per questo motivo, è stata condotta una serie di analisi funzionali in vitro in cellule primarie di GBM, dimostrando come ANXA2 sia un principale mediatore dell’aggressività di questo tumore attraverso la regolazione di processi quali motilità cellulare, proliferazione e differenziamento. Inoltre, basandoci sul profilo d’espressione genica di cellule di GBM in cui abbiamo inibito la funzione di ANXA2, abbiamo validato il potenziale prognostico di una signature “ANXA2down” (basata sui geni maggiormente down-regolati in cellule di GBM trattate con anticorpo neutralizzante ANXA2) in diversi dataset pubblici, dimostrando come l’espressione di geni regolati dai livelli di ANXA2 sia in grado di predire l’andamento dei pazienti. Infine, la signature precedentemente generata dai dataset TCGA e GSE13041 è stata mappata funzionalmente utilizzando il tool bioinformatico Connectivity Map con lo scopo di identificare composti in grado di revertire tale signature. I composti identificati sono stati analizzati successivamente per la loro abilità di inibire il processo di invasione in vitro in colture primarie di GBM. Inoltre, le signature ANXA2-dipendenti, ottenute dalle precedenti analisi (cellule inibite/silenziate per ANXA2), sono state applicate al tool QUADrATiC. Ciò ha permesso di approfondire i risultati grazie all’utilizzo di un database più ampio che si basa sullo studio di un numero maggiore di composti approvati in numerose linee cellulari.

Epidermolysis Bullosa encompasses a group of inherited heterogeneous diseases involving trauma induced blistering of the skin. Recessive Dystrophic Epidermolysis Bullosa (RDEB) is one of the most debilitating variants of the disease and patients are predisposed to developing aggressive cutaneous Squamous Cell Carcinoma (SCC). Unlike SCC in the general population, the primary cause of RDEB associated SCC is not UV-radiation. SCC in RDEB patients has poor prognosis due to a high frequency of recurrence and metastasis. 70% of all severe generalized RDEB patients die from SCC by the age of 45, compared to only 1.25% of all patients with UV-induced SCC in the general population (Fine et al. 2008), making SCC the leading cause of death in these RDEB patients. The aim of this investigation was to identify therapeutic targets for RDEB associated SCC. Global gene expression studies identified 36 candidate genes which were differentially regulated in RDEB SCC (n=4) compared with non-RDEB SCC (n=5) primary keratinocyte cultures. The validation of these genes by qRT-PCR in replicate cultures of RDEB SCC (n=3), non-RDEB SCC (n=3) keratinocytes and normal human keratinocytes as a control, deduced 5 genes to be significantly differentially regulated. Of particular interest, is the over-expression of SLCO1B3 by 6.25 fold in RDEB SCC keratinocytes (p = 0.035). SLCO1B3 encodes the organic anion transporter OATP1B3, which is normally exclusively expressed on the basolateral membrane of hepatocytes. qRT-PCR revealed the mRNA expression level of SLCO1B3 is reduced in RDEB SCC keratinocyte cultures when COL7A1, the causal gene mutated in RDEB, is re-expressed, suggesting that COL7A1 which encodes the Type VII Collagen protein and is secreted into the extracellular matrix, may suppress the transcription of SLCO1B3. Immunofluorescent staining of RDEB SCC keratinocytes and tissue identified OATP1B3 expression, whilst qRT-PCR using mRNA isolated from freshly frozen skin and SCC tissue samples from both RDEB and non-RDEB individuals identified increased SLCO1B3 mRNA expression in RDEB SCC in vivo. Over expression of SLCO1B3 and increased activity of OATP1B3 is associated with breast, colon and pancreatic cancer and is a known transporter of chemotherapeutic agents, such as Methotrexate and Paclitaxel. These observations have led to speculation that, as a transporter over expressed in cancer and capable of introducing drugs into cells, OATP1B3 represents a potential therapeutic target. Preliminary results from a cell viability assay suggest that exposing RDEB SCC cells to Microcystin-LR specifically reduces cell viability in a SLCO1B3 dependent manner. This supports the conclusion that SLCO1B3 represents a viable RDEB SCC specific therapeutic target and provides a pathway which can be exploited to deliver anti-cancer drugs directly to tumour cells whilst reducing the systemic toxicity of these agents.

Le mélanome uvéal (MU) est la tumeur intraoculaire la plus fréquente chez l’adulte. C’est une tumeur agressive qui dérive de la transformation maligne des mélanocytes. Les traitements du MU primaire reposent sur des techniques de radiothérapies, et sur la chirurgie. Malgré le succès du traitement primaire, plus de 50% des patients développent des métastases qui vont principalement envahir le foie, suggérant l’existence d’une sous-population cellulaire, responsable de la dissémination métastatique. Il n’existe aujourd’hui aucun traitement systémique pour soigner le MU métastatique. A ce stade, 80% des patients meurent dans un délai de 1 an. Il est donc capital de mieux comprendre la biologie du MU, afin de découvrir de nouvelles cibles thérapeutiques et ainsi, à terme, pouvoir proposer un traitement efficace aux patients. L’hétérogénéité intra-tumorale est reconnue pour être impliquée dans la résistance aux traitements et la dissémination métastatique dans de nombreux cancers, mais n’a encore jamais été démontrée dans le MU. Au cours de ma thèse, grâce au séquençage à l’échelle de la cellule unique, nous avons démontré l’existence d’une hétérogénéité intra-tumorale aux niveaux génomique et transcriptomique dans des MU primaires. Nous avons mis en évidence une nouvelle signature, associée à un mauvais pronostic, comprenant des gènes n’ayant encore jamais été décrits dans le MU. Nous avons également identifié trois états transcriptionnels distincts, exprimés par différentes sous populations cellulaires, associés à la différenciation cellulaire, à la prolifération ou à l’invasion. La signature génique que nous avons établie est fortement associée à l’état transcriptionnel invasif, et des cellules exprimant cette signature ont été détectées au sein de chacune des tumeurs analysées, même dans celles classées de bon pronostic par les pathologistes. Cette découverte peut avoir un impact capital sur le pronostic et le suivi des patients. Grâce à nos analyses bio-informatiques, nous avons identifié le facteur de transcription HES6 comme nouveau marqueur de mauvais pronostic du MU. L’expression hétérogène de HES6 au sein des tumeurs, même celles classées de bon pronostic, a été validée par des expériences de RNAscope. J’ai montré que l’inhibition d’expression de HES6 entraine une diminution de la prolifération, de la migration, et de l’invasion dans des lignées de MU primaires, et du développement métastatique in vivo, dans un modèle d’embryon de poulet. De plus, l’inhibition de HES6 bloque aussi la prolifération et la migration des cellules de MU métastatiques. L’ensemble de mes résultats met en évidence le rôle essentiel de HES6 dans la progression du MU, et fait de HES6 une nouvelle cible pour bloquer la croissance et les capacités motiles des cellules de MU
Uveal melanoma is an aggressive and deadly neoplasm, which develops from melanocytes in the choroid. Treatments of primary UM treatments rely on radiotherapy techniques and surgery. Despite successful treatment of the primary tumor, metastases, that display a pronounced liver tropism, develop in 50% of patients. This implies the existence of a cellular subpopulation, that disseminate early during tumor progression dissemination. Currently, there is no systemic treatment for metastatic UM. At this stage, 80% of patients die within one year. It is therefore essential to better understand the biology of UM, in order to discover new therapeutic targets and, ultimately, to develop effective treatments. Intra-tumor heterogeneity is known to be involved in metastatic spread and treatment resistance in many cancers, yet its role in UM remains to be elucidated. During my thesis, through single cell sequencing, we demonstrated the existence of intra-tumor heterogeneity at genomic and transcriptomic levels in primary UM. We highlighted a new gene signature, associated with a poor prognosis, including genes that have never been reported in the UM. We also identified three distinct transcriptional cell states, associated with cell differentiation, proliferation or invasion. The invasive transcriptional state strongly associates with the poor prognosis gene signature that we have established. Cells expressing this signature were detected within all six tumors that we analyzed, even in those classified by pathologists as of good prognosis. These observations can have a major impact on patients’ prognosis and follow-up. Through our bioinformatics analysis, we identified the transcription factor HES6 as a new marker of poor prognosis in UM. Heterogeneous expression of HES6 in tumors, even in those classified as of good prognosis, was validated by the RNAscope approach. We showed that the inhibition of HES6 expression leads to decrease in proliferation, migration, and invasion of primary UM cell lines, and metastatic development in vivo, in a chicken embryo model. In addition, HES6 inhibition also impairs the proliferation and migration of metastatic UM cell lines, suggesting that HES6 has also a critical role in the metastatic settings. Together, my findings highlight the essential role of HES6 in the progression of UM, and identify HES6 as new target to prevent UM growth and motile ability

Les Sarcomes indifférenciés pléomorphes (UPS pour Undifferentiated Pleomorphic Sarcoma) forment un groupehétérogène « par défaut ». Nous avons émis l’hypothèse qu’il existe un lien entre le niveau de dédifférenciation des UPS et l’infiltrat immun intra-tumoral, et que cette relation repose sur des altérations génomiques ainsi que sur l’activation de voies de signalisation spécifiques associées à un impact thérapeutique potentiel. Les objectifs de ce travail étaient de générer une classification des UPS intégrant une approche génomique, immunophénotypique, protéomique et radiomique, et d’identifier et tester de potentielles cibles thérapeutiques sur des lignées cellulaires et des xénogreffes dérivées de tumeurs de patients (PDX) caractérisées génomiquement. Nous avons réuni 135 tumeurs de patients de type UPS dans notre institution, parmi lesquels 25 ont été sélectionnées pour séquençage de l’ARN et de l’exome entier. L’analyse hiérarchisée non supervisée des données de séquençage ARN identifia trois groupes : A, B et C. Le groupe A était principalement enrichi en gènes jouant un rôle dans le développement et le phénotype « cellule souche », notamment LHX8, LRRN1, LGR5, BMP5 et FGFR2. Le group B était fortement enrichi en gènes impliqués dans l’immunité, incluant MARCO, TIMD4, TIGIT, CD27, IFNG, CD8B, PDCD1, CD3D et IDO1, ainsi que DKK1. Le groupe C était trop petit pour être caractérisable de façon fiable. Nous avons pu confirmer cette classification sur une cohorte indépendante de 41 UPS du consortium TCGA. Nous avons trouvé une forte corrélation entre l’expression génique et la densité protéique en IHC sur les échantillons tumoraux correspondants pour CD8, PD-1 et IDO1, amenant à appeler le groupe B « immune-high » et le groupe A « immune-low ». Dans une cohorte indépendante de validation de 110 UPS, la densité de CD8 était significativement associée à une meilleure survie sans métastase (p = 0.04). Nous avons retrouvé une fréquence significativement plus importante de variation du nombre de copies de gènes dans le groupe « immune-low », principalement des délétions, touchant notamment PTEN, RB1, FANCA, FAS, CDKN2A, TP53, AXIN1, NF2 et BRCA2. Les analyses protéomiques permirent de détecter deux principaux groupes - PA and PB – fortement corrélés avec les deux principaux groupes identifiés en transcriptomique – A « immune low » et B « immune-high ». Le groupe PB était significativement enrichi en protéines appartenant aux voies de la réponse immunitaire, et le groupe PA en protéines appartenant aux cibles de MYC et à la transition épithélio-mésenchymateuse. Nous avons ensuite développé des lignées cellulaires et des PDX issues de tumeurs provenant de l’étude de profilage moléculaire et obtenu des modèles représentant chaque groupe A, B et C. L’inhibiteur de FGFR JNJ-42756493 avait une activité anti-tumorale in vitro et in vivo dans les lignées et les modèles de PDX du groupe A, de façon sélective. Nous avons également montré in vitro une sensibilité sélective des lignées cellulaires du groupe A aux trois inhibiteurs de protéines à bromodomaines BET CBP/P300 : CPI637, NEO1132 and NEO2734. Enfin, nous avons identifié un set de neuf variables de texture issues de séquences d’IRM conventionnelles hautement corrélées à notre classification transcriptomique des UPS, permettant de poser la base d’une signature radiomique pour sélectionner les UPS « immune-high » sur leur imagerie pré-thérapeutique.Cette étude est la première à réaliser un portrait intégré d’UPS primaires non pré-traités. Nous avons pu identifier deux groupes principaux, avec des implications thérapeutiques potentielles : le groupe « immunehigh », fortement inflammé et probablement le meilleur candidat à l’immunothérapie, et le groupe « immunelow », avec un rationnel pour l’utilisation d’inhibiteurs de FGFR et de BET dans ce dernier
Undifferentiated Pleomorphic Sarcoma (UPS) are an heterogeneous group of poorly differentiated tumors made up ‘by default’. We hypothesized that there is a link between dedifferentiation state of UPS and immune infiltrate and that this relation relies on specific pathways activation and related genomics alterations with potential therapeutic impact. Objectives of this work were to generate a comprehensive Omics landscape of UPS, integrating genomic, immuno-phenotypic, proteomic and radiomic approach, and to identify and test potential targets for therapeutic approach on cell lines and patients tumor mouse xenografts (PDX). We analyzed a cohort of 135 UPS samples from patients in our institution, of whom 25 were selected for full exome and RNA-sequencing. Unsupervised consensus and hierarchical clustering of RNA-sequencing identified 3 groups, A, B and C. Group A was mainly enriched in genes that play a crucial role in both normal development and stemcellness, notably LHX8, LRRN1, LGR5, BMP5 and FGFR2. Group B was strongly enriched in genes involved in immunity, including MARCO, TIMD4, TIGIT, CD27, IFNG, CD8B, PDCD1, CD3D and IDO1, but also DKK1. Group C was too small to be analyzed with sufficient robustness. This classification was confirmed on an independent cohort of 41 UPS from TCGA consortium. We found a high correlation between gene expression and protein density by IHC on related tumor sample slides for CD8, PD-1 and IDO1, leading to call group B ‘immune-high’ and group A ‘immune-low’. In an independent validation cohort of 110 UPS patients, CD8 expression was significantly associated with metastase-free survival (p = 0.04). Copy numbers variations were significantly more frequent in the immune-low group. Main recurrent events were deletions, notably in PTEN, RB1, FANCA, FAS, CDKN2A, TP53, AXIN1, NF2 and BRCA2. Proteomic analysis allowed us to detect two main proteomic groups - PA and PB – that highly correlated with the two main transcriptomic groups - A and B. Group PB was significantly enriched in immune response pathways, whereas group PA was enriched in MYC targets and epithelial-mesenchymal transition pathways. We then further developed cell lines and PDX models from patient tumor samples included in the molecular profiling study for each class, A, B, C. We showed robust in vitro and in vivo anti-tumor activity of FGFR inhibitor JNJ-42756493 in cell lines and PDX models from group A, selectively. We also showed in vitro activity of three potent dual inhibitors of BET-proteins CBP/P300, CPI637, NEO1132 and NEO2734, in cell lines from group A, selectively. Finally, we showed that a set of 9 radiomic features from basic MRI conventional sequences correlated well with our UPS molecular classification and provided the basis for a radiomics signature that could select immune-high UPS on their pretherapeutic imaging. This study is the first to give a comprehensive genomic, immuno-phenotypic, proteomic and radiomic landscape of non-pretreated primary UPS. We identified two main groups of UPS with therapeutics potential: the immunehigh group, strongly inflamed and probably the best candidate for immunotherapy, and the immune-low group, with a rational for FGFR and BET inhibitors activity in this one

A shortlist of candidate tumour endothelial markers was generated by Microarray comparison of differential gene expression between multiple patient-matched colorectal cancer and normal colon samples. This list was narrowed through a process of literature review, real-time quantitative polymerase chain reaction and immunohistochemistry. Through siRNA knockdown and analysis in in vitro models of angiogenesis, it has been demonstrated that a decrease in an novel target’s expression significantly decreases cellular migration, communication and chemotaxis, without adversely affecting cell viability or proliferation in HUVEC. Vaccination with a murine Fc fusion protein in combination with Freund’s adjuvant stimulated a specific immune response to this self-antigen, by breaking immune tolerance. The resulting increase in specific IgG1 antibody titers, indicative of Th2 T-cell response, resulted in a significant reduction in physiological angiogenesis in the subcutaneous sponge assay, and a significant decrease in colorectal tumour growth in a murine subcutaneous CT26 tumour model. The gene of interest represents a novel tumour endothelial marker in colorectal cancer. A hypothesised mechanism for the observed effects is an inhibition of endothelial calcium influx, leading to decreased angiogenic potential in tumour endothelial cells.

La recherche dans le domaine de l'obésité a énormément progressé pendant les dernières décennies et a apporté une contribution fondamentale à la compréhension des mécanismes biologiques et physiologiques impliqués, de même que leurs interactions avec l'environnement obésogène. Les études génétiques et génomiques ont mis en évidence les traits héréditaires majeurs qui peuvent causer ou prédisposer à l'accumulation excessive de gras corporel et ils ont stimulé la caractérisation de nombreux gènes codant pour des protéines impliquées dans la physiologie du bilan énergétique. Malgré le progrès considérable des connaissances, les pharmacotherapies actuelles ne démontrent pas d'effets suffisamment efficaces sur la perte persistante de poids, et sont souvent suivies par des effets secondaires importants. Le travail présenté dans cette thèse a comme objectif principal d'identifier et de caractériser de nouvelles cibles thérapeutiques pour le traitement et la prévention de l'obésité et des maladies métaboliques associées. Plus spécifiquement, nous avons concentré nos études sur les mécanismes précoces régulant la prise alimentaire et le métabolisme énergétique. Nous nous sommes premièrement intéressés aux changements métaboliques précoces qui surviennent avec la menopause et qui peuvent prédisposer au développement de l'obésité. Nous avons ainsi analysé les effets aigus de la prévalence androgénique sur l'expression génique du tissu adipeux rétro-péritonéal, chez un modèle murin de menopause. Nos résultats démontrent qu'une seule injection de dihydrotestostérone induit des changements significatifs dans le profil transcriptionnel du tissu adipeux. Enfin, l'expression augmentée de plusieurs transcrits myogéniques dans ce tissu témoigne de sa plasticité exceptionnelle, une qualité qui pourrait être exploitée à des fins thérapeutiques. Nous avons ensuite analysé les effets rapides que la consommation de repas à haute teneur lipidique cause sur la perception de la satiété chez la souris. Selon plusieurs évidences, obtenues chez des sujets humains de même que chez des modèles animaux, les repas riches en gras ont des effets réduits et retardés sur la perception de la satiété, comparativement aux glucides ou aux protéines. Ils peuvent donc favoriser la surconsommation passive d'énergie et, à long terme, l'accumulation de poids corporel. Nous avons utilisé la méthode de l'analyse sérielle de l'expression génique (SAGE) et étudié les changements transcriptionnels induits par un seul repas dans des tissus clés de la souris, comme l'estomac et l'hypothalamus. Nous avons ainsi identifié plusieurs nouveaux transcrits qui avaient été spécifiquement et rapidement régulés par le repas riche en gras. Un certain nombre de ces gènes a été sélectionné pour caractériser ultérieurement leur potentiel dans le développement de l'obésité induite par la diète (OID). Cette thèse présente la première caractérisation in vivo des rôles du gène trefoil factor family member 2 (Tff2) dans la régulation du bilan énergétique et l'OID. Chez les souris, la déficience du gène Tff2 a altéré significativement le comportement alimentaire, ainsi que la prise énergétique et la dépense d'énergie après douze semaines de diète riche en gras. En conclusion, les souris Tff2 KO étaient moins efficaces dans l'accumulation de l'énergie ingérée et, par conséquent, plus résistantes à l'OID par rapport aux souris normales. Les résultats obtenus dévoilent des rôles totalement nouveaux pour Tff2 et indiquent pour la première fois son implication dans la régulation du bilan énergétique. Les évidences ici décrites suggèrent que Tff2 pourrait être une cible optimale pour la conception de molécules pharmacologiques, qui contrôleraient simultanément plusieurs points critiques pour la régulation du poids corporel et le traitement de l'obésité.

Leiomiosarcoma maligno e leiomioma benigno rappresentano le due controparti della trasformazione del miometrio. Tuttavia la diagnosi differenziale per discriminare tra lesioni benigne e maligne rappresenta un grande problema. Così vanno identificati nuovi markers per rendere la diagnosi differenziale piu’ accurata. Il ruolo pleiotropico dell’RKIP nel leiomiosarcoma non è ancora chiaro. In questa tesi tramite l’immunoistochimica si sono riscontrate la tendenza di cinque diverse varianti istologiche di lesioni benigne a essere positive all’RKIP e, al contrario, la tendenza delle lesioni maligne a essere negative all’RKIP. Ciò è stato molto evidente confrontando leiomiosarcoma maligno e leiomioma con nuclei bizzarri che, sebbene benigno, ha caratteristiche intermedie tra lesioni benigne e maligne. Per capire se l’RKIP ha un ruolo biologico nel leiomiosarcoma, sono stati eseguiti il knockout in vitro dell’RKIP e l’overespressione in vitro dell’RKIP nelle cellule di leiomiosarcoma. Si è visto che circa la vitalità delle cellule nel tempo e la capacità migratoria, sia le cellule RKIP knockout che le cellule che overesprimono RKIP hanno una tendenza simile a quella dei rispettivi controlli negativi. D'altra parte si è visto che le cellule RKIP knockout hanno una maggiore capacità di formare colonie rispetto al controllo negativo in cui l'espressione dell’RKIP non è stata modificata. In conclusione i risultati ottenuti suggeriscono che può esserci una perdita di RKIP nel leiomiosarcoma maligno e che ciò può favorire clonogenicità. Nel complesso questi risultati suggeriscono che l’RKIP potrebbe candidarsi come marker addizionale per l'analisi differenziale per discriminare se una preparazione simile al leiomioma benigno con nuclei bizzarri non sia, in realtà, una lesione maligna. Alla luce della rarità delle patologie studiate questi risultati costituiscono un punto di partenza per ulteriori ricerche per considerare l’RKIP un marker affidabile a tutti gli effetti.
Malign leiomyosarcoma and benign leiomyoma represent the two counterparts of the myometrium transformation. Nevertheless, the differential diagnosis to discriminate benign from malign lesions represents a remarkable problem. So, it is important to identify new markers making the differential diagnosis more accurate. The pleiotropic RKIP role on leiomyosarcoma is still unclear. In this study, the tendency of five different histological variants of benign lesions to be positive for RKIP and, conversely, the tendency of malign lesions to be negative for RKIP were found by immunohistochemistry. This was particularly evident comparing the malign leiomyosarcoma to the leiomyoma with bizarre nuclei that, although benign, shows intermediate features between benign lesions and malign ones. To understand if RKIP plays a biological role in leiomyosarcoma, in vitro knockout of RKIP and in vitro overexpression of RKIP in leiomyosarcoma cells were performed. It was found that about cells viability overtime and migratory capability both RKIP knockout cells and RKIP overexpressing cells show a trend similar to that of their respective negative controls. On the other hand, it was observed that RKIP knockout cells show a greater ability to form colonies when compared with negative control where the expression of RKIP was not modified. In conclusion, the obtained results suggest that there could be a RKIP loss in the malign leiomyosarcoma and this may favor the clonogenicity. Altogether these results suggest that RKIP may be a candidate to be considered as an additional marker for the differential analysis to discriminate if a preparation similar to the benign leiomyoma with bizarre nuclei, is not, actually a malign lesion. In light of the rarity of the studied pathologies, these results constitute a starting point for further researches in order to consider RKIP a reliable marker in all respects.

À l’heure actuelle, le paludisme reste un problème de santé majeur et demeure une des maladies parasitaires les plus menaçantes. Parmi les cinq espèces de malaria infectant l’homme, Plasmodium falciparum est la forme la plus mortelle. Lors de la phase érythrocytaire de son cycle de vie, causant tous les symptômes du paludisme, P.falciparum utilise les phospholipides pour créer les membranes nécessaires au développement de cellules filles. Chez P. falciparum, la phosphatidylcholine est principalement obtenue grâce à la voie de synthèse de novo, dite voie de Kennedy. Dans cette voie de biosynthèse, la seconde étape catalysée par la CTP:phosphocholine cytidylyltransferase [EC 2.7.7.15] est limitante et apparait essentielle pour la survie du parasite murin P. berghei lors de la phase sanguine. Les objectifs de mon travail de thèse ont été de caractériser structuralement cette enzyme et d’identifier des effecteurs, principalement grâce à des approches de « fragment-based drug design » (FBDD). Ainsi, la première structure cristalline du domaine catalytique de l’enzyme (PfCCT) a été déterminée avec une résolution de 2.2 Å. De plus, les structures de trois complexes enzyme-substrat (en présence de CMP, de phosphocholine ou de choline) et d’un complexe enzyme-produit (CDP-Choline) ont été déterminées. Ces structures cristallographiques apportent des informations détaillées sur la poche de liaison de l’enzyme et elles ont révélé des informations sur le mécanisme de la réaction catalytique à l’échelle atomique. La seconde partie de ma thèse présente les méthodes développées pour identifier des inhibiteurs potentiels de la PfCCT. Une approche de FBDD a été utilisée pour identifier et sélectionner de petites molécules (fragments, PM<300 Da) se liant à la PfCCT. Diverses techniques biophysiques (fluorescence-based thermal shift assay, différence de transfert de saturation par RMN, dénaturation chimique isotherme) ont permis la sélection de 23 fragments à partir du criblage d’une bibliothèque (~ 300 molécules). En parallèle, un criblage in silico de plus grandes bibliothèques de fragments (environ 15 000 composés) a permis d’identifier 100 fragments “hits”. Enfin, 5 composés déjà connus pour inhiber la croissance parasitaire (Malaria Box fournit par Medecines for Malaria Venture) ont été sélectionnés pour leur inhibition de l’activité de la PfCCT recombinante. L’ensemble de ces données ouvre la voie pour l’élaboration de futurs composés ciblant la PfCCT et inhibant la biosynthèse de phosphatidylcholine chez P. falciparum
Malaria remains a major global health problem and the most threatening parasitic disease. Among the 5 malaria species that affect humans, Plasmodium falciparum is the most deadly form. During its life cycle, in erythrocytic stage, which causes all the malaria symptoms, P. falciparum relies on phospholipids to build the membranes necessary for daughter cell development. Approximately 85% of parasite phospholipids consist of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesized by the parasite through the de novo Kennedy pathways. In the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by CTP:phosphocholine cytidylyltransferase [EC 2.7.7.15] is rate limiting and appears essential for the parasite survival at its blood stage. In this PhD thesis I focus on the structural characterization of this enzyme and the identification of effectors mainly by fragment-based drug design approach (FBDD). The first reported crystal structure of the catalytic domain of the enzyme target (PfCCT) has been solved at resolution 2.2 Å. Four other crystal structures of PfCCT in complex with substrates (CMP, phosphocholine and choline) or product (CDP-choline) have been determined. These structural data give detailed images of the binding pocket and reveal the enzyme structures at all catalytic steps that provide crucial information on the catalytic mechanism at atomic level. The second part of the project present the methods developed to identify potential PfCCT inhibitors. A FBDD approach was used in order to identify and select small molecules (fragments, MW< 300 Da) binding to the PfCCT. A combination of biophysical techniques (fluorescence-based thermal shift assay, saturation transfer difference NMR and isothermal chemical denaturation) allowed the selection of 23 fragment hits from the screenings of fragment library (~ 300 molecules). In parallel in silico screening of larger fragment libraries (~15,000 compounds) resulted in 100 selected hits. Finally, 5 compounds already known to inhibit parasite growth (Malaria Box from Medicines for Malaria Venture) were selected for their inhibition of the recombinant PfCCT activity. The results obtained within this thesis brought important knowledge and structural insights on the catalytic mechanism of PfCCT. Taken together, these results pave the way for future structure-based drug design to target PfCCT and to inhibit the essential phosphatidylcholine biosynthesis in P. falciparum

Colorectal cancer (CRC) is the third most common form of cancer in the Western world. Despite the emergence of new targeted agents and the use of various therapeutic combinations, none of the treatment options available is curative in patients with advanced cancer. A growing body of evidence is increasingly supporting the idea that malignancies originate from a small fraction of cancer cells, called Cancer Stem Cells (CSC), that show self-renewal and pluripotency and are capable of initiating and sustaining tumor growth. Several studies have shown that, with respect to the bulk of tumor cells, CSC posses a higher degree of resistance to chemotherapy and radiotherapy that could explain the inefficacy of current therapies. The ability to isolate and study these tumor cells provided a powerful tool for the investigation of drug-and radio-resistance mechanisms thus paving the way for the development of novel targeted therapies aimed at the tumor complete eradication. The aim of this PhD thesis was to use CSC lines, derived from CRC specimens, to individuate new potential molecular targets for the development of novel therapies. To this end four colon-CSC lines were subjected to phosphoproteomic analysis by RPPA (Reverse Phase Protein Array) technology. Through this analysis phosphorylation levels of various protein kinases and their substrates were evaluated in order to create an activation map of the main colon-CSC proliferation and cell survival pathways. In parallel colon-CSC lines have been screened in vitro to the action of 80 commercially available protein-kinase inhibitors. This screening has revealed a partial correlation between in vitro sensitivity and phosphoproteomic analysis, but in this study, was not possible to identify predictive factors to infer colon-CSC sensitivity to specific kinase inhibitors. Colon-CSC was sensitive to the inhibition of protein kinase C (PKC), known regulator of cell proliferation and survival. Among PKC inhibitors, the most interesting was the UCN-01, a staurosporine derivative that can also inhibit PDK1 and Chk1. This compound has been already used in clinical trials as antineoplastic agent in combination with conventional chemotherapy. The in vitro treatment of colon-CSC with UCN-01 has demonstrated its ability to enhance the irinotecan cytotoxicity by increasing the apoptotic response. The combined action of UCN-01 and irinotecan caused a marked reduction in the levels of antiapoptotic proteins such as Bcl-XL and Mcl-1 and the activation of caspase 3. The in vivo administration of UCN-01/irinotecan combination, in a mouse model of subcutaneous xenograft, confirmed the observations obtained in vitro, leading to a significant reduction in tumor growth compared to the single treatments. UCN-01 has also shown efficacy in the inhibition of Chk1, as demonstrated by the reduction of the phosphorylation of its target protein cdc25. Inhibition of Chk1, an important regulator of cell cycle, in combination with chemotherapy, could help in reducing the viability of colon-CSC, thus preventing cell cycle arrest and repair DNA damage induced by irinotecan. Although UCN-01 exerts its effect by inhibiting the activity of various protein kinases, this reduced selectivity could be the basis of its effectiveness. The present study demonstrated that it is possible to identify, among the commercially available compounds, those that interfere with processes that regulate colon-CSC survival or proliferation and therefore are potentially able to interfere with tumor growth. The use of newly developed inhibitors, combined with the analysis of genetic alterations or phosphoproteomic, will identify factors predictive of response to therapy and lead to the possibility of developing individualized therapeutic strategies, increasing the likelihood of success of targeted therapy.

Metastatic melanoma, a cancer historically refractory to chemotherapeutic strategies, has a poor prognosis and accounts for the majority of skin cancer related mortality. Although the recent approval of two new drugs combating this disease, Ipilimumab and Vemurafenib (PLX4032), has demonstrated for the first time in decades an improvement in overall survival; the clinical efficacy of these drugs has been marred by severe adverse immune reactions and acquired drug resistance in patients, respectively. Thus, understanding the etiology of metastatic melanoma will contribute to the improvement of current therapeutic strategies while leading to the development of novel drug approaches. In order to identify recurrently mutated genes of therapeutic relevance in metastatic melanoma, a panel of stage III local lymph node melanomas were extensively characterised using high-throughput genomic technologies. This led to the identification of mutations in TFG in 5% of melanomas from a candidate gene sequencing approach using SNP array analysis, 24% of melanomas with mutations in MAP3K5 or MAP3K9 though unbiased whole-exome sequencing strategies, and inactivating mutations in NF1 in BRAF/NRAS wild type tumours though pathway analysis. Lastly, this thesis describes the development of a melanoma specific mutation panel that can rapidly identify clinically relevant mutation profiles that could guide effective treatment strategies through a personalised therapeutic approach. These findings are discussed in respect to a number of important issues raised by this study including the current limitation of next-generation sequencing technology, the difficulty in identifying ‘driver’ mutations critical to the development of melanoma due to high carcinogenic exposure by UV radiation, and the ultimate application of mutation screening in a personalised therapeutic setting. In summary, a number novel genes involved in metastatic melanoma have been identified that may have relevance for current therapeutic strategies in treating this disease.

L’hypertension pulmonaire (HTP) est définie par une valeur de pression artérielle pulmonaire moyenne (PAPm) supérieure à 25 mmHg au repos. Il existe des formes sévères d’HTP (HTPs) avec des valeurs de PAPm encore plus élevées associées à des symptômes plus marqués et à l’apparition de lésions anatomo pathologiques spécifiques. Le diagnostic et le développement de nouvelles thérapies sont des enjeux majeurs pour une meilleure prise encharge de ces patients À l’aide de la tomodensitométrie, nos travaux observationnels chez l’homme atteint debroncho-pneumopathie chronique obstructive (BPCO) ont montré une corrélation de l’HTP avec le remodelage bronchique et non pas avec l’emphysème. L’HTPs peut être une complication rare mais grave de la BPCO. L’évaluation in vivo des modifications du lit vasculaire pulmonaire a permis de confirmer l’existence d’un phénotype particulier chez les patients atteints de BPCO compliquée d’HTPs. De plus, la définition d’un score combiné comprenant des paramètres non invasifs tomodensitométriques devrait permettre de sélectionner plus finement les patients devant subir un cathétérisme cardiaque droit.En parallèle, dans nos travaux expérimentaux, nous avons complété la caractérisation d’un modèle d’hypertension artérielle pulmonaire chez le rat développant un phénotype sévère(HTAPs) et présentant des lésions artérielles pulmonaires caractéristiques de la maladie humaine. Ce modèle, ainsi que des modèles animaux plus classiques d’HTP, nous ont permis d’identifier un rôle du facteur de croissance des nerfs NGF dans cette pathologie, dont le ciblage pourrait ouvrir de nouvelles perspectives thérapeutiques
Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure (mPAP) at or above 25 mmHg at rest. Severe forms of PH (sPH) are characterized by a stronger elevation of mPAP, more marked symptoms and specific pulmonary vascular lesions. Real challenges come from a better diagnosis for these patients and identification of new therapeutic targets to improve their therapeutic care. Our results show by computed tomography that PH associated to chronic obstructive pulmonary disease (COPD) is correlated to airway remodeling and not to emphysema. Severe PH is a rare and serious complication of COPD. We confirmed existence of a phenotype in COPD patients with sPH, by evaluating in vivo modifications of the pulmonary vascular bed in these patients. Moreover, we defined a combined score, which may be a non-invasive tool to select patients for right heart catheterization In parallel, we completed the characterization of a rat model of severe pulmonary arterial hypertension (sPAH) that developed a severe phenotype with pulmonary arterial specific and human-like lesions. In this model, as well as in more classical PH models, our results demonstrated an increased expression of the nerve growth factor NGF and its role in PH and sPAH pathophysiology. These results therefore suggest that NGF may be an interesting target to develop new therapeutic perspectives in this disease

Chordoma is a rare malignant bone tumour, showing notochordal differentiation, which occurs in the axial skeleton. Brachyury, a molecule involved in notochordal development, is a highly specific and sensitive marker for chordoma. It is hypothesised that brachyury or genes involved in its activation are implicated in the pathogenesis of chordoma. As there is currently no effective drug therapy for chordoma the aim of this study was to identify genetic events involved in chordoma pathogenesis with a view to identifying potential therapeutic targets. One hundred chordomas (50 skull-based, 50 non-skull based) were studied. Immunohistochemistry showed that the PI3K/AKT/TSC/mTOR pathway was activated in 65% of chordomas, thereby providing a rationale for testing mTOR inhibitors for the treatment of selected cases. DNA sequencing revealed no mutations in PI3KCA or RAS homologue enriched in brain (Rheb) in 23 tumours. Immunohistochemistry and Western blotting showed activation of the fibroblastic growth factor receptor (FGFR)/RAS/RAF/MEK/ERK/ETS2/brachyury pathway in more than 90% of cases, but no mutations were found in the genes analysed (FGFRs, KRAS, BRAF and brachyury) in 23 tumours. Three percent of cases revealed brachyury amplification but nearly half of the cases showed chromosomal abnormalities involving the brachyury locus. Knockdown of brachyury was achieved in the U-CH1 chordoma cell line using shRNA and resulted in premature cell senescence. These findings demonstrate that brachyury plays an important role in chordoma pathology. FISH analysis showed EGFR copy number gain in 45% of chordomas, including 6% with amplification and 39% with high level polysomy. The EGFR inhibitor, tyrphostin (AG1478) significantly inhibited growth of the chordoma cell line, and Western blotting showed this was associated with reduced phosphorylation of EGFR in a dose dependent manner. This study provides evidence for the first time that selected chordomas may be susceptible to treatment with EGFR inhibitors.

Mestrado em Biomedicina Molecular
Traumatic spinal cord injury (SCI) is a devastating event that leads to loss of neurological functions below the vertebral level of the lesion. As adult neurons from central nervous system (CNS) fail to regenerate when injured, the consequences of SCI are partially or totally irreversible. The lack of regeneration ability of CNS neurons has been studied for years but still no effective treatment was found for this pathology; only steroids are validated and recognized as a pharmacologic treatment attempt, but just limit the lesion extent. This work focused on finding putative candidate genes involved in regeneration that could be targeted for therapy. A bioinformatics analysis based on studies with rodent SCI models, where a regenerative treatment attempt was applied and functional recovery was observed, was performed and some common regulated genes were found in the analysed studies. KIF4A and MPP3 genes were highlighted for further experimental studies in a regenerative model: a rodent model of peripheral nervous system (PNS) injury, with crush or transection of the sciatic nerve. Our results demonstrated that KIF4A and MPP3 are expressed and regulated in the lesioned sciatic nerve and in the corresponding dorsal root ganglia (DRG). Moreover, these genes also showed protein distribution in spinal cord tissue sections, in sciatic nerve and in DRG cuts, revealing that they are neuronal specific. These results represent important remarks to instigate further studies regarding the role of these genes in regenerative processes of lesioned neuronal tissues and the possibility of becoming important therapeutic targets in spinal cord injuries or related pathologies affecting the spinal cord integrity
A lesão traumática da medula espinal é um evento devastador que leva à perda de funções neurológicas abaixo do nível vertebral da lesão. Devido à falta de capacidade regenerativa dos neurónios adultos do sistema nervoso central, quando lesionados, as consequências das lesões são parcial ou totalmente irreversíveis. A falta de capacidade de regeneração dos neurónios do SNC tem sido estudada há anos, mas ainda não foi encontrado um tratamento efetivo para esta patologia; apenas os esteroides são validados e reconhecidos como um tratamento farmacológico, mas só limitam a extensão da lesão. Este trabalho centrou-se na procura de genes hipoteticamente envolvidos em regeneração do sistema nervoso, que possam ser candidatos a alvos de terapia para lesões na medula. Foi realizada uma análise bioinformática baseada em estudos com modelos de roedores com lesão da medula espinal, onde uma tentativa de tratamento regenerativo foi aplicada e observou-se recuperação funcional, e foram levantados os genes regulados comuns aos três estudos. Os genes KIF4A e MPP3 foram destacados para estudos experimentais adicionais num modelo regenerativo: um modelo de roedor, de lesão do sistema nervoso periférico, com esmagamento ou corte do nervo ciático. Os resultados demonstraram que os genes KIF4A e MPP3 são expressos e regulados no nervo ciático lesionado e nos gânglios da raiz dorsal correspondentes. Além disso, estes genes também mostraram distribuição proteica em secções de tecido de medula espinhal, de nervo ciático e em cortes de DRG, desvendando que possam ser específicos de tecido neuronal. Estes resultados representam observações importantes para instigar estudos adicionais sobre o papel destes genes nos processos regenerativos de tecidos neuronais lesionados e a possibilidade de se tornarem alvos terapêuticos importantes para lesões ou patologias relacionadas que afetem a integridade da medula espinal.

La leucémie aiguë myéloïde (LAM) est une pathologie hématologique dont le pronostic reste très défavorable, malgré les progrès réalisés dans la compréhension des mécanismes physiopathologiques sous-tendant son développement. Identifier de nouvelles stratégies anti-leucémiques représente donc une étape clé dans la concrétisation des avancées thérapeutiques. Grâce à la combinaison de plusieurs approches de criblage génétiques et pharmacologiques, l’objectif de ma thèse a été de définir et valider de nouvelles cibles thérapeutiques dans les LAM. La première partie de ma thèse a eu pour but de transposer en clinique l’inhibition de la Glycogen Synthase Kinase 3 (GSK3). La stabilisation de la β-caténine secondaire à l'inhibition concomitante des deux paralogues de GSK3, représente un obstacle à l’utilisation clinique de cette classe thérapeutique. Mettant à profit la présence d'un «switch» Asp133 à Glu196 dans les domaines de liaison ATP de GSK3, nous avons identifié un inhibiteur sélectif du paralogue GSK3α et mené des études précliniques validant le BRD0705 comme nouveau traitement pro-différenciant dans les LAM. De plus, une combinaison de profilage métabolomique et d'approches de criblage haut débit à l’aide d’une banque de shRNA a permis d'identifier un nouveau lien entre EVI-1, la voie de la créatine kinase et la voie de signalisation GSK3. La deuxième partie de ma thèse a porté sur l'identification de nouvelles cibles thérapeutiques en utilisant une approche de criblage par banque de shRNA dans le modèle murin de LAM porteur de la translocation MLL-AF9. VCP, une AAA-ATPase, a ainsi été identifiée puis validée comme cible thérapeutique. Nous avons montré que VCP orchestre la génération d'une plateforme à ADN simple brin recouverte de RPA, ce qui entraîne l'activation de la kinase ATM et la HR. Dans leur ensemble, nos découvertes permettent une meilleure compréhension de la biologie des LAM et participeront ainsi à l’amélioration des traitements futurs de cette pathologie
Despite the significant progress made in understanding Acute Myeloid Leukemia oncogenesis over the last decades, this disease remains devastating and the overall five-year survival does not exceed 17%. Developing new translational research strategies focused on the identification of druggable oncogenic targets is critical to continued progress in AML treatment. The goal of this work was to define and validate novel leukemia-specific dependencies using small-molecule inhibitors and RNA-interference-based high-throughput screening methods.The first part of my thesis work aimed at translating Glycogen synthase kinase 3 (GSK3) inhibition into the clinic. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, were a concern in the clinical translation of this target candidate. Specific knock-down of GSK3α or GSK3β alone does not increase β-catenin, thereby offering a conceptual resolution to GSK3 targeting. The design of selective ATP-competitive inhibitors posed a drug discovery challenge due to the high homology in the GSK3α and GSK3β ATP binding domains. Taking advantage of an Asp133 ® Glu196 “switch” in the GSK3 paralog hinge binding domains, we identified a first-in-class GSK3α selective inhibitor and conducted preclinical studies validating BRD0705 as a promising new differentiation therapy in AML. In addition, a combination of a metabolomic profiling and a pooled shRNA screening method identified a new interplay between the oncogene EVI-1, the creatine kinase pathway and GSK3 signaling. The second part of my studies focused on identification of new therapeutic targets using an in vivo pooled shRNA screening approach in the MLL-AF9-driven AML mouse model. VCP, an AAA-ATPase, was thus identified and validated as a top target. We demonstrated that VCP orchestrates RPA-coated-single-stranded-DNA platform generation, resulting in ATM kinase activation and subsequent HR. Taken together, our discoveries increased our understanding of AML biology and may therefore contribute to novel and more efficacious treatments for this highly aggressive and lethal disease

Every year, more than one million of people worldwide are diagnosed with colorectal cancer (CRC). Even though preventive screenings have been able to reduce incidence and mortality, nearly half of the patients die following the diagnosis and the treatment. In particular, a not negligible part is due to recurrences after treatment, probably caused by a limited efficacy of the current therapies. To improve the therapeutic success and options, repositioned or new drugs and novel therapeutic targets should be taken in consideration. Moreover, in the last years, large cancer genome analyses projects have shed new light on previously unknown genetic and biological makeup of this disease. Starting from a collection of amplified genes identified in CRC samples by The Cancer Genome Atlas (TCGA), the aim is to identify whether these genes have an oncogenic activity by using a RNAi screening, with a drug discovery perspective. Using colon cancer cell lines, we set up a functional RNAi screening, to address the impact of downregulation of the putative oncogenes on cell viability. The preliminary results have shown that among the genes, the silencing of only one could indeed significantly reduce cell viability, that was STARD3. Further validations confirmed STARD3 activity on cell viability, suggesting its possible involvement in cell cycle regulation. In fact, cell cycle analyses revealed an alteration of the phases, showing a decrease of G1 and S phases and concomitant increase of G2/M phase when STARD3 was knockdown. The effect of STARD3 downregulation, that was a block of G2/M transition, also increased the population of cells in sub-G1, a status often associated with apoptosis. Programmed cell death was indeed analysed, showing that the amount of apoptosis was higher after silencing among all cell lines. Moreover, we shown that STARD3 is necessary for cancer cell lines capability to grow regardless their interaction with solid substrate, hence substantiating the general concept that STARD3 could be considered a proper oncogene. Whereas further investigation should be carried out, STARD3 could represent a novel oncogene that play a supporting role in colorectal cancer oncogenesis and development. Finally, harboring a cholesterol binding domain, through which it exerts its function, STARD3 could represent a suitable pharmacological target of a novel class of inhibitors or nucleic acids.

Development of resistance is the major hindrance to successful chemotherapy treatment in oesophago-gastric (OEG) cancers. Platinum based chemotherapy prolong the survival however only 20 - 30 % of patients survive 5- years since diagnosis of the disease. Therefore understanding of the mechanisms that underlie this phenomenon and identification of novel biomarkers/targets that could predict the response to the treatment and sensitize these tumours to current therapy is needed. We established a panel of human cancer cell lines of oesophagus (OE21 and OE33) and gastric cardia (AGS) resistant to oxaliplatin, cisplatin and docetaxel. To study mechanisms of drug resistance we performed gene expression profiling on resistant and parental lines and differentially expressed genes involved in sphingolipids / lysosomes metabolism have been shown as associated with drug resistance. Selected markers were validated on mRNA and protein levels among the panel of OEG cell lines and clinical specimens. Cellular levels of sphingolipid species were determined in drug sensitive and resistant lines using mass spectrometry. This study revealed a positive correlation between over-expression of sphingosine kinase 1 (SPHK1) and increased levels of sphingosine -1-phosphate (S1P) associated with drug resistance in gastric cancer cell lines. Moreover it showed the predictive value of SPHK1 as high level of this protein correlates with poor survival of OEG cancer patients treated with cisplatin based chemotherapy, in contrast to those patients that received surgery alone. Additionally, lipidomic profiling data showed possibly distinct mechanisms of drug resistance between gastric and oesophageal tumours, indicating that mechanisms of drug resistance are likely cell type, rather than drug specific. In conclusions, this study proofs the clinical relevance of our in vitro experimental models to study mechanisms of drug resistance in OEG tumours and provides the source of novel biomarkers for targeted therapy strategies in this disease.

Chronic myeloid leukemia (CML) has long served as a paradigm for new insights into the cellular origin, pathogenesis and treatment of human cancers. ABL tyrosine kinase inhibitor (TKI) therapies have had remarkable effects on treatment of early phase CML. However, TKI monotherapies are not curative, and initial and acquired TKI resistance remain clinically challenging. Particularly, CML stem/progenitor cells are insensitive to TKIs. Therefore, novel treatments and predictive biomarkers are clearly needed. In this work, I studied the biological effects of dual BCR-ABL and JAK2 suppressions on TKI-nonresponder stem/progenitor cells, and identified and characterized novel microRNA (miRNA) biomarkers in these cells. I examined the biological effects of a new JAK2 inhibitor, BMS-911543, in combination with TKIs on CD34⁺ CML cells from IM-nonresponders. I demonstrated that combination therapy significantly reduces JAK2/STAT5 and CRKL activities, induces apoptosis, inhibits colony growth, and eliminates leukemic stem cells in vitro, while sparing healthy counterparts. I further showed that oral BMS-911543 combined with dasatinib is more effective in eliminating leukemic cells in an aggressive mouse model of BCR-ABL⁺ human leukemia. Next, I identified differentially expressed miRNAs in CD34⁺ CML cells using RNA-seq analysis, and validated the results in additional samples using high-throughput qPCR. Potential miRNA target genes were also identified by integrating miRNA expression profiles with gene expression profiles using strand-specific RNA-seq. These studies revealed that expression of miR-185 is significantly reduced in CD34⁺ CML cells from TKI-nonresponders compared to TKI-responders. Restoration of miR-185 expression by lentiviral transduction in CD34⁺ TKI-nonresponder cells significantly impairs survival of these cells and sensitizes them to TKI treatment in vitro and in vivo. Additionally, I validated the target genes of miR-185 to rationalize its roles in CML. Lastly, I demonstrated that the expression levels of several miRNAs, including miR185, were restored in patients treated with nilotinib, suggesting their potential as biomarkers to predict clinical response to TKI therapies. These studies have uncovered the biological significance of JAK2 and miR-185 in regulation of the properties of drug-insensitive CML stem/progenitor cells, and their potential as therapeutic targets for improved treatments with TKIs especially in patients at risk of developing TKI resistance.
Medicine, Faculty of
Experimental Medicine, Division of
Medicine, Department of
Graduate

Tamoxifen, a competitive inhibitor of oestradiol binding to the oestrogen receptor ER, remains a key anti-hormonal treatment for ER ve breast cancer although its effectiveness is limited by development of resistance. It is hypothesised that in addition to blockade of pro-proliferative/anti-apoptotic genes, anti-oestrogens exert an early protective effect, inducing cell survival and pro-invasive genes that enable a subset of cells to escape the anti-tumour effects of the anti-oestrogens and facilitate disease progression. It was hoped that identification of such early compensatory events in this thesis and their subsequent therapeutic targeting in combination with anti-oestrogens would be able to improve anti-tumour response. Proof of this principle exists since co-targeting of anti-oestrogen-induced epidermal growth factor receptor EGFR alongside tamoxifen has been reported to improve growth inhibition in ER ve MCF-7 human breast cancer cells and delay acquisition of resistance. After filtering, hierarchical clustering and ontological investigation, 8 possible compensatory genes were identified as anti-oestrogen tamoxifen and faslodex-induced /oestrogen-suppressed at an early time point in MCF-7 cells in vitro using cDNA microarrays bearing 1200 cancer-related genes, comparing anti-oestrogen with oestrogen E2 treatment and control oestrogen deprived conditions. RT-PCR and protein investigation gave further insight into expression levels of these genes based on ER occupancy. Genes induced by both an anti-oestrogen-occupied ER and an unoccupied ER versus E2 treatment were the Rnd family member RhoE and nucleoside diphosphate kinase NME3 implicated in migration and cell survival respectively, and the anti-apoptotic transcription factor NFkBl. The adhesive junction protein 5-catenin, cell survival elements 14-3-3 and chaperone Bag-1 and nuclear serine/threonine kinase NDR implicated in progression were all induced by an anti-oestrogen occupied ER but not by an unoccupied ER versus E2 treatment. Co-treating with the NFkB inhibitor parthenolide and faslodex suppressed NFkBl DNA binding, transcriptional activity and improved growth inhibition of MCF-7 cells. These data demonstrate that several genes of adverse potential are induced during the anti-oestrogen-responsive phase, and that their co-targeting has promise to improve anti-tumour response.

Mutational activation of RAS is one of the most common molecular abnormalities associated with acute myeloid leukaemia (AML). Normal human haematopoietic progenitor cells (HPC) expressing mutant RAS overproduce ROS due to NADPH oxidase (NOX) activation and this promotes the proliferation of these cells as well as AML blasts. The mechanisms by which ROS promote proliferation is however unclear. The current study investigated the effect of RAS-induced ROS production on gene expression in normal HPC using gene expression profiling (GEP) and assessed whether ROS-induced gene expression changes contributed to the pro-proliferative phenotype. In order to determine the ROS-specific GEP, Affymetrix Human Exon 1.0ST arrays were used for the comparison of mutant RAS and control cells cultured in the presence or absence of the NOX inhibitor, DPI, which strongly suppressed the production of ROS. This analysis showed that RAS changed the expression of 342 genes. Of these, 24 genes were specifically altered in response to ROS production by these cells and amongst these increased expressions of genes of the glycolytic pathway were prominent. To establish the functional significance of up-regulated expression of glycolytic enzymes, aldolase C (ALDOC) was investigated since it showed greatest induction with ROS. ALDOC was directly induced by physiological levels of ROS in both HPC and AML cell lines. Further, overexpression of ALDOC demonstrated that its overexpression promoted the proliferation and serum-independent survival of leukaemic cell lines. Conversely, antiproliferative effects were observed when ALDOC was knocked-down in cells known to have high levels of constitutive ROS production. Given the high frequency of ROS production in AML, this study provides a plausible mechanism of enhanced glycolysis seen in this disease and suggests that agents restoring the redox environment could be used to correct metabolic imbalances which contribute to treatment resistance.

Les tumeurs rhabdoïdes (TR) constituent un rare cancer indifférencié du jeune enfant et du nourrisson, avec un âge médian au diagnostic de 20 mois. Ces tumeurs sont caractérisées par une inactivation biallélique du gène suppresseur de tumeur SMARCB1, un des membres du complexe SWI/SNF, acteur majeur du remodelage de la chromatine, sans autre altération génomique récurrente. Le pronostic des TR est péjoratif, le taux de survie globale atteignant 30% dans la plupart des séries, malgré des approches thérapeutiques conventionnelles particulièrement agressives. Les approches d’immunothérapies ont obtenu un succès certain dans certains cancers de l’adulte, et récentes analyses de l’infiltrat immun des cancers pédiatriques ne montrent pas un fort taux de tumeurs infiltrées à l’exception de rare types de cancers dont les TR intracrâniennes. Nous avons donc procédé à une analyse multimodale de l’infiltrat immun de cohortes de patients ainsi que d’un modèle de TR murines établi dans notre laboratoire. Nous avons identifié une forte proportion de tumeurs infiltrées dans certains sous-groupes de TR. Cet infiltrat était composé à la fois de cellules myéloïdes incluant des populations au phénotype immunosuppresseur, et lymphocytaires T notamment de phénotype résident mémoire caractérisées par une forte expansion clonale probablement spécifique d’un antigène tumoral. Nous avons identifié des cibles thérapeutiques communes aux tumeurs humaines et au modèle murin syngénique, et trouvé que cibler l’infiltrat lymphocytaire T ou myéloïde était susceptible d’induire une réponse tumorale complète avec induction d’une mémoire immunitaire, confirmant le caractère immunogénique des TR, et apportant de nouvelles stratégies thérapeutiques utiles en clinique. Enfin, nous avons identifié que les TR étaient le site d’une réexpression de rétrovirus endogènes, dépendante de celle de SMARCB1, avec activation des voies de l’interféron, apportant une base à une immunogénicité des TR issue du génome non codant
Rhabdoid tumors (RT) are highly undifferentiated cancers occurring in infancy and early childhood, with a median age at diagnosis about 20 months. These tumors are characterized by the biallelic inactivation of SMARCB1 tumor suppressor gene, core member of the SWI/SNF complex, one major chromatin remodeling actor, in an otherwise highly stable genome. The prognosis of RT is dismal with overall survival hardly reaching 30% in most series, despite particularly aggressive conventional treatment. Immunotherapy approaches has gained a striking success within some adult cancer types and recent analyses of immune cell content of pediatric cancers don’t reveal a high rate of infiltrated tumors, except in few tumor types such as intracranial rhabdoid tumors. Then, we conducted a comprehensive analysis of the immune context of both human RT cohorts and a mouse RT model, including at single cell level. We identified a high recurrence of infiltrated tumors, in a RT-subgroup related manner, composed of both myeloid cells including cells with immune suppressive phenotypes, and T cells with notably a tissue resident memory phenotype demonstrating a high clonal expansion highly suggestive of immunogenicity. We identified common targetable immune populations between human and mouse RTs, and found that targeting both T and myeloid infiltrating cells was able to induce complete anti-tumor response with induced memory, confirming the immunogenic properties of RTs, and identifying new therapeutic strategies of clinical relevance. We finally identified that RTs were the site of SMARCB1-dependent endogenous retroviruses reexpression, with subsequent activation of interferon signaling, likely triggering the immune response in the context of RT, and providing a basis of non-coding genome-driven immunogenicity for these tumors

Les DIPG représentent les tumeurs cérébrales pédiatriques les plus sévères. Aucun progrès dans leur prise en charge n’a été accompli au cours des 50 dernières années et la radiothérapie ne demeure que transitoirement efficace. Récemment, une mutation somatique de l’histone H3 (K27M) spécifique des DIPG a été trouvée chez environ 95% des patients. Elle est aujourd’hui considérée comme l'événement oncogénique initiateur de ces tumeurs. Deux sous-groupes majeurs de patients présentant des programmes oncogéniques et une réponse à la radiothérapie distincts peuvent être définis en fonction du gène dans lequel l’altération survient, codant les variantes protéiques H3.1 ou H3.3. Nous avons réalisé deux cribles de létalité synthétique par ARN interférence ciblant le kinome humain afin d'identifier d’une part les gènes nécessaires à la survie des DIPG et d’autre part les gènes dont l’inhibition sensibilise ces tumeurs à la radiothérapie. Le double objectif de ce projet était de mieux comprendre la biologie sous-jacente à l’oncogenèse des DIPG et de découvrir de nouvelles cibles thérapeutiques.Nous avons mis en évidence 41 gènes requis pour la survie des DIPG sans effet délétère majeur sur des cellules contrôles normales. Parmi eux, nous avons identifié VRK3 codant une serine thréonine kinase dont les fonctions restent peu décrites à ce jour et qui n'avait jamais été associée préalablement à l'oncogenèse de DIPG. Nous avons pu confirmer par la suite que son inhibition conduit à un arrêt total de la prolifération des cellules de DIPG associé à d’importants changements morphologiques, plus particulièrement dans les tumeurs mutées pour H3.3-K27M. VRK3 constitue par conséquent une nouvelle cible thérapeutique prometteuse dans cette pathologie à l’issue fatale pour la totalité des patients.En parallèle, un crible de survie similaire a été réalisé en conjonction avec l’irradiation des cellules. Très peu d’ARN interférents ont permis de sensibiliser les cellules H3.3-K27M à la radiothérapie contrairement aux cellules H3.1-K27M. Ce travail nous a permis de mettre en évidence une différence significative de radiosensibilité des modèles vitro de DMG en fonction du sous-groupe de tumeurs considéré, H3.1- ou H3.3-K27M muté, conformément à la survie des patients observée suite à la radiothérapie. Ces résultats inédits laissent entrevoir des perspectives d’amélioration du traitement de référence des patients atteints de DIPG actuellement identique quelle que soit leur génotype
DIPG is one of the most severe paediatric brain tumours. No progress has been made in their management over the past 50 years and radiotherapy remains only transiently effective. Recently, a specific somatic mutation in the histone H3 (K27M) has been found in approximately 95% of DIPG patients and can be considered as the oncogenic driver of these tumours. Two major subgroup of patients with distinct oncogenic program and response to radiotherapy can be defined according to the gene in which the alteration occurs, encoding the H3.1 or H3.3 protein variants. We performed two synthetic lethality screens by RNA interference targeting the human kinome in order to identify the genes responsible for DIPG cell survival, as well as those sensitizing tumour cells to radiotherapy after inhibition. The dual purpose of this project was to better understand the biology underlying oncogenesis of DIPGs and to discover new therapeutic targets.We identified 41 genes required for DIPG cell survival with no major deleterious effect on normal control cells. Among them, we identified VRK3, a serine threonine kinase never involved in DIPG oncogenesis with functions remaining poorly described to date. We have shown that its inhibition leads to a complete arrest of DIPG cell proliferation and is additionally associated with important morphological changes, more particularly in H3.3-K27M mutated tumours. VRK3 is therefore a promising new therapeutic target for all patients in this fatal pathology.In parallel, a similar survival screen was performed in conjunction to cell radiation and very few interfering RNAs enhance H3.3-K27M cell radiosensitivity, in contrast to H3.1-K27M cells. These data highlighted a significant difference in radiosensitivity of the DMG in vitro models in H3.1- versus H3.3-K27M mutated tumours, in a concordant way with patient survival following radiotherapy. These unprecedented results suggest new opportunities for improving the current treatment of DIPG patients regardless of their genotype

Metastatic prostate cancer is currently incurable. Metastasis is thought to result from changes in the expression of specific metastasis-driving genes, leading to a cascade of activated downstream genes setting the metastatic process in motion. As such, metastasis-driving genes could provide effective therapeutic targets and prognostic biomarkers for improved disease management. In search of potential metastasis-driving genes, genes with elevated expression in patient-derived metastatic LTL-313H prostate cancer tissues, as distinct from non-metastatic LTL-313B tissues, were identified. Among these genes, TIMELESS and DLX1 were promising. Unfortunately, their silencing and overexpression in prostate cancer cells did not lead to inhibition of metastatic properties, indicating that they were not metastasis-driving genes. A different, novel approach was used based on the notion that metastasis-driving genes can activate genes in an amplification cascade fashion. Accordingly, I used the IPA’s Upstream Regulator Analysis tool to analyze the differential gene expression profile of the metastatic and non-metastatic tissues to predict the upstream master regulatory (metastasis-driving) genes accountable for the differential expression. Six candidate genes were identified, including GATA2, a pioneer factor-encoding gene. Elevated GATA2 expression in clinical metastatic prostate cancer specimens correlated with poor patient prognosis. Furthermore, GATA2 gene silencing in human prostate cancer LNCaP cells led to marked reduction in cell proliferation, cell migration, tissue invasion, focal adhesion disassembly and a dramatic change in transcriptional activity, indicating that GATA2 plays a critical role in prostate cancer metastasis. As such, GATA2 could represent a metastasis-driving gene and a potential therapeutic target for inhibiting the growth and metastasis development in prostate cancer. Further analysis of GATA2-regulated genes led to the development of a GATA2-based metastatic gene signature. Its prognostic value was confirmed using two prostate cancer patient cohorts. In addition, it was shown to be a prognostic factor for risk assessment of metastasis development, independent of the widely used D’Amico prognostic classification system. However, a thorough validation is critical and, if successful, the GATA2-based gene signature could lead to a paradigm shift in the management of early prostate cancer. In conclusion, the findings of this study appear to be potentially useful for improved management of metastatic prostate cancer.
Medicine, Faculty of
Graduate

Le virus humain T lymphotrope de type 1 (HTLV-1) est l’agent étiologique de la leucémie/lymphome T de l’adulte (ATLL) qui se développe après plusieurs décennies et pour laquelle il n’existe à ce jour pas de traitement efficace. Parmi les protéines virales de HTLV-1, Tax et HBZ jouent un rôle déterminant dans le développement de l’ATLL. Si Tax participe au processus leucémogène dès les étapes précoces, HBZ jouerait plutôt un rôle dans le maintien du phénotype tumoral dans les étapes tardives. Dans ce contexte, là nous nous sommes intéressés à la régulation de l’expression des protéines anti-apoptotiques Bfl-1 et Bcl-xL, par les protéines virales Tax et HBZ. Nous avons montré que Tax induit l’expression des protéines anti-apoptotiques Bfl-1 et Bcl-xL de la famille Bcl-2 via la voie NF-κB, alors que HBZ n’a aucun effet sur leur expression. De plus, Tax coopère avec les facteurs de transcription c-Jun et JunD de la voie AP-1 pour augmenter l’expression de ces gènes anti-apoptotiques. En revanche, HBZ module uniquement la trans-activation de bfl-1 induite par Tax. L’ensemble de nos résultats indique donc que Tax joue un rôle prépondérant dans l’activation de l’expression de Bfl-1 et de Bcl-xL et suggère que Bfl-1 et Bcl-xL sont exprimées au cours des étapes précoces et tardives du développement de l’ATLL. Par une stratégie d’ARN interférence, nous avons ensuite montré que Bfl-1 et/ou Bcl-xL sont impliquées dans la survie de lignées cellulaires T infectées par HTLV-1, suggérant que Bfl-1 et Bcl-xL représentent des cibles thérapeutiques potentielles pour traiter l’ATLL. Actuellement, il existe des petites molécules ciblant les membres anti-apoptotiques de la famille Bcl-2, mais aucune ne cible spécifiquement Bfl-1. En collaboration avec la société IMAXIO, nous avons identifié par deux cribles à haut débit 83 molécules capables d’inhiber l’activité anti-apoptotique de Bfl-1. L’une de ces molécules induit spécifiquement la mort de lignées cellulaires T infectées par HTLV-1 pour lesquelles Bfl-1 représente un gène de survie. Ainsi, ce travail doit permettre à terme de développer de futurs médicaments dirigés contre Bfl-1 et de proposer une nouvelle stratégie thérapeutique ciblée contre l’ATLL
Human T lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) that develops after several decades and for which there is no effective treatment. Among the viral proteins of HTLV-1, Tax and HBZ play a major role in the development of ATLL. If Tax participates in the initiation of leukemogenesis from the early stages, HBZ rather plays a role in maintaining the tumor phenotype in the late stages. The aims of our study were to better understand the regulation of Bfl-1 and Bcl-xL anti-apoptotic protein expression by Tax and HBZ viral proteins, as well as their role in the survival of HTLV-1-infected T-cells to propose new therapeutic strategies. We showed that Tax induces Bfl-1 and Bcl-xL expression via the NF-κB pathway, whereas HBZ has no effect on their expression. Tax also cooperates with c-Jun and JunD transcription factors of AP-1 family to increase the expression of these anti-apoptotic genes. By contrast, HBZ modulates the Tax-induced bfl-1 trans-activation. Altogether, our data indicate that Tax plays a key role in activating Bfl-1 and Bcl-xL expression and suggests that Bfl-1 and Bcl-xL are potentially expressed during the early and the late stages of ATLL development. Using short hairpin RNA strategy, we then showed that Bfl-1 and/or Bcl-xL are involved in HTLV-1-infected T-cell line survival, indicating that Bfl-1 and Bcl-xL represent potential therapeutic targets in the case of ATLL. One approach currently being developed in anti-cancer drug discovery is to search for small inhibitory compounds targeting anti-apoptotic proteins of the Bcl-2 family. But so far, no drug specifically targeting Bfl-1 is available. In collaboration with the IMAXIO Company, we have identified 83 molecules able to inhibit Bfl-1 anti-apoptotic activity using two high-throughput screening. One of these molecules specifically induced the death of HTLV-1-infected T-cell for which Bfl-1 represents a survival gene. This work provides new insight for long-term development of future drugs directed against Bfl-1 and should allow us to propose new therapeutic strategy for ATLL treatment

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, June 2009.
"April 2009." Cataloged from PDF version of thesis.
Includes bibliographical references.
Tamoxifen resistance is the biggest problem in endocrine treatment against hormone receptor positive breast cancer patients. HER2 is a membrane receptor tyrosine kinase that is known to correlate with poor disease outcome and unresponsiveness to endocrine treatment. Although much work has been done over the past decades to elucidate pathways involved in HER2 receptor signaling, the map of network-wide signaling events that contributes to the resistance to Tamoxifen treatment has not been characterized, making it difficult to pin-point the downstream drug target to revert the Tamoxifen resistance. To gain a molecular understanding of the mechanisms by which cells gain drug resistance, we have employed a proteomic analysis by mass spectrometry to quantitatively analyze cellular tyrosine phosphorylation signaling events in breast cancer model systems and human tumor samples. As a result of research, we have identified the major differences in downstream signaling pathways between Tamoxifen sensitive and Tamoxifen resistant breast cancer cell line models. These findings were further analyzed in Tamoxifen sensitive, and Tamoxifen treated/recurred patient samples to study clinical relevance. Specifically, we determined that P13K/Akt, MEK/ERK, and Src/FAK/Abl pathways are major components of the Tamoxifen resistance. We further showed that they signaling components are possible drug targets to revert Tamoxifen resistance. This study revealed cell-context specific network-wide changes in signaling events in response to use of therapeutic drugs. This is, to our first knowledge, the first phosphoproteomic analysis of the signaling network in breast cancer to address Tamoxifen resistance. We believe that same approach is applicable to other drug resistance problems in various disease settings.
by Hideshiro Saito-Benz.
Ph.D.

Cancer signaling pathways have been extensively investigated. However, how cross-talk processes and integrates pathway responses in cancer is still far from being completely elucidated. Genetic and epigenetic alterations lead cells to aberrant proliferation and escapement from physiological mechanism controlling cell growth, survival and migration. In this context, specific mutations transform cellular proto-oncogenes to oncogenes, triggering hyperactivation of signaling pathways, whereas inactivation of tumor suppressors removes critical negative regulators of signaling. MAPK and PI3K/AKT pathways often present mutated genes in different types of cancer, and are strongly involved in intensive cross-talk. There is an ever-increasing awareness that computational modeling and simulation are more than helpful in improving the understanding at cellular and molecular levels, in speeding-up the drug discovery process through the identification of alternative strategies with the aim to overcome drug resistance in cancer. The main objective of this thesis is to reveal biochemical and genetic mechanisms underlying drug resistance in melanoma and thyroid cancer through the application of ordinary differential equations based models coupled with algorithmic approaches. These tumors share both MAPK and PI3K/AKT signaling pathway, with the presence of BRAF V600E mutation. Computational approaches developed in this PhD project were demonstrated to be able to find novel therapeutic targets and prognostic biomarkers for a more effective treatment in melanoma and thyroid cancer.

De nos jours, la communauté scientifique s'accorde sur la nécessité de diagnostics et de thérapies personnalisés pour les patients atteints de cancer, conçus par des études translationnelles combinant approches expérimentales et statistiques. Les défis actuels incluent la validation de modèles expérimentaux précliniques et leur profilage multi-omiques, ainsi que la conception de méthodes bioinformatiques et mathématiques dédiées pour identifier les combinaisons de médicaments optimales pour chaque patient.Cette thèse a visé à concevoir de telles approches statistiques pour analyser différents types de données à grande échelle et les intégrer afin d'identifier les vulnérabilités ciblables des lignées cancéreuses. Nous nous sommes focalisés sur les altérations du complexe de remodelage de la chromatine SWI/SNF, muté dans ~20 % des cancers, pour lesquels aucune thérapie efficace n'est disponible. Nous avons utilisé un panel de lignées cellulaires isogéniques HAP1 mutées pour les sous-unités du complexe SWI/SNF ou d'autres enzymes épigénétiques, pour lesquelles des données de transcriptomique, protéomique et de criblage de médicaments étaient disponibles.Nous avons travaillé sur quatre axes méthodologiques. Premièrement, nous avons conçu une méthodologie optimisée d'enrichissement pour détecter les voies de régulation différentiellement activées entre mutants et type sauvage. Ensuite, nous avons croisé les résultats des criblages de médicaments et les bases d'interaction gène-médicament, pour inférer des voies de régulation ciblables spécifiquement chez les lignées mutantes. Ensuite, la validation de ces cibles potentielles a été réalisée à l'aide d'une nouvelle méthode détectant la létalité synthétique à partir de données transcriptomiques et CRISPR de lignées cancéreuses indépendantes du projet DepMap. Enfin, en vue de l'optimisation de thérapies multi-agents, nous avons conçu une première représentation digitale des voies de régulation ciblables pour les tumeurs mutées SMARCA4, en construisant un réseau dirigé d'interaction protéine-protéine reliant les cibles inférées des analyses multi-omiques HAP1 et CRISPR DepMap. Nous avons utilisé la base de données OmniPath pour récupérer les interactions protéiques directes et ajouté les protéines liant celles présentes dans le réseau avec l'algorithme Neko.Ces développements méthodologiques ont été appliqués aux ensembles de données disponibles pour le panel HAP1. En utilisant notre méthodologie d'enrichissement optimisée, nous avons identifié le Métabolisme des protéines comme la catégorie de voies de régulation la plus fréquemment dérégulée dans les lignées SWI/SNF-KO. Ensuite, l'analyse de criblage de médicaments a révélé des médicaments cytotoxiques et épigénétiques ciblant sélectivement les mutants SWI/SNF, notamment les inhibiteurs de CBP/EP300 ou de la respiration mitochondriale, également identifiés comme létaux synthétiques par notre analyse CRISPR DepMap. Ces résultats ont été validés dans deux modèles expérimentaux isogéniques indépendants. L'analyse CRISPR DepMap a également été utilisée pour identifier des interactions létales synthétiques dans le glioblastome, qui se sont révélées pertinentes pour des lignées cellulaires dérivées de patients et sont en cours de validation.En résumé, nous avons développé des méthodes computationnelles pour intégrer des données d'expression multi-omiques avec des criblages de médicaments et des tests CRISPR, et identifié de nouvelles vulnérabilités chez les mutants SWI/SNF, qui ont été validées expérimentalement. Cette étude était limitée à l'identification de monothérapies efficaces. Pour l'avenir, nous proposons de concevoir des modèles mathématiques représentant les réseaux de protéines ciblables à l'aide d'équations différentielles et de les utiliser dans des procédures d'optimisation numérique et d'apprentissage automatique pour étudier les cibles médicamenteuses concomitantes et personnaliser les combinaisons de médicaments
Nowadays the cancer community agrees on the need for patient-tailored diagnostics and therapies, which calls for the design of translational studies combining experimental and statistical approaches. Current challenges include the validation of preclinical experimental models and their multi-omics profiling, along with the design of dedicated bioinformatics and mathematical pipelines (i.e. dimension reduction, multi-omics integration, mechanism-based digital twins) for identifying patient-specific optimal drug combinations.To address these challenges, we designed bioinformatics and statistical approaches to analyze various large-scale data types and integrate them to identify targetable vulnerabilities in cancer cell lines. We developed our pipeline in the context of alterations of the SWItch Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex. SWI/SNF mutations occur in ~20% of all cancers, but such malignancies still lack efficient therapies. We leveraged a panel of HAP1 isogenic cell lines mutated for SWI/SNF subunits or other epigenetic enzymes for which transcriptomics, proteomics and drug screening data were available.We worked on four methodological axes, the first one being the design of an optimized pathway enrichment pipeline to detect pathways differentially activated in the mutants against the wild-type. We developed a pruning algorithm to reduce gene and pathway redundancy in the Reactome database and improve the interpretability of the results. We evidenced the bad performance of first-generation enrichment methods and proposed to combine the topology-based method ROntoTools with pre-ranked GSEA to increase enrichment performance .Secondly, we analyzed drug screens, processed drug-gene interaction databases to obtain genes and pathways targeted by effective drugs and integrated them with proteomics enrichment results to infer targetable vulnerabilities selectively harming mutant cell lines. The validation of potential targets was achieved using a novel method detecting synthetic lethality from transcriptomics and CRISPR data of independent cancer cell lines in DepMap, run for each studied epigenetic enzyme. Finally, to further inform multi-agent therapy optimization, we designed a first digital representation of targetable pathways for SMARCA4-mutated tumors by building a directed protein-protein interaction network connecting targets inferred from multi-omics HAP1 and DepMap CRISPR analyses. We used the OmniPath database to retrieve direct protein interactions and added the connecting neighboring genes with the Neko algorithm.These methodological developments were applied to the HAP1 panel datasets. Using our optimized enrichment pipeline, we identified Metabolism of proteins as the most frequently dysregulated pathway category in SWI/SNF-KO lines. Next, the drug screening analysis revealed cytotoxic and epigenetic drugs selectively targeting SWI/SNF mutants, including CBP/EP300 or mitochondrial respiration inhibitors, also identified as synthetic lethal by our Depmap CRISPR analysis. Importantly, we validated these findings in two independent isogenic cancer-relevant experimental models. The Depmap CRISPR analysis was also used in a separate project to identify synthetic lethal interactions in glioblastoma, which proved relevant for patient-derived cell lines and are being validated in dedicated drug screens.To sum up, we developed computational methods to integrate multi-omics expression data with drug screening and CRISPR assays and identified new vulnerabilities in SWI/SNF mutants which were experimentally revalidated. This study was limited to the identification of effective single agents. As a future direction, we propose to design mathematical models representing targetable protein networks using differential equations and their use in numerical optimization and machine learning procedures as a key tool to investigate concomitant druggable targets and personalize drug combinations

Lysosomal storage disorders (LSDs) are rare diseases caused by inherited mutations in genes coding for proteins of the endolysosomal system. The lysosome is an organelle responsible for the degradation of dysfunctional organelles and for the catabolism, and subsequent recycling, of macromolecules within the cell. When this process becomes defective the substrates of lysosomal catabolism accumulate; these can include lipids, proteins, polysaccharides, nucleotides and diverse combinations of all three. The phenotypic spectrum of these diseases in isolation, and even more so as a group, is extremely broad but an almost universal consequence of lysosomal dysfunction is severe, early onset neurodegeneration. Neurodegenerative diseases of ageing such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease represent a major challenge to the provision of human healthcare in light of an ageing global population. Whilst some commonalities exist between these three diseases a myriad of hypotheses for the onset of pathology has been proposed. There is growing evidence for involvement of the lysosome in all three of these diseases. We have been looking at specific lysosomal pathologies such as lipid storage, endocytosis and Ca2+ dysregulation in forms of these three neurodegenerative diseases of ageing whilst using LSDs as models to inform our study. We have found that lysosomal alkalisation in familial models of Alzheimer’s disease results in changes to lipid and Ca2+ homeostasis in this compartment and identified a lysosomal ion channel, transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1), as a key constituent of this process. Our study of models of Huntington’s disease have implicated the Niemann-Pick type C1 protein (NPC1) in the pathogenesis of this disease and identified ways in which this could be therapeutically targeted. Finally, we have found evidence of Ca2+ dyshomeostasis throughout the cell in genetic models of Parkinson’s disease which have defects in lysosomal proteins. Taken together, these studies strengthen the evidence for lysosomal involvement in neurodegenerative diseases of ageing, albeit with different mechanisms in each case, whilst expanding on the molecular basis for these processes. Accordingly, our understanding of the mechanisms underlying the pathogenesis of these diseases has improved and new therapeutic targets have been identified by these studies.

Prostate cancer (PC), is initially androgen dependent due to the androgenic nature of the organ. Hence, initial therapy comprises androgen depletion via chemical castration in conjunction with an anti-androgen therapeutic. However, patients relapse and the tumours aggressively re-grow in a castrate resistant (CRPC) manner. In CRPC, androgen receptor (AR) signaling remains functional via numerous mechanisms hence the AR remains a viable therapeutic target. However, treatment with current AR targeting therapeutics also results in relapse indicating the potential of targeting AR signaling indirectly by targeting AR co-factors. Recently, KMT5A, a lysine methyltransferase, has been identified as an AR co-activator exclusively in models of CRPC. A number of KMT5A inhibitors have been identified recent years, which would enhance the possibility of targeting KMT5A in PC. This thesis aims to determine the signature of genes that are regulated directly by KMT5A or by combined activities of AR and KMT5A in PC cell lines and to further identify biomarkers for KMT5A activity. These aims were approached using Illumina Human HT-12 arrays to detect KMT5A gene expression profiles in an in vitro cell line model of androgen independent PC (LNCaP-AI cells). Microarray data analysis revealed a number of androgenregulated genes to be modulated by KMT5A concurrently, and other genes that were found to be regulated by KMT5A activity, and a further cohort of genes that were found to be regulated solely by KMT5A. CDC20 was selected for further study from the identified KMT5A regulated genes as a possible biomarker for KMT5A activity in aggressive PC. KMT5A was found to regulate CDC20 mRNA and protein expression. The enzymatic activity of KMT5A was demonstrated to affect CDC20 expression through the enrichment of the H4K20me1 mark at the CDC20 promoter in androgen-sensitive (LNCaP) and androgen-independent (LNCaP-AI) cells. The regulation of CDC20 by KMT5A expression, therefore identifies CDC20 as putative biomarker for KMT5A activity. KMT5A was also shown to influence CDC20 expression via p53. Knockdown of KMT5A inhibited the mono-methylation of p53 at K382 to enhance p53 activity, demonstrated by increased p21 expression which negatively regulated CDC20 ii expression. These findings were confirmed using commercially available KMT5A inhibitors Ryuvidine and UNC0379. In summary, KMT5A inhibition in PC cells using small molecule inhibitors may provide benefit to patients that have relapsed on AR- targeting therapeutics and as such requires further investigation as a potential therapeutic target. CDC20 was identified as a putative biomarker for KMT5A activity which may prove useful to detect effective KMT5A inhibition in these studies.

Acute myeloid leukaemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify novel therapeutic targets in AML, I have optimized a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. This work led to the identification of 492 AML-specific cell-essential genes, including several established therapeutic targets such as that represent new clinically actionable candidates. I have validated selected genes using DOT1L, BCL2, MEN1 and many other genes genetic and pharmacological inhibition, and chose candidates for downstream studies. Both the epigenetic modifier KAT2A and SRPK1 as promising KAT2A and spliceosome kinase SRPK1 inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs while sparing normal hemopoietic stem-progenitor cells. My findings propose that KAT2A and SRPK1 inhibition should be investigated as new therapeutic strategies in AML and also provide a large number of novel genetic vulnerabilities of this leukaemia that can be pursued in downstream studies. As these screens were performed in immortalised AML cell lines, I then went on to develop a method for the performance of dropout screens in genotypically-defined primary murine AMLs developed in our lab and arising in Cas9-expressing mice. Through this work, I successfully carried out the first such screen in AML cells driven by mutant Npm1 (NPM1c) and Flt3-ITD, the commonest two-mutation combination in human AML. Downstream analysis of the results revealed the excellent potential of this type of screen and enabled me to investigate the molecular effects on mutant Npm1, which are currently poorly understood. Overall, my results demonstrate that unbiased CRISPR dropout screens can identify novel therapeutic targets in cancer while, in parallel, revealing novel biological insights.

Lymphoid neoplasms are a subgroup of haematological malignancies that affect circulating lymphocytes. The clinical and biological heterogeneity of lymphoid neoplasms can lead to difficulty in accurate diagnosis in this group of diseases. The advancement in effective and feasible detection platforms has enabled novel biomarkers to improve diagnosis and prognosis, in addition to assist in patient stratification and personalised treatments for these diseases. Although there have been improvements in high-throughput diagnostic techniques, the conventional immunohistochemistry (IHC) remains the most widely used platform for biomarker assessment in the field of tissue pathology. As this conventional technique has certain limitations, multiplex IHC (MIHC) approaches have found ways to overcome these challenges, therefore becoming the main focus of immunotherapy for lymphoid neoplasms. This particular effective and proficient technology can simultaneously target multiple molecule/protein of interest within the tumor microenvironment to determine the status of immune cell activation and the presence of protein expression. MIHC is advantageous in providing information about the underlying immune evasion mechanisms, which play a vital role in the development of prognostic and diagnostic biomarkers. This thesis focuses on the novel use of IHC/MIHC approaches and their current role in biomarker development to be used in diagnosis, prognosis, and the potential treatment strategies within haematological malignancies in specific lymphoid neoplasms.

Wilms tumor (WT) is a pediatric kidney tumor that accounts for 6-7% of children’s malignant tumors. WT cells arise from embryonic mesenchymal renal progenitors, not differentiated, remaining in the organ after birth. The classic triphasic WT contains a mixture of undifferentiated blastemal, differentiated epithelial cells, and stromal elements, whereas individual cell types may predominate in some tumors. Moreover, as human mesenchymal stem cells (MSCs), these cells have the ability to differentiate in many tissue lineages. In particular, stromal-like WT (str-WT) display morphological, phenotypic, and biological features comparable to MSCs, suggesting they could play a significant role in the interactions with immune cells in the tumor microenvironment. The role of immune cells in the response against WT is still unclear, because of the very few data available; it is of note, however, that the presence of leukocyte infiltrate and a pro-inflammatory milieu have been described. In view of the similarity between MSCs and stromal-like WT cells, the aim of this study is to investigate the immunoregulatory properties of WT cells towards Natural Killer (NK) cells and the immunoregulatory mechanisms taking place in the tumor microenvironment. Our data showed the molecular interactions between str-WT cells and NK lymphocytes, resulting in different outcomes presumably occurring in the WT microenvironment.

Colorectal cancer is a disease caused by genetic and epigenetic changes. Inactivation of tumor suppressor genes and activation of oncogenes are key landmarks in tumor progression. However, the list of tumor suppressor genes and oncogenes is far from complete, even in the case of the tumor types that are best characterized, such as colorectal cancer. Colorectal cancer is the second most frequent cause of cancer-related death in the Western world and is a serious health issue for the European Union. Patients having stage III or IV cancer undergo surgery followed by chemotherapy. However, the clinical management of these patients is far from optimal, and only about 30 % of the patients show an objective response to even the best chemotherapeutic agents available. In this study genome-wide high throughput assays were used to better characterize important aspects of the oncogenic progression such as deregulation of proliferation and aberrant expression caused by epigenetic mechanisms. Because rapid tumor proliferation is associated with poor patient prognosis, here we characterized the transcriptional signature of rapidly proliferating colorectal cancer cells in an attempt to identify genes important to sustain tumor growth and that could be used as novel therapeutic targets. The proliferation rate of 52 colorectal cancer cell lines was determined and genome-wide expression profiling of a subset of these lines was assessed by microarray analysis. The expression of 1,290 genes was significantly correlated with the growth rates of colorectal cancer cells. These included genes involved in cell cycle, RNA processing/splicing and protein transport. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and protoporphyrinogen oxidase (PPDX) were shown to have higher expression in faster growing cancer cells. Importantly, pharmacological and genetic inhibition of GAPDH or PPDX reduced the growth of colon cancer cells in vitro and in vivo. To better understand the mechanisms underlying the profound transcriptional reprogramming observed in cancer cells, we investigated the association between the levels of DNA methylation in the promoters of >14,000 genes and the levels of expression of these genes in a panel of 45 colorectal cancer cell lines. A group of cell lines with significantly higher methylation levels was observed, supporting the notion that there is a group of colorectal tumors with a CpG methylator phenotype (CIMP+). A significant negative regulation between methylation and expression levels was observed for 1,409 genes, suggesting that these genes are silenced during the tumorigenic process through this epigenetic mechanism. A significant number of these genes were zinc finger proteins, suggesting an important role of these DNA-binding proteins on the tumorigenic process. Strikingly, approximately one fourth of these genes are not associated with CpG islands, indicating that DNA methylation outside these CpG rich regions is an important mechanism regulating gene expression and significantly contribute to tumor progression. In addition, we postulate that at least some of those genes have tumor suppressor activity. As a proof-of-concept, we show that restoration of the expression of ZNF238, a gene showing a significant methylation/expression correlation, resulted in reduced growth of colon cancer cells in vitro and in vivo. In conclusion, in this study we shed new light on the mechanisms underlying the uncontrolled proliferation of colon cancer cells and the expression reprograming imposed in these cells through CpG methylation. The results of this study may contribute to the identification of novel chemotherapeutic targets for patients with colorectal cancer, and the characterization of novel genes/pathways with tumor suppressor activity, that are epigenetically silenced.
El càncer colorectal és una malaltia causada per canvis genètics i epigenètics. La inactivació de gens supressors de tumors i l'activació d'oncogens són fites clau en la progressió tumoral. Els pacients amb càncer en estadi III o IV són sotmesos a cirurgia seguida de quimioteràpia. No obstant això, només el 30% dels pacients mostren una resposta objectiva fins i tot als millors agents quimioterapèutics disponibles. En aquest estudi es van utilitzar assajos d'alt rendiment de tot el genoma per caracteritzar millor els aspectes importants de la progressió oncogènica, com la desregulació de la proliferació i l'expressió aberrant causada per mecanismes epigenètics. La ràpida proliferació tumoral s'associa al pitjor pronòstic del pacient, aquí hem caracteritzat la signatura transcripcional de les cèl.lules de càncer colorectal amb ràpida proliferació de cèl.lules en un intent d'identificar els gens importants per sostenir el creixement tumoral i que podrien ser utilitzats com a dianes terapèutiques. Per comprendre millor els mecanismes subjacents a la profunda reprogramació transcripcional observada en les cèl.lules canceroses, es va investigar l'associació entre els nivells de metilació de l'ADN en els promotors i els nivells d'expressió d'aquests gens en línies cel.lulars de càncer colorectal. Els resultats d'aquest estudi poden contribuir a la identificació de noves dianes quimioterapèutics per a pacients amb càncer colorectal, i la caracterització de nous gens / vies amb activitat supressora de tumors, que són silenciats epigeneticament.

Dietary flaxseed is a Manitoban crop rich in the n3 fatty acid alpha-linolenic acid, fibre and antioxidant lignans. Addition of flaxseed to the diet decreased brachial blood pressure in patients with hypertension and peripheral artery disease over one year (n=110). With the addition of flaxseed to standard of care, 21% of patients improved from blood pressure above goal (>140/90 mmHg) to blood pressure within goal (<140/90 mmHg). Dietary flaxseed may have induced these anti-hypertensive effects through the reduction of vascular constriction and inflammation. Healthy older adults, who consumed flaxseed every day for 4 weeks, exhibited significantly decreased concentrations of plasma pro-inflammatory oxylipins such as 5-hydroxyeicosatetraenoic acid and trihydroxyoctadecenoic acid. In patients with hypertension and peripheral artery disease, flaxseed consumption for 1 year significantly decreased plasma oxylipins that are responsible for propagating inflammation and vascular constriction. The oxylipins that decreased in the flaxseed group were all produced by the same enzyme, soluble epoxide hydrolase. Alpha-linolenic acid decreased soluble epoxide hydrolase activity in an activity assay. Thus, flaxseed may exert its anti-hypertensive effects through an inhibition of soluble epoxide hydrolase by alpha-linolenic acid. Flaxseed also induced a significant decrease in central aortic blood pressure without cardiac or arterial elasticity involvement as measured by pulse wave analysis. Some of the same oxylipins that decreased with flaxseed consumption were significantly associated with higher central aortic blood pressure and a higher prevalence of cardiovascular and cerebrovascular events in patients with peripheral artery disease. Every 1 nM increase in plasma 16-hydroxyeicosatetraenoic acid increased the odds of a stroke by 55-fold, thus indicating the potential of particular oxylipins to act as diagnostic markers or therapeutic targets. New research is currently investigating if dietary flaxseed can lower blood pressure and prevent the need for anti-hypertensive medications in those newly diagnosed with hypertension. The implications of this research may change how standard of care is implemented for patients with hypertension and cardiovascular disease. The goal is to offer patients an additional effective strategy beyond anti-hypertensive medications for the management of hypertension in order to reduce the risk of cardiovascular events and to improve patient care and quality of life.
October 2016

INTRODUCTION: In 1966, Rett syndrome (RTT, OMIM#312750) was for the first time described as a clinical issue by Dr. Andreas Rett, an Austrian pediatrician. He has observed in 22 patients similar unique symptoms. A few years later Hagberg and colleagues described further the syndrome in 35 girls. Rett syndrome is the cause of mental retardation that affects 1 in 10.000 female births, which makes it the second cause of mental retardation in girls. In 1999 Zoghbi lab found out the genetic basis of Rett disease. Mutation in MeCP2 is in 95% cases the reason of classical Rett. MeCP2 is a nuclear protein, expressed widely in different tissues, but is most abundant in neurons of the mature nervous system. A few years earlier Bird and coworkers indentified MeCP2 as a new protein that binds to the methylated CpG dinucleotides. Although the function of the MeCP2 is still unknown, it is considered likely to regulate gene expression, either through the silencing or activation of the specific genes or by more global regulation of transcriptional processes. RTT was the first neurodevelopmental disorder related to epigenetics. AIM OF THE STUDY Hypotesis: The main aim of this thesis is to perform preclinical evaluation of drugs that are known to target pathways which are altered in Rett syndrome. It is also necessary to investigate new mechanisms associated with the development of Rett syndrome, aiming to find new pathways related to Rett phenotype that can be manipulated through the pharmacological approach. Aims: 1. Determine tests that can reflect the difference at the behavioral and molecular levels between the Mecp2 KO and WT littermate mice. 2. Optimize study design protocol for evaluation of in vivo drug treatments. 3. Identify candidate drugs against selected targets in order to improve Rett disease, with the goal to reverse the symptoms, prolong the life span or ameliorate dysfunctions based on inflammation and neural mechanisms. 4. Determine the potential effects of novel therapeutic approaches for newly discovered pathways dysregulated in Rett syndrome. Methods: Animals knockout (KO) to the MeCP2 gene are a well-established murine model that mimics RTT human disease. This model is an excellent tool for the study of the consequences of the loss of MeCP2 in neuronal function. Once we know the dose administered, use two experimental groups of treated and untreated animals. Treatment is initiated when the animals are 4 weeks old and start showing symptoms characteristic of this model: reduced mobility, retraction of the hind legs, tremors, hair frizzing and respiratory abnormalities, difficulty walking, retraction of the rear legs. During the test, the mice will be evaluated according to a set of symptoms resulting from MeCP2 deficiency. Results: Part 1 • We performed a comprehensive panel of experiments investigating the differences between Mecp2 KO and wt littermate mice at the behavioral and molecular levels. This was further extended to evaluate the specific drug treatments efficacy. Part 2 • Combined administration of Levodopa and a Dopa decarboxylase inhibitor in Rett syndrome mouse models was well tolerated, diminished Rett syndrome-associated symptoms and increased lifespan. • The use of L-Dopa + Ddci in the Mecp2 KO mice induced dendritic growth mediated by dopaminergic neurons. • L-Dopa + Ddci-treated group exhibited higher Th and pTh expression and dopamine levels in comparison to the vehicle treated group. Part 3 • The results presented here reveal an important role for the relationship between Mecp2 and GSK3 signaling in Rett syndrome disease • Inhibitor of GSK3, SB216763, improved life span and reduced single and total symptoms scores, as well as motor deficiency in Mecp2 KO mice • Inhibition of GSK3 is a possible line of stimulation of neuronal dendritic growth, supported by elevated levels of D2- receptor after drug administration. • Treatment with SB216763 decreased inflammation and strengthened antioxidant defense in the brain of Mecp2KO mice Part 4 • Treatment with copaxone, a BDNF analogue reflect rather low improvement in Mecp2 KO studied mice, displaying also very variable results between studied animals. • Treatment with dexamethasone, an example of glucocorticoids intervention display rather low improvement – approximately 20% when compared to untreated mice. • Ropirinol treatment confirmed that dopminergic pathway is dysregulated in Rett. Both life span and the phenotype were improved. However, the efficiency was lower compared to L-dopa + Ddci treatment. • Injections of bromperidol, a serotonin modulator, were toxic for Mecp2 KO mice. Even though the selected doses were very low, they caused significant reduction of body weight in the KO treated group. • Previously described positive effect of cysteamine was not reproduced in our laboratory (Roux et al., 2012). • Gabapentin, one of GABA modulators, improved the phenotype, but not life span of Mecp2 KO animals, being more efficient with the low dose. • TDZD8 treatment confirmed previously a described role of GSK3 inhibition in Rett. Both life span and phenotype were improved. However, the efficacy was lower than after SB216763 treatment. • Antioxidants studied in this thesis displayed improvement of 30% in Rett syndrome phenotype and some of them prolonged the life span by 25% when compared to the vehicle group. • Epigenetics drugs evaluated in this thesis, even though some of them were suggested to be relevant for Rett syndrome, did not show any specific improvement. Conclusions: The treatment with L-Dopa + Ddci is promising to overcome the dopaminergic defects observed in the preclinical model of Rett syndrome (Szczesna et al., 2014) The new data indicate selected inhibitor, SB216763, as a being potential drug treatment for Rett disease. The drug can be adressed to have neuroprotection properties via the improvement of synaptic plasticity, reduced oxidant and inflammation damage or partial rescue of mobility dysfunctions (Szczesna et al in preparation).
INTRODUCCIÓN: En 1966, el síndrome de Rett (RTT, OMIM#312750) fue por primera vez descrito como un problema clínico por el pediatra austriaco Andreas Rett. Observó síntomas similares en 22 pacientes. Pocos años después, Hagberg y colaboradores describieron el síndrome en 35 niñas más. El síndrome de Rett causa retraso mental en 1 de cada 10000 niñas, lo que hace que sea la segunda causa de retraso mental en niñas. En 1999 en el laboratorio de Huda Zoghbi descubrieron las bases genéticas de la enfermedad. El 95% de los casos de Rett clásico se produce por mutaciones en MeCP2. MeCP2 es una proteína nuclear, que se expresa en diferentes tejidos, pero es especialmente abundante en neuronas del sistema nervioso maduro. Pocos años antes Bird y colaboradores habían identificado MeCP2 como proteína con capacidad para unirse a dinucleótidos CpG. Aunque la función de MeCP2 todavía no se conoce con exactitud, se considera que probablemente actúa como regulador de la expresión génica, tanto mediante el silenciamiento o activación de genes específicos como actuando de manera más global sobre la transcripción. El síndrome de Rett fue la primera enfermedad del desarrollo neuronal relacionada con la epigenética. OBJETIVOS DEL ESTUDIO Hipótesis: El principal objetivo de esta tesis es realizar la evaluación preclínica de fármacos que actúan sobre las diferentes rutas alteradas en el síndrome de Rett. Es necesario también investigar nuevos mecanismos asociados al desarrollo de la enfermedad, con el fin de descubrir nuevas rutas que puedan estar relacionadas con la patología y que sean susceptibles de ser manipuladas mediante la utilización de fármacos específicos. Objetivos: 1. Determinar qué tests pueden reflejar las diferencias tanto a nivel conductual como molecular entre ratones knockout de MeCP2 y salvajes de la misma camada. 2. Optimización de protocolos de evaluación de tratamientos farmacológicos in vivo. 3. Identificación de fármacos dirigidos a dianas específicas para la mejora del curso de la enfermedad, con la finalidad de revertir la sintomatología, aumentar la supervivencia o mejorar disfunciones relacionadas con mecanismos neuronales o inflamación. 4. Determinar el potencial de las nuevas terapias desarrolladas para las rutas que se descubran alteradas en el síndrome de Rett. Métodos: El knockout de MeCP2 es un modelo murino bien establecido que mimetiza el síndrome de Rett humano. Este es un excelente modelo para el estudio de las consecuencias relacionadas con la pérdida de MeCP2 en las funciones neuronales. Una vez conocida la dosis que se ha de administrar, se utilizaron dos grupos experimetales, el grupo de tratados y no tratados. El tratamiento se inicia cuando los animales tienen 4 semanas y empiezan a mostras síntomas característicos del modelo: movilidad reducida, retracción de las patas traseras, temblores, pelo encrespado y anormalidades respiratorias, dificultas al andar, retracción de las patas delanteras. Durante el test, los ratones serán evaluados según los síntomas derivados de la deficiencia de MeCP2. Resultados: Parte 1 • Hemos realizado una serie de experimentos investigando las diferencias entre ratones MeCP2 KO y sus hermanos de camada salvajes, tanto a nivel de comportamiento como molecular. Posteriormente, se evaluó la eficacia de una serie de tratamientos. Parte 2 • La administración combinada de Levodopa y un inhibidor de la Dopa decarboxylasa en ratones con síndrome de Rett fue bien tolerada por los animales, disminuyendo los síntomas asociados al síndrome y aumentando la supervivencia. • El uso de L-Dopa + Ddci en el los ratones Mecp2 KO indujo el crecimiento dendrítico mediado por neuronas dopaminérgicas. • El grupo tratado con L-Dopa + Ddci muestra exhibió un aumento en los niveles de expression Th y pTh y de los niveles de dopamina en comparación con el grupo tratado con el vehículo. Parte 3 • Los resultados presentados revelan que la relación entre Mecp2 y la ruta de señalización de GSK3 juega un papel importante en el síndrome de Rett. • El inhibidor de GSK3, SB216763, mejoró la supervivencia y redujo la gravedad de los síntomas, así como la deficiencia motora ,de los ratones Mecp2 KO • La inhibición de GSK3 es una posible vía de estimulación del crecimiento de las neuronas dendríticas, avalado por los elevados niveles del receptor D2 encontrados tras la administración de la droga. • El tratamiento con SB216763 disminuyó la inflamación y reforzó las defensas antioxidantes en el cerebro de los ratones Mecp2 KO. Parte 4 • El tratamiento con copaxona, un análogo de BDNF dio lugar a una menor mejora de los ratones Mecp2 KO, mostrando además una gran variabilidad entre los ratones estudiados. • El tratamiento con dexamethasona, un ejemplo de tratamiento con glucocorticoides, mostró una pequeña mejora, aproximadamente un 20% cuando se comparan los ratones tratados con los no tratados. • El tratamiento con Ropirinol confirmó que la vía dopaminérgica está alterada en Rett. Tanto la supervivencia como la sintomatología mejoró tras la administración de la droga. Sin embrago, la eficiencia fue menor a la obtenida con la tratamiento combinado de Dopa + Ddci. • Las inyecciones de bromperidol, un modulador de la serotonina, resultaron ser tóxicas en ratones Mecp2. Incluso aunque las dosis administradas fueran muy bajas, los ratones sufrían pérdida de peso acusado. • Resultados previos describiendo un efecto positive en los ratones Mecp2 tras la administración de cysteamina no pudieron ser reproducidos en nuestro laboratorio (Roux et al., 2012). • Gabapentin, un modulador de GABA, mejoró los síntomas pero no tuvo ningún efecto en la supervivencia de los animales Mecp2. • El tratamiento con TDZD8 confirmó resultados los previos describiendo el efecto positivo de la inhibición de GSK3 en Rett. Se mejoró tanto la supervivencia como la sintomatología. Sin embrago, la eficiencia fue menor a la obtenida con SB216763. • Los antioxidantes estudiados en esta tesis mostraron una mejora del 30% en la sintomatología de Rett, siendo algunos también eficaces en la mejora de la supervivencia. • Las drogas epigenéticas evaluadas en esta tesis no mostraron ninguna mejora significativa en el tratamiento del síndrome de Rett. Conclusiones: El tratamiento con L-Dopa + Ddci es muy prometedor para sobreponer los defectos dopaminérgicos observados en el modelo preclínico del síndrome de Rett utilizado en esta tesis (Szczesna et al., 2014). Los nuevos datos indican que el inhibidor de GSK3, SB216763, es un posible fármaco para el tratamiento del síndrome de Rett. La droga ha mostrado propiedades neuroprotectoras mediante la mejora de la plasticidad sináptica y la reducción del daño oxidativo e inflamación, así como la mejora de las disfunciones motoras (Szczesna et al, artículo en preparación).