Rôle du microenvironnement dans le maintien et la résistance des cellules souches leucémiques de la Leucémie Myéloïde Chronique. voie BMP et contraintes mécaniques

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

An epigenetic approach to fatty acid metabolism in haematological malignancies

[eng] The role of fatty acids to overcome stress and contribute to disease progression is becoming increasingly evident in haematological diseases. Further, epigenetic factors play an important role in the aetiology of myelodysplastic syndromes (MDS) and the transformation to secondary acute myeloid leukaemia (sAML). To investigate this in the MDS/sAML cell line, SKK-1, we employed a shRNA knockdown screen to target 912 epigenetic regulators. We then coupled this loss-of-function approach to a fatty acid metabolism-based assay with which we were able to cell-sort the SKK-1 cells based on the fatty acid uptake. Here I describe the methodology of this epigenetics-metabolism approach and our efforts to validate candidate hits from the screen that were predicted to be modulators of fatty acid uptake. Following testing using single gene knockdowns of the top genes from the screen, we were not able to identify epigenetic regulators that significantly alter fatty acid uptake. In parallel, we characterised metabolic and genetic parameters of triple-sorted low (TS LOW) and high (TS HIGH) fatty acid uptake sub- populations. However, during the course of the study, we discovered latent contamination by another myeloid cell line, U-937, in our SKK-1 parental cells and TS LOW and TS HIGH sub-populations. Therefore, we interpreted results from the characterisation study with the knowledge that we had mixed cellular populations. I describe the steps we took to first identify the cell line and then our further characterisation of single cell clones of TS LOW and TS HIGH. Interestingly, we observed distinct cytogenetic profiles between single clones of TS LOW and TS HIGH, namely trisomy 8, which is a highly relevant chromosomal aberration in myeloid malignancies. Overall, this study provides a novel approach to investigate epigenetic and metabolic interactions in blood malignancies. We also find metabolically distinct sub-populations that differ by a disease-relevant karyotype

Identification and Characterization of the P53-Induced Long Noncoding RNA Isoform Pvt1b and Its Role in Stress-Specific Growth Inhibition via Myc Repression

The tumor suppressor p53 and proto-oncogenic Myc transcription factors are frequently deregulated in cancer, with common loss-of-function and gain-of-function mutations observed in the p53 and Myc networks, respectively. Referred to as the ‘guardian of the genome,’ p53 regulates genes important for curtailing cellular proliferation and tumorigenesis under conditions of stress, while the proto-oncogene Myc induces genes that, in contrast, promote cellular growth and can, in overcoming growth inhibitory signals, support cancer development. While previous literature has documented decreased Myc expression in response to cellular stress, researchers have long puzzled over identifying the specific regulatory lever responsible. The work presented here identifies a novel regulatory axis positioned at the intersection of the p53 and Myc pathways, which represses Myc and restricts cellular proliferation downstream of p53 activation. Long noncoding RNAs (lncRNAs) are a diverse class of transcripts lacking protein-coding potential and implicated in gene expression regulation. Here I present my work on the identification of an isoform of the lncRNA Plasmacytoma variant translocation 1 (Pvt1) and the characterization of its role in the p53-mediated response to stress. I found that the stress-specific Pvt1b, expressed 50 Kb downstream of the Myc locus, is induced by p53 in response to oncogenic and genotoxic stress and accumulates at its site of transcription. I demonstrated that production of the Pvt1b RNA is necessary and sufficient to repress Myc transcription in cis without altering the chromatin organization of the locus. I investigated the functional outputs of Pvt1b-mediated Myc downregulation and found that inhibition of Pvt1b increased both Myc levels and transcriptional activity and promoted cellular proliferation. Notably, Pvt1b loss accelerated tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. Further examination of the Pvt1b mechanism of action failed to identify Pvt1b-specific sequences required for its function, but uncovered a potential role for histone deacetylation in Pvt1b regulation of Myc. Finally, I initiated development of a suite of genetically engineered Pvt1 mouse models, the characterization of which will shed light on Pvt1 function in vivo and benefit future mechanistic studies. Taken together, this work conceptually advances our understanding of stress-induced growth inhibition orchestrated by p53. Specifically, I identify Pvt1b as the primary mediator of stress-specific Myc repression, providing insight into the long-standing question of how p53 activation triggers Myc downregulation. As such, this work has far-reaching implications not only for our understanding of cis-acting lncRNAs, which can fine-tune local gene expression downstream of broadly active transcription programs, but also for the exciting therapeutic possibility of restricting Myc levels in cancer via Pvt1b modulation

Quantitative Approaches to the Genomics of Clonal Evolution

Many problems in the biological sciences reduce to questions of genetic evolution. Entire classes of medical pathology, such as malignant neoplasia or infectious disease, can be viewed in the light of Darwinian competition of genomes. With the benefit of today's maturing sequencing technologies we can observe and quantify genetic evolution with nucleotide resolution. This provides a molecular view of genetic material that has adapted, or is in the process of adapting, to its local selection pressures. A series of problems will be discussed in this thesis, all involving the mathematical modeling of genomic data derived from clonally evolving populations. We use a variety of computational approaches to characterize over-represented features in the data, with the underlying hypothesis that we may be detecting fitness-conferring features of the biology. In Part I we consider the cross-sectional sampling of human tumors via RNA-sequencing, and devise computational pipelines for detecting oncogenic gene fusions and oncovirus infections. Genomic translocation and oncovirus infection can each be a highly penetrant alteration in a tumor's evolutionary history, with famous examples of both populating the cancer biology literature. In order to exert a transforming influence over the host cell, gene fusions and viral genetic programs need to be expressed and thus can be detected via whole transcriptome sequencing of a malignant cell population. We describe our approaches to predicting oncogenic gene fusions (Chapter 2) and quantifying host-viral interactions (Chapter 3) in large panels of human tumor tissue. The alterations that we characterize prompt the larger question of how the genetics of tumors and viruses might vary in time, leading us to the study of serially sampled populations. In Part II we consider longitudinal sampling of a clonally evolving population. Phylogenetic trees are the standard representation of a clonal process, an evolutionary picture as old as Darwin's voyages on the Beagle. Chapter 4 first reviews phylogenetic inference and then introduces a certain phylogenetic tree space that forms the starting point of our work on the topic. Specifically, Chapter 4 describes the construction of our projective tree space along with an explicit implementation for visualizing point clouds of rescaled trees. The Chapter finishes by defining a method for stable dimensionality reduction of large phylogenies, which is useful for analyzing long genomic time series. In Chapter 5 we consider medically relevant instances of clonal evolution and the longitudinal genetic data sets to which they give rise. We analyze data from (i) the sequencing of cancers along their therapeutic course, (ii) the passaging of a xenografted tumor through a mouse model, and (iii) the seasonal surveillance of H3N2 influenza's hemagglutinin segment. A novel approach to predicting influenza vaccine effectiveness is demonstrated using statistics of point clouds in tree spaces. Our investigations into clonal processes may be extended beyond naturally occurring genomes. In Part III we focus on the directed clonal evolution of populations of synthetic RNAs in vitro. Analogous to the selection pressures exerted upon malignant cells or viral particles, these synthetic RNA genomes can be evolved against a desired fitness objective. We investigate fitness objectives related to reprogramming ribosomal translation. Chapter 6 identifies high fitness RNA pseudoknot geometries capable of inducing ribosomal frameshift, while Chapter 7 takes an unbiased approach to evolving sequence and structural elements that promote stop codon readthrough