Pharmacological induction of a progenitor state for the efficient expansion of primary human hepatocytes

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.

A Tale of Two Tumors: Unraveling the Complexities of Epigenetic Dysregulation in Cancer

Maria Christine Donaldson

Epigenetics plays an important role in cancer development and progression. Cancer cells hijack the epigenome by modifying the histone protein units responsible for packaging DNA, or by modifying the DNA itself, resulting in changes to chromatin topology and transcriptional programming within the cell. In this thesis, I report our investigation to uncover the mechanisms of epigenetic regulation and how epigenetic control of transcriptional programming goes awry in cancer. We investigate these processes in two separate contexts. In our first study, we investigate the mechanisms by which EZH2 oncogenic mutations alter structure and function of topologically associating domains in non-Hodgkin lymphoma. The process of chromatin folding leads to a systematic arrangement of hierarchical structural and regulatory elements found within the nucleus. A topologically associating domain (TAD) is a regulatory unit of self interacting DNA, where the transcription of the genes within a TAD is usually dictated by the presence of active (H3K36me3) or inactive (H3K27me3) histone marks. In cancer, the somatic point mutation in EZH2Y646X leads to a genome wide increase in H3K27me3, associated with transcriptional repression. While this alteration leads to changes in the global epigenetic status of the cell, its impact on chromatin organization and TAD-related function remain unclear. By combining transcriptomics and epigenetics with analyses of chromatin structure, we demonstrate a functional interplay between TADs and the epigenetic and transcriptional program of the genes found within them. This balance is altered by EZH2Y646X, leading to the synergistic silencing of entire domains directly targeting cell differentiation and tumor suppressive programs. A closer look reveals that the silencing of tumor suppressive TADs are coupled with structural modifications and changes in promoter interactions within EZH2Y646X target TAD6.139. Impressively, the TADâs transcriptional and epigenetic programs are restored by pharmacological inhibition of EZH2Y646X. Our results indicate that EZH2Y646X alters the topology and function of chromatin domains to promote synergistic oncogenic programs. In our second study, we dissect the epigenetic, transcriptomic, and metabolic signaling dependencies using a novel model of central nervous system primitive neural ectodermal tumors (CNS-PNETs). Central nervous system (CNS) tumors are the leading cause of cancer-associated death in children. Primitive neural-ectodermal tumors (CNS-PNETs) are a particularly aggressive subtype of embryonal CNS-tumor, with a five-year overall survival rate in less than 50% of patients. Despite sharing a similar cell of origin of other CNS tumors, CNS-PNETs have a different anatomical location, unique genetic and epigenetic features, and significantly worse clinical outcome. In addition, a lack of in vivo models for studying CNS tumors challenges the opportunity to dissect the genetic variables that underlie the origin of these tumors. We developed a novel CNS-PNET mouse model, called CNS-NPCs, using neural progenitor cells derived from human-iPS cells. Through histological, DNA methylation, and RNA sequencing analyses, we find that the CNS-NPC model recapitulates the the morphologic, epigenetic, and transcriptomic features of primary CNS-PNETs. In addition, through in vivo metabolic analyses of CNS-NPCs and the patient derived cell line, PFSK-1, we identified dysregulation in the neurotransmitter...

EPFL2019

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.

EPFL2013

Notch Signaling - a Booster of Chronic Lymphocytic Leukemia During Initiation and Disease Progression

Delphine Maude Tardivon

Chronic lymphocytic leukemia (CLL) is a B cell malignancy and represents the most common leukemia in adults within the Western world. This disease mainly affects the elderly, with a median age of diagnosis over 70. The disease course is highly variable among patients; some experience rapidly progressing leukemia which becomes resistant to treatment, while others bear indolent disease and will die from other causes. Morbidity correlates with the chromosomal aberration and mutational burdens observed in patients. NOTCH1 gain-of-function mutations are among the most frequently observed mutations in patients. They are found in 10-15% of CLL patients at diagnosis and their prevalence increases up to 30% in late-stage disease. Many correlative studies have shown that patients harboring NOTCH1 mutations exhibit a reduced survival compared to those with unmutated NOTCH1. Although the presence of NOTCH1 mutations is associated with poor prognosis, it remains unclear by which mechanisms NOTCH signaling might influence disease development. Here, we assessed the role of Notch signaling in CLL using genetic loss- and gain-of-function studies in a transgenic mouse model (IgH.TEµ) that faithfully recapitulates human disease phenotypes. We found that B cell specific knock-out of RBP-J significantly decreased prevalence of CLL induction in RBP-J depleted mice compared to control animals. Reciprocal Notch gain-of-function experiments revealed that mice with enforced Notch1 signaling had an earlier CLL onset compared to controls. Recipients of sorted CLL-like cells over-expressing Notch1 had reduced survival compared to control infusion. Taken together, these genetic studies strongly suggest a role for Notch signaling in disease initiation. To study the molecular mechanisms by which Notch1 signaling enhanced CLL aggressiveness, global gene expression as well as global epigenetic analyses were performed on sorted CLL-like cells from Notch1 gain-of-function and control animals. Cell cycle related pathways in gene set enrichment analysis were the most significantly induced signature in the Notch1 gain-of function CLL-like cells compared to control CLL-like cells. This result was further confirmed by functional cell cycle assays in vivo, strongly suggesting that Notch signaling accelerates CLL initiation by promoting proliferation.

EPFL2020