Roles of Epigenetic Dysregulation in Cancer Progression and Metastasis

Nearly all the cells of an organism share the same DNA sequence or genome, and yet they show different phenotypes and carry out different functions. This diversity is made possible by a verity of molecular modifications acting on the DNA sequence that collectively define the cell's epigenome. Failure in the proper regulation of epigenome can lead to abnormal activation or inhibition of vital signaling pathways, which contributes to the initiation and progression of multiple diseases, including cancer. In this thesis, I used a combination of in silico, in vitro, and in vivo techniques to explore the roles of epigenetic dysregulation in tumor progression and metastasis. In the first part, I performed a systematic and unbiased investigation of cancer-associated DNA methylation and gene expression changes across human cancers. I designed a novel algorithmic approach (RESET) to identify aberrant DNA methylation and associated gene expression changes across >6,000 human tumors. We identified a DNA Methylation Instability (DMI) phenotype in tumors acquiring numerous hyper and hypomethylated transcription start sites, which is associated with mutations of chromatin remodeling factors and Wnt-signaling. We showed that silenced genes coalesced in specific pathways including apoptosis, transcriptional regulation, and cell metabolism. The majority of the enhanced genes belonged to cancer-germline antigens (CG), whose expression correlated with response to anti-PD-1 in melanoma patients. Finally, we demonstrated the potential of RESET to explore aberrant DNA methylation in pediatric tumors; pediatric Wilms tumors with diffuse anaplasia exhibit DMI, and specific silencing events predict a worse outcome in cases with favorable histology. These results established a new approach to explore pan-cancer epigenetic modifications and provided a resource of candidate oncogenic events for future functional and therapeutic studies. In the second part, I studied the dynamics of cancer progression and metastasis in Pancreatic Neuroendocrine Tumors (PanNET). This rare tumor is the second most common form of pancreatic cancer. Two principal subtypes of PanNET has been identified: insulinomas (IT) and metastasis-like primaries (MLP), corresponding to the low and high grade of human PanNETs, respectively. From the mouse model of PanNET (RT2), we profiled the single-cell, bulk mRNA and miRNA transcriptomes, and the proteomes of primary and metastasis specimens. We demonstrated that the tumor progression from IT to MLP follows the reverse embryonic and postnatal developmental path. Transcriptomic data from human patients showed that aggressive human PanNETs also follow the same reverse developmental trajectory of dedifferentiation. We established one possible mechanism underlying IT-to-MLP transition. Over-expression of the miR-181cd cluster in IT-like cancer cell-lines resulted in acquisition of the MLP phenotypes. miR-181cd mediated its effect through suppressing the expression of Meis2 and, indirectly, inducing the expression of Hmgb3 and Mycn transcription factors. Inhibiting the expression of Hmgb3 in MLP-like cancer cell-lines resulted in a significant growth decrease both in vitro and in vivo, demonstrating the importance of Hmgb3 in the maintenance of MLP-like cell state. These data presented dedifferentiation as a mechanism by which malignant neuroendocrine cancer cells acquire progenitor-like features, enabling them to become more aggressive and metastatic.

Pan-Cancer Landscape of Aberrant DNA Methylation across Human Tumors

Giovanni Ciriello, Douglas Hanahan, Marco Mina, Mohammad Sadegh Saghafinia

The discovery of cancer-associated alterations has primarily focused on genetic variants. Nonetheless, altered epigenomes contribute to deregulate transcription and promote oncogenic pathways. Here, we designed an algorithmic approach (RESET) to identify aberrant DNA methylation and associated cis-transcriptional changes across >6,000 human tumors. Tumors exhibiting mutations of chromatin remodeling factors and Wnt signaling displayed DNA methylation instability, characterized by numerous hyper- and hypo-methylated loci. Most silenced and enhanced genes coalesced in specific pathways including apoptosis, DNA repair, and cell metabolism. Cancer-germ line antigens (CG) were frequently epigenomically enhanced and their expression correlated with response to anti-PD-1, but not anti-CTLA4, in skin melanoma. Finally, we demonstrated the potential of our approach to explore DNA methylation changes in pediatric tumors, which frequently lack genetic drivers and exhibit epigenomic modifications. Our results provide a pan-cancer map of aberrant DNA methylation to inform functional and therapeutic studies.

CELL PRESS2018

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

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

In mammals the circadian clock drives daily behavioural and physiological changes that resonate with environmental cues, which can be observed, for example, in the intricate timing of rest during the night and activity during the day in humans. The circadian clock consists of a network of hierarchical clocks in which the central pacemaker is located in the suprachiasmatic nucleus, which is sensitive to external light and propagates time to the peripheral organ-based clocks. Within organs, clocks tick in each individual cell and are molecularly encoded in the genome as a network of dynamic feedback loops. By mediating the expression of the appropriate gene products at the correct moment of the day, the molecular clocks op-timize physiological functions of virtually every cell in our body, allowing us to synchronize with daily fluc-tuations in the environment. In mammalian cells the chromatin organization in the nucleus consists of a topological network, in which chromatin interactions between distal regulatory elements such as enhancers and target genes are essen-tial to regulate gene expression. Important questions remain: is the complex chromatin folding dynamic and if so on which genomic and temporal scales? In fact, dynamic rearrangements of the chromatin have been reported on multiple genomic and time scales, from the entire genome to enhancer-promoter con-tacts, across developmental transitions, cellular differentiation, and transcription cycles. With this per-spective in mind, the circadian oscillator provides a unique model to study the dynamics of genomic inter-actions in the context of fluctuating transcription. Here, we explored chromatin contacts of core-clock and clock-controlled gene promoters in the mouse. We aimed at evaluating the putative dynamics of chromatin contacts, at estimating their conservation across tissues, and at exploring the contribution of the circadian clock to this interaction network. To achieve this goal, we performed 4C-sequencing assays at two circadian time points in the liver and kidney of WT and clock-deficient animals. Overall, we observe that chromatin contacts are largely conserved across circadian time points and genetic backgrounds, and are tissue-specific. Our experiments reveal that the promoters of core-clock and clock-controlled genes contact distal genomic regions containing en-hancer chromatin signatures, suggesting a functional role of these distal sites in target gene regulation. Our data also suggest a clock-driven module of rhythmic genes that are colocalized in the nucleus. However, we also observe dynamic contacts between oscillating gene promoter and enhancer-like ge-nomic regions. In this thesis, we discuss in detail two examples of dynamic chromatin looping that are ob-served at the Cry1 and Gys2 loci. Our data reveal not only a dynamic coupling between the chromatin loop and the transcriptional state but also that the dynamic of chromatin looping depends on a functional clock. Furthermore, genome editing experiments using CRISPR-Cas9 tools reveal that the Cry1 distal site is essential for the transcriptional regulation of Cry1 and contributes to the circadian clock robustness both in-vivo and in cultured cells. These results demonstrate how local chromatin rearrangements take place in a 24-hour time-scale and set chromatin looping between gene promoters and distal regions as a new reg-ulatory layer for circadian gene expression.