A systematic pan-cancer analysis of somatic variants reveals epigenetic drivers of intratumor heterogeneity

Abstract

Increasing evidence supports the existence of intratumor heterogeneity (ITH) in many cancer types, with potential clinical implications for both cancer diagnosis and treatment. Expansion of genetically distinct cell populations within a tumor creates a subclonal architecture that varies dynamically throughout cancer progression. This acquired cancer trait, ITH is the substrate for Darwinian evolution to act upon, selecting the subclones that carry beneficial phenotypes. The outgrowth of such subclones impacts cancer development, drug resistance and tumor relapse. Nevertheless, despite the key role ITH plays in cancer, important questions regarding its magnitude, origin and genetic drivers across different cancer types remain largely unanswered. By facilitating the emergence of nucleotide sequence mutations, copy-number alterations (CNA), chromosomal translocations or aneuploidies, genomic instability has been regarded as the major source of ITH. However, discrepancies in the rates of genomic instability and ITH observed in previous comprehensive studies suggest that additional events congregate to increase genetic heterogeneity in tumors. It is widely accepted that genetic alterations and disruption of epigenetic regulatory mechanisms are hallmarks of cancer. Nonetheless, while the genetic contribution for cancer development is easily illustrated by mutations in tumor suppressors or oncogenes, the epigenetic involvement and its functional relevance is far more complex and has only recently become a major focus of cancer research. Besides genetic mutations, cancer cells invariably present with some degree of epigenetic alterations that contribute to the acquisition of the cancer hallmarks. Indeed, there is evidence that epigenomic reprogramming plays a seminal role in tumorigenesis by creating a progenitor-like cell state that facilitates expression of driver mutations and tumor initiation. High throughput genome sequencing (HTS) efforts have identified driver mutations in genes that regulate the epigenome, namely genome-wide chromatin and deoxyribonucleic acid (DNA) methylation. While epigenetic deregulation in cancer arises primarily as a consequence of DNA mutations, the view that altered epigenomes may also change DNA mutations rates highlights reciprocal interactions that contribute to cancer development. Accordingly, epigenomic disruption should favour the development of genetically diverse tumor cell populations, fuelling ITH. In fact, a possible relationship between genomic and epigenomic alterations during clonal evolution of tumors has been suggested in esophageal squamous cell carcinoma and glioma where high concordance was observed between the evolution of genetic and epigenetic diversification. In this PhD thesis, I conducted an exhaustive characterization of ITH across 2,807 tumor samples from 16 different carcinomas using whole-exome sequencing (WES) datasets from The Cancer Genome Atlas (TCGA). Integration of ITH scores and somatic variants detected in each tumor sample revealed that mutations in epigenetics modifier genes are the stronger determinants of ITH and display an association with increased clonal evolution across several cancer types. To investigate whether epigenomic deregulation drives the development of tumors with high levels of ITH, we focused our analysis on kidney renal clear cell carcinoma (KIRC), the cancer type with the highest frequency of mutations in epigenetic modifiers. The important role of epigenomic deregulation in the development and progression of KIRC is illustrated by the finding that patients with mutations in epigenetic modifiers have worse overall survival than those without mutations in these genes. In particular, genes that regulate genome-wide histone and DNA methylation emerged as candidate drivers of ITH. Knockout of histone methyltransferase SETD2 or DNA methyltransferase DNMT3A using the CRISPR/Cas9 system on renal carcinoma cells led to significant expansion of genetically distinct clones and culminated in highly heterogeneous cell populations which recapitulate the heterogeneity levels observed in the TCGA patients. We thus reasoned that the increased ITH observed after DNMT3A or SETD2 knockouts likely underpins variations in mitochondrial metabolism upon which natural selection can act. Indeed, changes in mitochondrial metabolism constitute an important source of variability for natural selection during cancer evolution. Upon DNMT3A or SETD2 knockout we observed that positively selected clones displayed similar mutational spectra and increased mitochondrial bioenergetic performance under stress conditions, suggesting that specific patterns of genotypic and phenotypic variation emerge upon epigenomic deregulation. Our work provides new insights on tumor development and unravels new drivers of ITH, showing an unprecedented pan-cancer portrait of the major determinants of ITH. This work validates experimentally the role of specific epigenetic modifier genes and lays a foundation for more effective cancer prognosis and treatment.