EPCO-13. MULTIOMIC SINGLE NUCLEUS RNA- AND ATACseq PROFILING REVEALS REGULATORS OF GLIOMA CELL STATE DIVERSITY

Multiomic single nucleus RNA- and ATACseq profiling reveals regulators of glioma cell state diversity. The extensive intra- and intertumoral heterogeneity observed in glioma reflects the resistance to therapy and poor prognosis observed clinically. Single-cell sequencing studies have highlighted that glioma heterogeneity reflects the co-existence of cell subpopulations with distinct cell states. Prior studies have also shown that EGFR-amplifying extrachromosomal DNA (ecDNA) elements in IDH-wild-type gliomas can contribute to heterogeneity by driving oncogene amplification through long range chromatin contacts. However, single cell studies have largely focused on analyses of transcriptional profiles, and the epigenetic mechanisms underlying the contribution of ecDNA elements to tumor cell state diversity remain poorly understood. To further our understanding of the regulatory programs that contribute to transcriptional diversity and mediate the distribution of tumor cell states, we profiled primary-recurrent tumor pairs from 18 patient samples with multiomic single-nucleus RNA- and ATACseq, resulting in 86,135 cells identified with linked chromatin accessibility and gene expression profiles. Integrative clustering of the tumor cells identified tumor cell states ranging from a stem-like to differentiated- phenotype that were also associated with differences in chromatin accessibility and inferred transcription factor binding activity. Analyses of chromatin accessibility resulted in the identification of ecDNA, and integrative clustering of ecDNA+ cells highlighted distinct cell states with increased copy number burden, oncogene amplification, and differential chromatin accessibility. These results suggest that a better understanding of extrachromosomal contributions to tumor diversity would aid in development of more efficient therapies.

Principles of ecDNA random inheritance drive rapid genome change and therapy resistance in human cancers

The foundational principles of Darwinian evolution are variation, selection, and identity by descent. Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumour growth, drug resistance, and shorter survival in patients. Currently, the impact of non-chromosomal oncogene inheritance - random identity by descent - is not well understood. Neither is the impact of ecDNA on variation and selection. Here, integrating mathematical modeling, unbiased image analysis, CRISPR-based ecDNA tagging, and live-cell imaging, we identify a set of basic rules for how random ecDNA inheritance drives oncogene copy number and distribution, resulting in extensive intratumoural ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted cancer treatment. Observed ecDNAs obligatorily benefit host cell survival or growth and can change within a single cell cycle. In studies ranging from well-curated, patient-derived cancer cell cultures to clinical tumour samples from patients with glioblastoma and neuroblastoma treated with oncogene-targeted drugs, we show how these ecDNA inheritance rules can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are entailed by their ability to rapidly change their genomes in a way that is not possible for cancers driven by chromosomal oncogene amplification. These results shed new light on how the non-chromosomal random inheritance pattern of ecDNA underlies poor outcomes for cancer patients.

Frequent extrachromosomal oncogene amplification drives aggressive tumors

Extrachromosomal DNA (ecDNA) amplification promotes high oncogene copy number, intratumoral genetic heterogeneity, and accelerated tumor evolution1–3, but its frequency and clinical impact are not well understood. Here we show, using computational analysis of whole-genome sequencing data from 1,979 cancer patients, that ecDNA amplification occurs in at least 26% of human cancers, of a wide variety of histological types, but not in whole blood or normal tissue. We demonstrate a highly significant enrichment for oncogenes on amplified ecDNA and that the most common recurrent oncogene amplifications arise on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number matched linear DNA, coupled with enhanced chromatin accessibility. Patients whose tumors have ecDNA-based oncogene amplification showed increase of cell proliferation signature activity, greater likelihood of lymph node spread at initial diagnosis, and significantly shorter survival, even when controlled for tissue type, than do patients whose cancers are not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification plays a central role in driving the poor outcome for patients with some of the most aggressive forms of cancers.