DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma.

DNA methylation is crucial for chromatin structure and gene expression and its aberrancies, including the global "hypomethylator phenotype", are associated with cancer. Here we show that an underlying mechanism for this phenotype in the large proportion of the highly lethal brain tumor glioblastoma (GBM) carrying receptor tyrosine kinase gene mutations, involves the mechanistic target of rapamycin complex 2 (mTORC2), that is critical for growth factor signaling. In this scenario, mTORC2 suppresses the expression of the de novo DNA methyltransferase (DNMT3A) thereby inducing genome-wide DNA hypomethylation. Mechanistically, mTORC2 facilitates a redistribution of EZH2 histone methyltransferase into the promoter region of DNMT3A, and epigenetically represses the expression of DNA methyltransferase. Integrated analyses in both orthotopic mouse models and clinical GBM samples indicate that the DNA hypomethylator phenotype consistently reprograms a glutamate metabolism network, eventually driving GBM cell invasion and survival. These results nominate mTORC2 as a novel regulator of DNA hypomethylation in cancer and an exploitable target against cancer-promoting epigenetics.

CoRAL accurately resolves extrachromosomal DNA genome structures with long-read sequencing.

Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in approximately 15% of early stage cancers and 30% of late-stage cancers. EcDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy-number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies. Paired-end short-read (Illumina) sequencing and mapping have been utilized to represent ecDNA amplifications using a breakpoint graph, where the inferred architecture of ecDNA is encoded as a cycle in the graph. Traversals of breakpoint graph have been used to successfully predict ecDNA presence in cancer samples. However, short-read technologies are intrinsically limited in the identification of breakpoints, phasing together of complex rearrangements and internal duplications, and deconvolution of cell-to-cell heterogeneity of ecDNA structures. Long-read technologies, such as from Oxford Nanopore Technologies, have the potential to improve inference as the longer reads are better at mapping structural variants and are more likely to span rearranged or duplicated regions. Here, we propose CoRAL (Complete Reconstruction of Amplifications with Long reads), for reconstructing ecDNA architectures using long-read data. CoRAL reconstructs likely cyclic architectures using quadratic programming that simultaneously optimizes parsimony of reconstruction, explained copy number, and consistency of long-read mapping. CoRAL substantially improves reconstructions in extensive simulations and 9 datasets from previously-characterized cell-lines as compared to previous short-read-based tools. As long-read usage becomes wide-spread, we anticipate that CoRAL will be a valuable tool for profiling the landscape and evolution of focal amplifications in tumors. Availability: https://github.com/AmpliconSuite/CoRAL.

Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for induction of ecDNA with extensive characterization of newly formed ecDNA to examine ecDNA biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of a 1 Mb and 1.8 Mb ecDNA both reaching 15%. We show non-homologous end joining and microhomology mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

Metabolic and epigenetic reprogramming in the pathogenesis of glioblastoma: Toward the establishment of "metabolism-based pathology".

Molecular genetic approaches are now mandatory for cancer diagnostics, especially for brain tumors. Genotype-based diagnosis has predominated over the phenotype-based approach, with its prognostic and predictive powers. However, comprehensive genetic testing would be difficult to perform in the clinical setting, and translational research is required to histologically decipher the peculiar biology of cancer. Of interest, recent studies have demonstrated discrete links between oncogenotypes and the resultant metabolic phenotypes, revealing cancer metabolism as a promising histologic surrogate to reveal specific characteristics of each cancer type and indicate the best way to manage cancer patients. Here, we provide an overview of our research progress to work on cancer metabolism, with a particular focus on the genomically well-characterized malignant tumor glioblastoma. With the use of clinically relevant animal models and human tissue, we found that metabolic reprogramming plays a major role in the aggressive cancer biology by conferring therapeutic resistance to cancer cells and rewiring their epigenomic landscapes. We further discuss our future endeavor to establish "metabolism-based pathology" on how the basic knowledge of cancer metabolism could be leveraged to improve the management of patients by linking cancer cell genotype, epigenotype, and phenotype through metabolic reprogramming.

Coordinated inheritance of extrachromosomal DNA species in human cancer cells.

The chromosomal theory of inheritance has dominated human genetics, including cancer genetics. Genes on the same chromosome segregate together while genes on different chromosomes assort independently, providing a fundamental tenet of Mendelian inheritance. Extrachromosomal DNA (ecDNA) is a frequent event in cancer that drives oncogene amplification, dysregulated gene expression and intratumoral heterogeneity, including through random segregation during cell division. Distinct ecDNA sequences, herein termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells. However, how multiple ecDNA species within a tumor cell are assorted and maintained across somatic cell generations to drive cancer cell evolution is not known. Here we show that cooperative ecDNA species can be coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. EcDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy number gains in multiple ecDNA species prior to any selection. Computational modeling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Finally, we show that coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Transcriptional immune suppression and upregulation of double stranded DNA damage and repair repertoires in ecDNA-containing tumors.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood. We examined gene expression patterns of 870 tumors of varied histological types, to identify transcriptional correlates of ecDNA. Here we show that ecDNA containing tumors impact four major biological processes. Specifically, ecDNA containing tumors upregulate DNA damage and repair, cell cycle control, and mitotic processes, but downregulate global immune regulation pathways. Taken together, these results suggest profound alterations in gene regulation in ecDNA containing tumors, shedding light on molecular processes that give rise to their development and progression.

Integrated analysis of single-cell chromatin state and transcriptome identified common vulnerability despite glioblastoma heterogeneity.

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

A Cell Type Selective YM155 Prodrug Targets Receptor-Interacting Protein Kinase 2 to Induce Brain Cancer Cell Death.

Glioblastoma (GBM) is the most prevalent and aggressive primary central nervous system (CNS) malignancy. YM155 is a highly potent broad-spectrum anti-cancer drug that was derived from a phenotypic screen for functional inhibitors of survivin expression, but for which the relevant biomolecular target remains unknown. Presumably as a result of its lack of cell-type selectivity, YM155 has suffered from tolerability issues in the clinic. Based on its structural similarity to the GBM-selective prodrug RIPGBM, here, we report the design, synthesis, and characterization of a prodrug form of YM155, termed aYM155. aYM155 displays potent cell killing activity against a broad panel of patient-derived GBM cancer stem-like cells (IC50 = 0.7-10 nM), as well as EGFR-amplified and EGFR variant III-expressing (EGFRvIII) cell lines (IC50 = 3.8-36 nM), and becomes activated in a cell-type-dependent manner. Mass spectrometry-based analysis indicates that enhanced cell-type selectivity results from relative rates of prodrug activation in transformed versus non-transformed cell types. The prodrug strategy also facilitates transport into the brain (brain-to-plasma ratio, aYM155 = 0.56; YM155 = BLQ). In addition, we determine that the survivin-suppressing and apoptosis-inducing activities of YM155 involve its interaction with receptor-interacting protein kinase 2 (RIPK2). In an orthotopic intracranial GBM xenograft model, aYM155 prodrug significantly inhibits brain tumor growth in vivo, which correlates with cell-type selective survivin-based pharmacodynamic effects.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Breakage fusion bridge cycles drive high oncogene copy number, but not intratumoral genetic heterogeneity or rapid cancer genome change.

Oncogene amplification is a major driver of cancer pathogenesis. Breakage fusion bridge (BFB) cycles, like extrachromosomal DNA (ecDNA), can lead to high copy numbers of oncogenes, but their impact on intratumoral heterogeneity, treatment response, and patient survival are not well understood due to difficulty in detecting them by DNA sequencing. We describe a novel algorithm that detects and reconstructs BFB amplifications using optical genome maps (OGMs), called OM2BFB. OM2BFB showed high precision (>93%) and recall (92%) in detecting BFB amplifications in cancer cell lines, PDX models and primary tumors. OM-based comparisons demonstrated that short-read BFB detection using our AmpliconSuite (AS) toolkit also achieved high precision, albeit with reduced sensitivity. We detected 371 BFB events using whole genome sequences from 2,557 primary tumors and cancer lines. BFB amplifications were preferentially found in cervical, head and neck, lung, and esophageal cancers, but rarely in brain cancers. BFB amplified genes show lower variance of gene expression, with fewer options for regulatory rewiring relative to ecDNA amplified genes. BFB positive (BFB (+)) tumors showed reduced heterogeneity of amplicon structures, and delayed onset of resistance, relative to ecDNA(+) tumors. EcDNA and BFB amplifications represent contrasting mechanisms to increase the copy numbers of oncogene with markedly different characteristics that suggest different routes for intervention.

Extrachromosomal DNA (ecDNA): an origin of tumor heterogeneity, genomic remodeling, and drug resistance.

The genome of cancer cells contains circular extrachromosomal DNA (ecDNA) elements not found in normal cells. Analysis of clinical samples reveal they are common in most cancers and their presence indicates poor prognosis. They often contain enhancers and driver oncogenes that are highly expressed. The circular ecDNA topology leads to an open chromatin conformation and generates new gene regulatory interactions, including with distal enhancers. The absence of centromeres leads to random distribution of ecDNAs during cell division and genes encoded on them are transmitted in a non-mendelian manner. ecDNA can integrate into and exit from chromosomal DNA. The numbers of specific ecDNAs can change in response to treatment. This dynamic ability to remodel the cancer genome challenges long-standing fundamentals, providing new insights into tumor heterogeneity, cancer genome remodeling, and drug resistance.

Systematic discovery and functional dissection of enhancers needed for cancer cell fitness and proliferation.

A scarcity of functionally validated enhancers in the human genome presents a significant hurdle to understanding how these cis-regulatory elements contribute to human diseases. We carry out highly multiplexed CRISPR-based perturbation and sequencing to identify enhancers required for cell proliferation and fitness in 10 human cancer cell lines. Our results suggest that the cell fitness enhancers, unlike their target genes, display high cell-type specificity of chromatin features. They typically adopt a modular structure, comprised of activating elements enriched for motifs of oncogenic transcription factors, surrounded by repressive elements enriched for motifs recognized by transcription factors with tumor suppressor functions. We further identify cell fitness enhancers that are selectively accessible in clinical tumor samples, and the levels of chromatin accessibility are associated with patient survival. These results reveal functional enhancers across multiple cancer cell lines, characterize their context-dependent chromatin organization, and yield insights into altered transcription programs in cancer cells.

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

The evolutionary dynamics of extrachromosomal DNA in human cancers.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumor growth, drug resistance and shorter survival. Currently, the impact of nonchromosomal oncogene inheritance-random identity by descent-is poorly understood. Also unclear is the impact of ecDNA on somatic variation and selection. Here integrating theoretical models of random segregation, unbiased image analysis, CRISPR-based ecDNA tagging with live-cell imaging and CRISPR-C, we demonstrate that random ecDNA inheritance results in extensive intratumoral ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted treatment. Observed ecDNAs benefit host cell survival or growth and can change within a single cell cycle. ecDNA inheritance can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are facilitated by the ability of ecDNA to rapidly adapt genomes in a way that is not possible through chromosomal oncogene amplification. These results show how the nonchromosomal random inheritance pattern of ecDNA contributes to poor outcomes for patients with cancer.

Leveraging extrachromosomal DNA to fine-tune trials of targeted therapy for glioblastoma: opportunities and challenges.

Glioblastoma evolution is facilitated by intratumour heterogeneity, which poses a major hurdle to effective treatment. Evidence indicates a key role for oncogene amplification on extrachromosomal DNA (ecDNA) in accelerating tumour evolution and thus resistance to treatment, particularly in glioblastomas. Oncogenes contained within ecDNA can reach high copy numbers and expression levels, and their unequal segregation can result in more rapid copy number changes in response to therapy than is possible through natural selection of intrachromosomal genomic loci. Notably, targeted therapies inhibiting oncogenic pathways have failed to improve glioblastoma outcomes. In this Perspective, we outline reasons for this disappointing lack of clinical translation and present the emerging evidence implicating ecDNA as an important driver of tumour evolution. Furthermore, we suggest that through detection of ecDNA, patient selection for clinical trials of novel agents can be optimized to include those most likely to benefit based on current understanding of resistance mechanisms. We discuss the challenges to successful translation of this approach, including accurate detection of ecDNA in tumour tissue with novel technologies, development of faithful preclinical models for predicting the efficacy of novel agents in the presence of ecDNA oncogenes, and understanding the mechanisms of ecDNA formation during cancer evolution and how they could be attenuated therapeutically. Finally, we evaluate the feasibility of routine ecDNA characterization in the clinic and how this process could be integrated with other methods of molecular stratification to maximize the potential for clinical translation of precision medicines.