The study of extrachromosomal DNA (ecDNA), an element existing beyond classical chromosomes, contributes to creating a more comprehensive map of the cancer genome. In hematological malignancies, research on ecDNA has lacked comprehensive investigation into its frequency, structure, function, and mechanisms of formation. We re-analyzed WGS data from 208 hematological cancer samples across 11 types, focusing on ecDNA characteristics. Amplification of ecDNA was observed in 7 of these cancer types, with no instances found in normal blood cells. Patients with leukemia carrying ecDNA showed a low induction therapy remission rate (<30 %), a high relapse rate (75 %) among those who achieved complete remission, and a significantly lower survival rate compared to the general leukemia population, even those with complex chromosomal karyotypes. Among the 55 identified ecDNA amplicons, 268 genes were detected, of which 38 are known cancer-related genes exhibiting significantly increased copy numbers. By integrating RNA-Seq data, we discovered that the increased copy number, resulting in a higher amount of available DNA templates, indeed leads to the elevated expression of genes encoded on ecDNA. Additionally, through the integration of H3K4me3/H3K27ac chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin with sequencing, and high-throughput chromosome conformation capture data, we identified that ecDNA amplifications can also facilitate efficient, copy number-independent amplification of oncogenes. This process is linked to active histone modifications, improved chromatin accessibility, and enhancer hijacking, all of which are effects of ecDNA amplification. Mechanistically, chromothripsis and dysfunction of the DNA repair pathway can, to some extent, explain the origin of ecDNA.

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 up-regulate DNA damage and repair, cell cycle control, and mitotic processes, but down-regulate 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.

Transposable elements (TEs) are mobile DNA elements that are particularly abundant in the plant genomes. They have long been considered as junk DNA; however, a growing body of evidence suggests that TE insertions promote genetic diversity that is essential for the adaptive evolution of a species. Thus far, studies have mainly investigated the cis-acting regulatory roles of TEs generated by their insertions nearby or within the host genes. However, the trans-acting effects of TE-derived RNA and DNA remained obscure to date. TEs contain various regulatory elements within their sequences that can accommodate the binding of specific RNAs and proteins. Recently, it was suggested that some of these cellular regulators are shared between TEs and the host genes, and the competition for the common host factors underlies the fine-tuned developmental reprogramming. In this review, we will highlight and discuss the latest discoveries on the biological functions of plant TEs, with a particular focus on their competitive binding with specific developmental regulators.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and this is one of the first analyses of these events using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes and only one BFB breakpoint showed chromothripsis. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Indeed, YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that cervical cancer patients with YAP1/BIRC2/BIRC3-amplification, especially those of African American ancestry, might benefit from targeted therapy. In summary, we uncovered new insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

UNASSIGNED: Medulloblastoma is the most common type of malignant pediatric brain tumor with group 4 medulloblastomas (G4 MBs) accounting for 40% of cases. However, the molecular mechanisms that underlie this subgroup are still poorly understood. Point mutations are detected in a large number of genes at low incidence per gene while the detection of complex structural variants in recurrently affected genes typically requires the application of long-read technologies.
UNASSIGNED: Here, we applied linked-read sequencing, which combines the long-range genome information of long-read sequencing with the high base pair accuracy of short read sequencing and very low sample input requirements.
UNASSIGNED: We demonstrate the detection of complex structural variants and point mutations in these tumors, and, for the first time, the detection of extrachromosomal DNA (ecDNA) with linked-reads. We provide further evidence for the high heterogeneity of somatic mutations in G4 MBs and add new complex events associated with it.
UNASSIGNED: We detected several enhancer-hijacking events, an ecDNA containing the MYCN gene, and rare structural rearrangements, such a chromothripsis in a G4 medulloblastoma, chromoplexy involving 8 different chromosomes, a TERT gene rearrangement, and a PRDM6 duplication.

Super-enhancers are large, densely concentrated swaths of enhancers that regulate genes critical for cell identity. Tumorigenesis is accompanied by changes in the super-enhancer landscape. These aberrant super-enhancers commonly form to activate proto-oncogenes, or other genes upon which cancer cells depend, that initiate tumorigenesis, promote tumor proliferation, and increase the fitness of cancer cells to survive in the tumor microenvironment. These include well-recognized master regulators of proliferation in the setting of cancer, such as the transcription factor MYC which is under the control of numerous super-enhancers gained in cancer compared to normal tissues. This Review will cover the expanding cell-intrinsic and cell-extrinsic etiology of these super-enhancer changes in cancer, including somatic mutations, copy number variation, fusion events, extrachromosomal DNA, and 3D chromatin architecture, as well as those activated by inflammation, extra-cellular signaling, and the tumor microenvironment.

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.

Telomeres are structures that cap the ends of linear chromosomes and play critical roles in maintaining genome integrity and establishing the replicative lifespan of cells. In stem and cancer cells, telomeres are actively elongated by either telomerase or the alternative lengthening of telomeres (ALT) pathway. This pathway is characterized by several hallmark features, including extrachromosomal C-rich circular DNAs that can be probed to assess ALT activity. These so-called C-circles are the product of ALT-associated DNA damage repair processes and simultaneously serve as potential templates for iterative telomere extension. This bifunctional nature makes C-circles highly sensitive and specific markers of ALT. Here, we describe a C-circle assay, adapted from previous reports, that enables the quantitation of C-circle abundance in mammalian cells subjected to a wide range of experimental perturbations. This protocol combines the Quick C-circle Preparation (QCP) method for DNA isolation with fluorometry-based DNA quantification, rolling circle amplification (RCA), and detection of C-circles using quantitative PCR. Moreover, the inclusion of internal standards with well-characterized telomere maintenance mechanisms (TMMs) allows for the reliable benchmarking of cells with unknown TMM status. Overall, our work builds upon existing protocols to create a generalizable workflow for in vitro C-circle quantitation and ascertainment of TMM identity.

The advent of tyrosine kinase inhibitors (TKIs) for treating epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) has been a game changer in lung cancer therapy. However, patients often develop resistance to the drugs within a few years. Despite numerous studies that have explored resistance mechanisms, particularly in regards to collateral signal pathway activation, the underlying biology of resistance remains largely unknown. This review focuses on the resistance mechanisms of EGFR-mutated NSCLC from the standpoint of intratumoral heterogeneity, as the biological mechanisms behind resistance are diverse and largely unclear. There exist various subclonal tumor populations in an individual tumor. For lung cancer patients, drug-tolerant persister (DTP) cell populations may have a pivotal role in accelerating the evolution of tumor resistance to treatment through neutral selection. Cancer cells undergo various changes to adapt to the new tumor microenvironment caused by drug exposure. DTP cells may play a crucial role in this adaptation and may be fundamental in mechanisms of resistance. Intratumoral heterogeneity may also be precipitated by DNA gains and losses through chromosomal instability, and the role of extrachromosomal DNA (ecDNA) may play an important role. Significantly, ecDNA can increase oncogene copy number alterations and enhance intratumoral heterogeneity more effectively than chromosomal instability. Additionally, advances in comprehensive genomic profiling have given us insights into various mutations and concurrent genetic alterations other than EGFR mutations, inducing primary resistance in the context of tumor heterogeneity. Understanding the mechanisms of resistance is clinically crucial since these molecular interlayers in cancer-resistance mechanisms may help to devise novel and individualized anticancer therapeutic approaches.