Close

Live-cell imaging shows uneven segregation of extrachromosomal DNA elements and transcriptionally active extrachromosomal DNA clusters in cancer

Eunhee Yi, Amit D. Gujar, Molly Guthrie, Hoon Kim, Kevin C. Johnson, Samirkumar B. Amin, Sunit Das, Patricia A. Clow, Albert W. Cheng, Roel GW Verhaak

Posted on: 28 October 2020

Preprint posted on 21 October 2020

Extrachromosomal DNA drives intratumor genetic heterogeneity

Selected by Sree Rama Chaitanya

Background1-3

Extrachromosomal DNAs (ecDNAs) are genetic elements present inside the nucleus independent of a linear chromosome that are about 50kb-5Mb in size, harboring a set of genes and regulatory elements. ecDNAs are formed when broken linear DNA ligate end-to-end. ecDNAs are found in many eukaryotic species like yeast, D. melanogasterC. elegans, and humans. In cancer cells, ecDNAs predominantly accommodate oncogenes or fused oncogenes to facilitate oncogene amplification. However, it is not clear how ecDNA propagate intratumor genetic heterogeneity, a characteristic feature of multidrug-resistant cancer colonies. Unequivocal segregation of ecDNA to daughter cells could impel intratumor genetic heterogeneity. To address this, the authors of the current study used cutting-edge CRISPR based imaging techniques to investigate the dynamics of ecDNA in glioblastoma.

Key findings

1. To gain insights into the dynamics of ecDNA in glioblastoma, the investigators used four glioblastoma samples and a pair of neurospheres derived from the same patients. They evaluated fluorescent in situ hybridization (FISH) of EGFR(an oncogene of glioblastoma) to score ecDNA based on their earlier studies4 and a chromosome 7 (Chr7) loci for linear intact chromosome. They found that EGFR-containing ecDNA numbers vary among samples than Chr7 that was rather evenly distributed. They further report that unequivocal EGFR ecDNA amplification corroborated with heterogeneity in protein expression. In other cell lines, they also found heterogenous gene amplification of ecDNA-associated genes than genes harbored in linear chromosomes. Thus, they report that ecDNA could drive genetic heterogeneity in cancer cells (fig.1).

Taken directly from Eunhee Yi et. al., 2020 under a CC-BY 4.0 international license.

2. To address how ecDNA heterogeneity is dispersed in cancer cells, the authors implemented a CRISPR-based DNA labeling system (fig.2). For this purpose, they used nuclease dead Cas9, provided a single guide-RNAs with 25 Pumilio/FBF (PUF) RNA-binding sites that would bind to the unique fusion sequences at the ecDNA breakpoints, and recruit fluorescently tagged PUF proteins to identify ecDNA in real-time. They identified four unique ecDNAs – ecEGFRx1, ecEGFR, ecCCAT1, and ecCCDC26– harboring an EGFR exon1, a full-length EGFR, and non-coding genes CCAT and CCDC26(using sequencing, FISH, and PCR-based techniques). They detected all four ecDNA outside chromosomes using breakpoint-specific FISH imaging in metaphase spreads. They were able to demonstrate all four ecDNA using the CRISPR-based system in neurosphere cells but not in prostate cancer cells that acted as a control. Moreover, they found that CRISPR labeling coincided with the breakpoint-specific FISH signal suggesting the reliability of the signal. They also found heterogeneous ecDNA copy numbers than linear chromosome locales (Chr7 and MUC4) in the neurospheres.

3. The authors then tracked the ecDNA live using the same CRISPR-based DNA labeling system and found uneven segregation of the ecDNA to daughter cells even when followed for 48hrs, unlike Chr7and MUC4. They also found that ecDNAs tend to coalesce in at least 50% of neurospheres in the 48hr time frame. Furthermore, they show that these ecDNA clusters co-localized with nuclear bodies5 (Cajal and PML bodies) that act as a macromolecular hub, but there was no significant linear correlation between the number of ecDNAs and the number of nuclear bodies, suggesting that ecDNAs generate its own hub. Intriguingly, they also report ecDNA clusters co-localized with RNA polymerase II in 60% of the cells and the size of ecEGFRfoci indicating its clustering positively correlated with EGFR mRNA levels.

Conclusion 

Recent studies revealed intratumor genetic heterogeneity as a critical factor for the perpetuation of multidrug-resistant cancer colonies. Intratumor genetic heterogeneity poses a great challenge to personalized medicine. Here the authors report that intratumor genetic heterogeneity is promoted by ecDNAs through their uneven segregation during cell divisions and clustering to facilitate oncogene expression or amplification (fig.1). The current study raises many interesting hypotheses on how genome instability drives oncogenesis and cancer evolution.

 Acknowledgments

I am grateful to all the authors for their support, especially Eunhee Yi and Roel GW Verhaak for being open to discuss the work and replying promptly.

References

  1. https://doi.org/10.1038/s41568-019-0128-6
  2. https://doi.org/10.1016/j.molonc.2014.06.005
  3. https://doi.org/10.1016/j.annonc.2020.03.303
  4. https://doi.org/10.1038/s41588-018-0105-0
  5. https://doi.org/10.1016/S0962-8924(99)01606-2

Tags: extra chromosomal dna, glioblastoma, intratumor heterogeneity, non-canonical transcription

doi: https://doi.org/10.1242/prelights.25485

Read preprint (No Ratings Yet)

Author's response

Eunhee Yi and other authors shared

1. The authors’ data suggest the impacts of oncogenic ecDNA on intratumor genetic heterogeneity and the evolution of resistant cancer cells. The authors also detected ecDNA specifically using CRISPR-based technology. Did the authors tested or considered genetically manipulating oncogenic ecDNA and evaluate the causative impact?

EY: Manipulating oncogenic ecDNA to study its role and impact in cancer progression is the most important quest that we hope to pursue since we believe that controlling oncogenic ecDNA will allow us to prove current therapeutic approaches. However, there was a technical limitation on the ecDNA-specific manipulation because most of the ecDNA is identical to chromosomal DNA. In this study, we not only tried to demonstrate the role of ecDNA on intratumoral heterogeneity but also successfully tested a CRISPR-based genome imaging strategy to generate ecDNA specific signals by targeting the breakpoint junction region that is unique to ecDNA. Thus, this technology with appropriate modifications might be able to apply for genetically manipulating oncogenic ecDNA.

2. DNA breakage and end-to-end ligation lead to ecDNA formation, suggesting that genome instability plays a crucial role in the formation of ecDNA. Do mutations in DNA damage response (DDR) pathway genes induce ecDNA formation in cancer cells?

EY: There are several pieces of evidence to support the implication of the DDR pathway in ecDNA formation. For example, inhibition of DNA-PKcs, a nonhomologous end joining (NHEJ) protein, reduced ecDNA formation in methotrexate (MTX)-resistance colon cancer cell line1. In the same cell line, BRCA1 silencing inhibits the homologous recombination (HR) pathway and decreased ecDNA copies2. However, the complexities of ecDNA structure within a single tumor and various genomic composition of ecDNA in different cancer types suggest that there are multiple processes, including chromothripsis, involved in the generation of ecDNA.

3. Transcription of ecDNA that harbors fused genes or part of genes could lead to the production of chimeric or fusion RNA that may or may not be functional. This is actually reflected by the authors’ data where EGFRtranscript signals correlate with ecEGFRbut, not ecEGFRx1 (Fig 4E and Fig S9B). However, ecDNAs could facilitate the aberrant production of chimeric RNA or chimeric oncoproteins. It would be interesting to hear the authors’ perspective on how such ecDNA affects cancer cell evolution?

EY: Since we used an EGFR-specific FISH probe that only generates visible signals when the full transcripts of EGFR available, we could potentially miss transcripts of chimeric oncoproteins fused with EGFR exon 1 in this particular example. A previous study from our lab showed fusion gene CAPZA2-MET amplified on ecDNA in glioblastoma patient tumor, and the inhibition of MET improved survival in the PDX model3. Thus, I believe that functional chimeric oncoproteins can be transcribed as long as there is an accessible promoter in the proximal region from the chimeric gene.

4. Are there any hotspots in linear chromosomes that evolve as ecDNAs?

EY: The hotspots of ecDNA have not been discovered yet. A previous study tried to see sequence homologies around the junctions of ecDNA. However, there was no sequence homologies found4. And they found 1-2 bp of microhomologies in 43% of the junctions and small insertion in 30% of the junctions. We also observed microhomologies (<5bp) in the most circular amplicon breakpoint5.

5. Cytosolic DNA triggers an inflammatory response via the cGAS-STING pathway6. Do the authors know the impact of ecDNA on the inflammatory response, if any? This could be relevant, especially for combinatorial immune-therapy.

EY: Excellent point. The aim we are currently focusing on is entrapping ecDNAs from their original place, nucleus, to cytoplasm. Then cGAS-STING, a cytoplasmic DNA-sensing pathway, targets entrapped ecDNA-harboring cells by activating an antitumor response. We expect that a combination of the STING agonist with the agent that recruits ecDNA to cytoplasm will be a potential therapeutic strategy.

References:

  1. https://doi.org/10.1136/jmedgenet-2014-102703
  2. https://doi.org/10.1002/ijc.31781
  3. https://doi.org/10.1038/s41588-018-0105-0
  4. https://doi.org/10.1093/nar/gku1101
  5. https://doi.org/10.1101/859306
  6. https://doi.org/10.1186/s13045-020-00916-z

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cancer biology category:

Mitochondria-derived nuclear ATP surge protects against confinement-induced proliferation defects

Ritobrata Ghose, Fabio Pezzano, Savvas Kourtis, et al.

Selected by 16 May 2024

Teodora Piskova

Cell Biology

Spatial transcriptomics elucidates medulla niche supporting germinal center response in myasthenia gravis thymoma

Yoshiaki Yasumizu, Makoto Kinoshita, Martin Jinye Zhang, et al.

Selected by 27 March 2024

Jessica Chevallier

Immunology

Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

Chrystian Junqueira Alves, Theodore Hannah, Sita Sadia, et al.

Selected by 13 February 2024

Jade Chan

Cancer Biology

Also in the cell biology category:

Mitochondria-derived nuclear ATP surge protects against confinement-induced proliferation defects

Ritobrata Ghose, Fabio Pezzano, Savvas Kourtis, et al.

Selected by 16 May 2024

Teodora Piskova

Cell Biology

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery

Aslihan Terzi, Tiger Liao, Adrian Jacobo

Selected by 09 May 2024

Roberto Rodríguez-Morales

Genetics

Fetal brain response to maternal inflammation requires microglia

Bridget Elaine LaMonica Ostrem, Nuria Dominguez Iturza, Jeffrey Stogsdill, et al.

Selected by 24 April 2024

Manuel Lessi

Neuroscience

Also in the molecular biology category:

Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments

Martina Begnis, Julien Duc, Sandra Offner, et al.

Selected by 10 April 2024

Silvia Carvalho

Cell Biology

Nanos2+ cells give rise to germline and somatic lineages in the sea anemone Nematostella vectensis

Andreas Denner, Julia Steger, Alexander Ries, et al.

Selected by 30 January 2024

Isabella Cisneros

Developmental Biology

Plant plasmodesmata bridges form through ER-driven incomplete cytokinesis

Ziqiang P. Li, Hortense Moreau, Jules D. Petit, et al.

AND

Plasmodesmata act as unconventional membrane contact sites regulating inter-cellular molecular exchange in plants

Jessica Pérez-Sancho, Marija Smokvarska, Marie Glavier, et al.

Selected by 09 January 2024

Gwendolyn K. Kirschner

Plant Biology

Also in the neuroscience category:

Fetal brain response to maternal inflammation requires microglia

Bridget Elaine LaMonica Ostrem, Nuria Dominguez Iturza, Jeffrey Stogsdill, et al.

Selected by 24 April 2024

Manuel Lessi

Neuroscience

Transcriptional profiling of human brain cortex identifies novel lncRNA-mediated networks dysregulated in amyotrophic lateral sclerosis

Alessandro Palma, Monica Ballarino

Selected by 16 April 2024

Julio Molina Pineda

Genomics

RIPK3 coordinates RHIM domain-dependent inflammatory transcription in neurons

Sigal B. Kofman, Lan H. Chu, Joshua M. Ames, et al.

Selected by 08 April 2024

Zoie Magri

Immunology

preLists in the cancer biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Anticancer agents: Discovery and clinical use

Preprints that describe the discovery of anticancer agents and their clinical use. Includes both small molecules and macromolecules like biologics.

 



List by Zhang-He Goh

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

Also in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the molecular biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Also in the neuroscience category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve
Close