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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

Preprint posted on 21 October 2020 https://www.biorxiv.org/content/10.1101/2020.10.20.335216v1

Extrachromosomal DNA drives intratumor genetic heterogeneity

Selected by Sree Rama Chaitanya

Categories: cancer biology, cell biology, molecular biology, neuroscience

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. melanogaster, C. 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

Posted on: 28 October 2020

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

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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

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