Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Hung, King L.; Luebeck, Jens; Dehkordi, Siavash R.; Colón, Caterina I.; Li, Rui; Wong, Ivy Tsz-Lo; Çoruh, Ceyda; Dharanipragada, Prashanthi; Lomeli, Shirley H.; Weiser, Natasha E.; Moriceau, Gatien; Zhang, Xiao; Bailey, Chris; Houlahan, Kathleen E.; Yang, Wenting; González, Rocío Chamorro; Swanton, Charles; Curtis, Christina; Jamal‐Hanjani, Mariam; Henssen, Anton; Law, Julie A.; Greenleaf, William J.; Lo, Roger S.; Mischel, Paul S.; Bafna, Vineet; Chang, Howard Y.

doi:10.1038/s41588-022-01190-0

Cited by 68 publications

(53 citation statements)

References 53 publications

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“…Moreover, we validated a recurrent ecDNA by direct isolation and high-depth sequencing [ NRAS amplification, CN 13, Pt9-double drug-disease progression (DD-DP)1; Fig. 2A ] using a new approach referred to as CRISPR-CATCH ( 24 ). This alternative technique confirmed the circularized junctions of an 890-kb driver ecDNA within this acquired-resistant clinical tumor sample.…”

Section: Resultsmentioning

confidence: 99%

Blocking Genomic Instability Prevents Acquired Resistance to MAPK Inhibitor Therapy in Melanoma

et al. 2023

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Blocking cancer genomic instability may prevent tumor diversification and escape from therapies. We show that, after MAPK inhibitor (MAPKi) therapy in patients and patient-derived xenografts (PDXs), acquired-resistant genomes of metastatic cutaneous melanoma specifically amplify resistance-driver, non-homologous end-joining (NHEJ), and homologous recombination repair (HRR) genes via complex genomic rearrangements (CGRs) and extrachromosomal DNAs (ecDNAs). Almost all sensitive and acquired-resistant genomes harbor pervasive chromothriptic regions with disproportionately high mutational burdens and significant overlaps with ecDNA and CGR spans. Recurrently, somatic mutations within ecDNA- and CGR-amplicons enrich for HRR signatures, particularly within acquired-resistant tumors. Regardless of sensitivity or resistance, breakpoint-sequence analysis suggests NHEJ as critical to double-stranded DNA break repair underlying CGR and ecDNA formation. In human melanoma cell lines and PDXs, NHEJ-targeting by a DNA-PKCS inhibitor prevents/delays acquired MAPKi resistance by reducing the size of ecDNAs and CGRs early on combination treatment. Thus, targeting the cause(s) of genomic instability prevents acquired resistance.

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Section: Resultsmentioning

confidence: 99%

Blocking Genomic Instability Prevents Acquired Resistance to MAPK Inhibitor Therapy in Melanoma

et al. 2023

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“…In some ecDNA+ patient-derived samples and cell lines, a deletion exists in the corresponding native chromosomal locus, which we term the “excision scar” ( Fig 1A )(5,6,18). While the end-swapping model predicts that the excised circle and the excision scar left behind should form at a 1:1 ratio, we previously observed that excision scar formation was less efficient than circularization when generating DHFR ecDNA ( Fig 2E )(6).…”

Section: Resultsmentioning

confidence: 99%

“…Potent oncogenes are frequently encoded on ecDNA and can become massively amplified in copy number and upregulated in gene expression (2,4). ecDNA is circular and acentric, ranging in size from 100 kb to several Mb (2,5). Due to their lack of centromeres, ecDNA are randomly segregated at cell division and drive intratumoral heterogeneity, facilitating rapid genomic changes to adapt to treatment or metabolic stress (6).…”

Section: Introductionmentioning

confidence: 99%

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair

Rose,

Wong,

Daniel

et al. 2023

Preprint

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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.

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“…The limited understanding of ecDNA stems from its large size, making it difficult to analyze. A recent study used CRISPR-CATCH and in vitro CRISPR-Cas9 treatment, along with pulsed-field gel electrophoresis of agarose-entrapped genomic DNA, to isolate megabase-sized human ecDNAs [ 169 ]. Urgent need exists for new research tools to investigate the amplification of oncogenes through ecDNA and understand ecDNA’s role in cancer development, as it may significantly contribute to cancer’s accelerated evolution [ 170 ].…”

Section: Discussionmentioning

confidence: 99%

Unraveling the Impact of Intratumoral Heterogeneity on EGFR Tyrosine Kinase Inhibitor Resistance in EGFR-Mutated NSCLC

Kobayashi

Tan

2023

IJMS

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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.

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Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Cited by 68 publications

References 53 publications

Blocking Genomic Instability Prevents Acquired Resistance to MAPK Inhibitor Therapy in Melanoma

Blocking Genomic Instability Prevents Acquired Resistance to MAPK Inhibitor Therapy in Melanoma

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair

Unraveling the Impact of Intratumoral Heterogeneity on EGFR Tyrosine Kinase Inhibitor Resistance in EGFR-Mutated NSCLC

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