Biology subjects

Hung, K. L.

Publications and source records attributed to Hung, K. L..

3 recordsLinked to original sources

bioRxiv2021

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 present a method for targeted purification of megabase-sized ecDNA by combining in-vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA (CRISPR-CATCH). We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells. Targeted purification of ecDNA versus chromosomal DNA enabled phasing of genetic variants and provided definitive proof of an EGFRvIII mutation on ecDNA and wild-type EGFR on chromosomal DNA in a glioblastoma neurosphere model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNA compared to the native chromosomal locus in the same cells. Finally, separation of ecDNA species by size and sequencing allowed accurate reconstruction of megabase- sized ecDNA structures with base-pair resolution. CRISPR-CATCH is a new addition to the toolkit for studying focal amplifications in cancer and will accelerate studies aiming to explore the genetic and epigenetic landscapes of ecDNA.

cancer biology↗

bioRxiv2021

Principles of ecDNA random inheritance drive rapid genome change and therapy resistance in human cancers

The foundational principles of Darwinian evolution are variation, selection, and identity by descent. Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumour growth, drug resistance, and shorter survival in patients1-4. Currently, the impact of non-chromosomal oncogene inheritance--random identity by descent--is not well understood. Neither is the impact of ecDNA on variation and selection. Here, integrating mathematical modeling, unbiased image analysis, CRISPR-based ecDNA tagging, and live-cell imaging, we identify a set of basic "rules" for how random ecDNA inheritance drives oncogene copy number and distribution, resulting in extensive intratumoural ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted cancer treatment. Observed ecDNAs obligatorily benefit host cell survival or growth and can change within a single cell cycle. In studies ranging from well-curated, patient-derived cancer cell cultures to clinical tumour samples from patients with glioblastoma and neuroblastoma treated with oncogene-targeted drugs, we show how these ecDNA inheritance "rules" can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are entailed by their ability to rapidly change their genomes in a way that is not possible for cancers driven by chromosomal oncogene amplification. These results shed new light on how the non-chromosomal random inheritance pattern of ecDNA underlies poor outcomes for cancer patients.

cancer biology↗

bioRxiv2020

EcDNA hubs drive cooperative intermolecular oncogene expression

Extrachromosomal DNAs (ecDNAs) are prevalent in human cancers and mediate high oncogene expression through elevated copy number and altered gene regulation1. Gene expression typically involves distal enhancer DNA elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs, comprised of ~10-100 ecDNAs clustered in the nucleus of interphase cells, drive intermolecular enhancer input for amplified oncogene expression. Single-molecule sequencing, single-cell multiome, and 3D enhancer connectome reveal subspecies of MYC-PVT1 ecDNAs lacking enhancers that access intermolecular and ectopic enhancer-promoter interactions in ecDNA hubs. ecDNA hubs persist without transcription and are tethered by BET protein BRD4. BET inhibitor JQ1 disperses ecDNA hubs, preferentially inhibits ecDNA oncogene transcription, and kills ecDNA+ cancer cells. Two amplified oncogenes MYC and FGFR2 intermix in ecDNA hubs, engage in intermolecular enhancer-promoter interactions, and transcription is uniformly sensitive to JQ1. Thus, ecDNA hubs are nuclear bodies of many ecDNAs tethered by proteins and platforms for cooperative transcription, leveraging the power of oncogene diversification and combinatorial DNA interactions. We suggest ecDNA hubs, rather than individual ecDNAs, as units of oncogene function, cooperative evolution, and new targets for cancer therapy.

cancer biology↗