Biology subjects

Rose, J. C.

Publications and source records attributed to Rose, J. C..

5 recordsLinked to original sources

bioRxiv2026

scAmp analyzes focal gene amplifications at single-cell resolution

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common driver of tumor progression and is associated with acquired drug resistance and poor patient survival. While whole genome sequencing (WGS) studies have revealed the landscape of genes amplified on ecDNA in tumors, it remains challenging to study the subclonal heterogeneity and functional (e.g., transcriptomic) consequences of ecDNA on tumors. To address this, we introduce scAmp: a probabilistic algorithm for detecting and analyzing ecDNA from single-cell datasets. We demonstrate scAmps improved accuracy over WGS approaches on well-characterized cell-lines and its applicability to clinical histopathology. We further showcase scAmp by analyzing 73 patient tumors profiled with single-cell ATAC-seq, where we analyze the subclonal evolution of ecDNA+ subclones and identify the effect of ecDNA amplifications on the chromatin accessibility landscape of cancer cells. Together, we anticipate that scAmp will broadly enable further studies - both retrospective and prospective - that dissect critical questions of how ecDNA affect cancer cells and the tumors in which they reside.

cancer biology↗

bioRxiv2023

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair

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. SIGNIFICANCEOur study harnesses a CRISPR-based method to examine ecDNA biogenesis, uncovering efficient circularization between DSBs. ecDNAs and their corresponding chromosomal scars can form via NHEJ or MMEJ, but the ecDNA and scar formation processes are distinct. Based on our findings, we establish a mechanistic model of excisional ecDNA formation.

cancer biology↗

bioRxiv2022

Designed sensors reveal normal and oncogenic Ras signaling in endomembranes and condensates

Genetically encoded biosensors have accelerated biological discovery, however many important targets such as active Ras (Ras-GTP) are difficult to sense as strategies to match a sensors sensitivity to the physiological range of target are lacking. Here, we use computational protein design to generate and optimize intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity: Ras-LOCKR-S) and proximity labelers of the signaling environment of Ras (LOCKR-based, Ras activity-dependent Proximity Labeler: Ras-LOCKR-PL). We demonstrate that our tools can measure endogenous Ras activity and environment at subcellular resolution. We illustrate the application of these tools by using them to identify Ras effectors, notably Src-Associated in Mitosis 68 kDa protein (SAM68), enriched in oncogenic EML4-Alk granules. Localizing these sensors to these granules revealed that SAM68 enhances Ras activity specifically at the granules, and SAM68 inhibition sensitizes EML4-Alk-driven cancer cells to existing drug therapies, suggesting a possible therapeutic strategy.

cell 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↗

bioRxiv2019

Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

CRISPR/Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report off-target sites can be shielded from the active Cas9*single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating \"scarless\" editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a novel and flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

molecular biology↗