Biology subjects

Au, V.

Publications and source records attributed to Au, V..

10 recordsLinked to original sources

bioRxiv2026

Chromosomal mutational signatures of DNA damaging agents at single cell resolution

The chromosomal-scale mutational spectrum of small molecules that interact with DNA has been hard to study at scale, as mutational events are distributed in location and occur in parallel in different cells. Here, we present a framework that pairs phylogenetic ancestry reconstruction with mutational signature decomposition to characterise recent, cell-private copy number alteration (CNA) mutational patterns at single-cell resolution. We used this framework to characterise the cell-wise mutational spectrum of contemporaneous CNAs generated by double-strand-break-inducing chemotherapeutic drugs. We demonstrate that platinum salts, G-quadruplex stabilizers and topoisomerase II inhibitors, although mechanistically distinct, converge on a mutational signature dominated by telomere-bounded copy-number gains and losses. This signature is observed in different genetic backgrounds and in vivo in drug-treated patient-derived xenografts. We also observe a high rate of endogenous telomere-bounded mutational foreground in BRCA1 deficient cells. We show that the single cell genome derived signature exposures are drug dose-dependent, and use this to identify the decay of mutational load after drug withdrawal. We observe that both cisplatin and a G4 binder molecule (CX5461) exhibit foreground mutational signature persistence for at least 3 weeks after drug withdrawal, suggesting that residual effects of exposure may last longer than anticipated. Finally, extending the framework to serially drug-treated patient-derived xenograft (PDX) models, we show that telomere-bounded CNA signature exposure is associated with tumoural response to drug, consistent with loss of mutational activity on the genome after acquired resistance emerges. Together, our results show that our framework applied on scWGS identifies contemporaneous chromosomal mutation patterns induced by small molecules in human tissues.

cancer biology↗

bioRxiv2026

Minimally invasive monitoring of clonal evolution through integrated single cell and ctDNA analysis

Circulating cell-free DNA (cfDNA) offers a minimally invasive lens into tumor evolution. However, the accurate quantification of clonal composition from cfDNA remains challenging. Existing methods for clone tracking using liquid biopsies are constrained by issues such as their reliance on bulk tissue references, incomplete representations of clonal architecture or limited breadth of mutation panels. To address these limitations, we developed cfClone, a Bayesian framework that integrates single-cell whole-genome sequencing (scWGS) derived clonal structures with cfDNA whole-genome sequencing (cfDNA-WGS) data to enable high-resolution tissue-informed clonal tracking. By jointly modeling local copy-number alterations and haplotype-specific signals via Bayesian model selection and Markov chain Monte Carlo (MCMC) sampling, the algorithm yields uncertainty-aware estimates of clonal prevalence and tumour fraction (TF). Applied to longitudinal clinical cohorts, cfClone can be used to reconstruct evolutionary trajectories and uncovers clonal selection driving therapeutic resistance, including the de novo detection of emergent clonal populations. We demonstrate that cfClone achieves accurate TF estimates and circulating tumor DNA (ctDNA) detection in malignancies with varying degrees of copy-number variant (CNV) burden using semi-synthetic data. We then compare to the state of the art scWGS informed panel based approach, and demonstrate cfClone provides comparable accuracy while allowing for the tracking of more clones and detection of novel clones. Finally, we show how cfClone can be used to quantitatively track clonal dynamics in response to treatment in high grade serous ovarian cancer. Github link: https://github.com/Roth-Lab/cfclone

cancer biology↗

bioRxiv2026

PD-1 blockade enhances T cell activation by reorchestrating CD28 and CTLA4 ligand interactions

The network of interactions comprising CD28, CTLA4 and PD-1 and their ligands CD80, CD86, and PD-L1 have profound implications for T cell activation. Despite their critical role, factors that determine their receptor occupancy when all three ligands are present remains incompletely understood. Using a supported lipid bilayer to replicate the antigen-presenting cell membrane facilitated a quantitative analysis of CD80, CD86, and PD-L1 interactions. Our observations reveal that PD-1 blockade operates through a previously unrecognized competitive mechanism beyond conventional checkpoint inhibition: liberated PD-L1 molecules redistribute to form CD80-PD-L1 heterodimers that strengthen CD28-CD80 interactions and outcompete CD86 for CD28 binding. This preferential binding enhances T cell activation through superior CD80 co-stimulation. Furthermore, the redistribution by PD-1 blockade reduces CD80 homodimer availability, limiting CTLA4 binding to CD80 and enhancing CD28 signaling. These findings provide vital insights into the competitive principles that allow B7-CD28 family receptors to regulate T cell activation through network-level effects.

immunology↗

bioRxiv2025

Somatic copy number mutations contribute to fitness in transplantation models of spontaneous human breast cancer metastasis

The contribution of somatic gene dosage mutations (CNA) to breast cancer metastasis remains poorly defined. Using 9 transplantable human neoadjuvant-naive triple-negative breast cancer xenografts, we studied the fitness of copy number clones in spontaneous metastasis from orthotopic transplant sites. Metastatic site preference was strongly patient-dependent, and the emergence of metastases exhibited a general trend toward slower growth at the orthotopic site. In our models, single-cell whole-genome sequencing of primary and metastatic sites showed that distant metastases were most often the result of minor prevalence clones at the orthotopic site, suggesting that some metastatic phenotypes may be weakly negatively fit at the primary site. We validated the existence of a fitness hierarchy of copy number clones using a previously established paradigm of remixing and retransplanting clones. Single-cell clone analysis of competitive repopulation and re-emergence of metastases showed that CNAs arising in cancer evolution can mediate metastatic fitness. Moreover, some clones displaying strong metastatic tendency exhibited weaker survival at the primary site, consistent with the notion that metastatic phenotypes could have a fitness cost at the primary site. Finally, we conducted RNA-seq analysis combined with DriverNet analysis to dissect the contribution of CNA-mediated versus genome-independent transcriptional states. CNA mutations appeared to contribute strongly to transcriptional differences between clones. Among clones of high metastatic potential, we observed CNA-mediated and CNA-independent convergence on pathways such as epithelial-mesenchymal transition (EMT), established as mediators of metastatic cell survival at distant sites. Taken together, our data point to a contribution of CNA-mediated cancer evolution to metastatic states and identify distant-site context as a key determinant of CNA-mediated fitness.

cancer biology↗

bioRxiv2024

Luminal breast epithelial cells from wildtype and BRCA mutation carriers harbor copy number alterations commonly associated with breast cancer

Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation and evolution is not well understood. Here, using single cell DNA sequencing, we describe the landscape of CNAs in >42,000 breast epithelial cells from women with normal or high risk of developing breast cancer. Accumulation of individual cells with one or two of a specific subset of CNAs (e.g. 1q gain and 16q, 22q, 7q, and 10q loss) is detectable in almost all breast tissues and, in those from BRCA1 or BRCA2 mutations carriers, occurs prior to loss of heterozygosity (LOH) of the wildtype alleles. These CNAs, which are among the most common associated with ductal carcinoma in situ (DCIS) and malignant breast tumors, are enriched almost exclusively in luminal cells not basal myoepithelial cells. Allele-specific analysis of the enriched CNAs reveals that each allele was independently altered, demonstrating convergent evolution of these CNAs in an individual breast. Tissues from BRCA1 or BRCA2 mutation carriers contain a small percentage of cells with extreme aneuploidy, featuring loss of TP53, LOH of BRCA1 or BRCA2, and multiple breast cancer-associated CNAs in addition to one or more of the common CNAs in 1q, 10q or 16q. Notably, cells with intermediate levels of CNAs are not detected, arguing against a stepwise gradual accumulation of CNAs. Overall, our findings demonstrate that chromosomal alterations in normal breast epithelium partially mirror those of established cancer genomes and are chromosome- and cell lineage-specific.

cancer biology↗

bioRxiv2020

Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans.

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome edited integrants, often circumventing the need for visual screening to identify genome edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome edited animals without the need for screening or scoring by eye. Author summaryThe ability to directly manipulate the genome and observe the resulting effects on the traits of an organism is a powerful approach to investigate gene function. CRISPR-based approaches to genome engineering have revolutionized such functional studies across model organisms but still face major challenges that limit the scope and complexity of projects that can be achieved in practice. Automating genome engineering and phenotyping would enable large-scale investigations of genome function in animals. Here, we describe the adaptation of peel-1 negative selection to an optimized dual-marker selection cassette CRISPR-Cas9 genome engineering method in C. elegans and combine it with automated machine vision phenotyping to achieve functional studies without the need for screening or scoring by eye. To demonstrate the applicability of the approach, we generated novel deletion alleles in two understudied genes, pbs-1 and K04F10.3, and used machine vision to characterize their phenotypic profiles, revealing homozygous lethal and heterozygous behavioral phenotypes. Our results open the door to systematic investigations of genome function in this model organism.

genomics↗

bioRxiv2019

Novel, non-nitrocatechol COMT inhibitors modulate dopamine neurotransmission in the frontal cortex and improve cognitive flexibility

Cognitive impairment is a primary feature of many neuropsychiatric disorders and there is a need for new therapeutic options. Catechol-O-methyltransferase (COMT) inhibitors modulate cortical dopaminergic function and have been proposed as potential cognitive enhancers. Unfortunately, currently available COMT inhibitors are not good candidates due to either poor blood-brain barrier penetration or severe toxicity. To address the need for safe, brain-penetrant COMT inhibitors, we tested multiple novel COMT inhibitors in a set of preclinical in vivo efficacy assays to determine their viability as potential clinical candidates. We found that multiple COMT inhibitors, exemplified by LIBD-1 and LIBD-3, significantly modulated dopaminergic function measured as decreases in homovanillic acid (HVA) and increases in 3,4-Dihydroxyphenylacetic acid (DOPAC), two dopamine metabolites, in cerebrospinal fluid (CSF) and the frontal cortex. Additionally, we found the LIBD-1 significantly improved cognitive flexibility in a rat attentional set-shifting assay (ASST), an effect previously seen with the COMT inhibitor tolcapone. These results demonstrate that LIBD-1 is a novel COMT inhibitor with promising in vivo activity and the potential to serve as a new therapy for cognitive impairment.

neuroscience↗

bioRxiv2018

CRISPR-Cas9 human gene replacement and phenomic characterization in Caenorhabditis elegans to understand the functional conservation of human genes and decipher variants of uncertain significance

Our ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole exome and whole genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision. We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene Phosphatase and Tensin Homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosenory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E) and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss-of-function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance.

molecular biology↗

bioRxiv2018

Optimizing guide RNA selection and CRISPR/Cas9 methodology for efficient generation of deletions in C. elegans.

The Caenorhabditis elegans Gene Knockout (KO) Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately15,000 ORFs have associated putative null alleles. A directed approach using CRISPR/Cas9 methodology is the most promising technique to complete the task. While there has been substantial success in using CRISPR/Cas9 in C. elegans, there has been little emphasis on optimizing the method for generating large insertions/deletions in this organism. To enhance the efficiency of using CRISPR/Cas9 to generate gene knockouts in C. elegans we have developed an online species-specific guide RNA selection tool (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust, efficient and effective protocol for generating deletions. Debate and speculation in the larger scientific community about off- target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude this modified protocol does not generate off-target events to any significant degree in C. elegans.

genetics↗

bioRxiv2017

MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature Sensitive Mutants via Molecular Inversion Probes

Temperature sensitive (TS) alleles are important tools for the genetic and functional analysis of essential genes in many model organisms. While isolating TS alleles is not difficult, determining the TS-conferring mutation can be problematic. Even with whole-genome sequencing (WGS) data there is a paucity of predictive methods for identifying TS alleles from DNA sequence alone. We assembled 173 TS lethal mutants of Caenorhabditis elegans and used WGS to identify several hundred mutations per strain. We leveraged single molecule molecular inversion probes (MIPs) to sequence variant sites at high depth in the cross-progeny of TS mutants and a mapping strain with identified sequence variants but no apparent phenotypic differences from the reference N2 strain. By sampling for variants at ~1Mb intervals across the genome we genetically mapped mutant alleles at a resolution comparable to current standards in a process we call MIP-MAP. The MIP-MAP protocol, however, permits high-throughput sequencing of multiple TS mutation mapping libraries at less than 200K reads per library. Using MIP-MAP on a subset of TS mutants, via a competitive selection assay and standard recombinant mutant selection, we defined TS-associated intervals of 3Mb or less. Our results suggest this collection of strains contains a diverse library of TS alleles for genes involved in development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes. The MIPs protocol should allow high-throughput tracking of genetic variants in any mixed population.

genomics↗