Biology subjects

Rusk, N.

Publications and source records attributed to Rusk, N..

12 recordsLinked to original sources

bioRxiv2026

Evolution of oncogene amplification across 86,000 cancer cell genomes

High-level copy-number (CN) amplification (HLAMP) is a major mechanism of oncogene activation in human cancer. Despite progress in therapeutically targeting amplified oncogenes, the processes underlying amplicon evolution remain incompletely understood, leaving critical knowledge gaps in their etiology and mechanisms of therapeutic response. To address this, we analyzed the evolutionary trajectories of HLAMPs using single-cell whole-genome sequencing data from 86,239 cancer cells across 93 patients and 9 experimental systems. We found that cell-to-cell CN variability provides a quantifiable readout of HLAMP mechanism, clearly distinguishing extrachromosomal circular DNA (ecDNA) from intrachromosomal amplification (ICamp) through characteristic CN distributions that reflect distinct modes of segregation and correspond to clonal architecture. Notably, ICamp events frequently showed multiple amplitude peaks specific to subclones, indicating punctuated shifts in oncogene dosage through numeric or structural modulatory mechanisms with transcriptional impact. In contrast, ecDNAs exhibited broad, continuous CN distribution with extreme high-copy outliers, consistent with asymmetric segregation. The CN and structural diversity of ecDNA regions enabled systematic deconvolution of ecDNA subspecies and estimation of their per-cell abundance, revealing the history of ecDNA-mediated oncogenesis at single-nucleotide resolution. We observed ecDNA diversification through internal rearrangements across cases and, notably, convergent evolution in glioblastoma cases marked by multiple, recurrent acquisition of EGFR-targeting ecDNAs. Finally, single-cell genome-based identification of ecDNAs showed substantial discrepancy with bulk genome graph-based predictions and reliably distinguished actively maintained ecDNAs from historical genomic footprints after chromosomal re-integration. These findings reveal marked tissue-type specificity of ecDNAs, suggesting that ecDNA-mediated oncogenesis may depend on a permissive tissue context.

genomics↗

bioRxiv2025

Complementary modes of resistance to EGFR TKI in lung adenocarcinoma through MAPK activation and cellular plasticity

EGFR-mutant lung adenocarcinoma (LUAD) represents 20% of all non-small cell lung carcinomas, with most patients presenting with incurable metastatic disease. Treatment with mutant-selective EGFR tyrosine kinase inhibitors (TKIs) results in initial tumor reduction, yet nearly all patients eventually relapse. The mechanisms driving drug resistance are incompletely understood, creating significant barriers to curing metastatic disease. We integrated clinical genomic and single-nuclei RNA (snRNA) sequencing from a cohort of 62 EGFR-mutant LUAD patients treated with the third generation EGFR TKI, osimertinib, and compared treatment-naive (TN), minimal residual disease (MRD), and progressive disease (PD) tumors. We found that disease progression is associated with a marked decrease in alveolar lineage fidelity, coincident with reduced MAPK signaling and adenocarcinoma identity. PD tumors with sustained MAPK pathway activity, such as those with EGFR or MET amplifications, tended to retain adenocarcinoma identity. In contrast, MAPK-low tumors were more likely to undergo histological transformation to squamous or neuroendocrine lineages. Remarkably, we observed rare tumor cell populations prior to treatment that were poorly differentiated, in some cases with neuroendocrine or squamous features. At progression, these histologically divergent tumor cells increased in prevalence, both in cases with overt histological transformation, and in others with sub-clinical histological plasticity. These findings suggest that pre-existing capacity for histologic plasticity may be a substrate for therapy induced selection. Taken together, our results illuminate genomically encoded MAPK signaling and lineage plasticity as complementary mechanisms of acquired resistance to EGFR TKI in lung adenocarcinoma.

cancer biology↗

bioRxiv2025

Disrupted priming within draining lymph nodes drives immune quiescence in gastric cancer

The gastric mucosa is characterized by continuous innate immune surveillance and inflammatory signaling, yet a high proportion of gastric carcinomas (GCs) are recalcitrant to immune-directed therapies. The mechanisms by which GCs evade adaptive immune surveillance within the highly antigenic microenvironment of the gastric mucosa remains unknown. To address this, we collected patient-matched tumor tissue, distant normal tissue, metastasis, and draining lymph nodes to generate a large-scale single-cell immune profiling dataset from 64 patients (n=179 samples, >150,000 cells). From single cell analysis, we identified two distinct sources of impaired tumor surveillance within tumor draining lymph nodes. First, we observed that a significant fraction of tumor draining lymph nodes had undergone cytokine-driven reprogramming, leading to reduced dendritic cell homing and limited T cell priming. Second, T cells undergoing successful activation exhibited limited expansion and constrained differentiation, marked by expression of the quiescence-associated transcription factor Kruppel-like Factor 2 (KLF2). Overexpression of KLF2 in primary T cells limited both their differentiation and cytotoxic capacity. These findings implicate both impaired T cell priming and KLF2-dependent T cell quiescence in limiting T cell immunity in gastric adenocarcinoma. We suggest these findings represent an emerging model for immune silencing in tumors developing from tissues with chronic inflammation.

immunology↗

bioRxiv2024

Tracking clonal evolution of drug resistance in ovarian cancer patients by exploiting structural variants in cfDNA

Drug resistance is the major cause of therapeutic failure in high-grade serous ovarian cancer (HGSOC). Yet, the mechanisms by which tumors evolve to drug resistant states remains largely unknown. To address this, we aimed to exploit clone-specific genomic structural variations by combining scaled single-cell whole genome sequencing with longitudinally collected cell-free DNA (cfDNA), enabling clonal tracking before, during and after treatment. We developed a cfDNA hybrid capture, deep sequencing approach based on leveraging clone-specific structural variants as endogenous barcodes, with orders of magnitude lower error rates than single nucleotide variants in ctDNA (circulating tumor DNA) detection, demonstrated on 19 patients at baseline. We then applied this to monitor and model clonal evolution over several years in ten HGSOC patients treated with systemic therapy from diagnosis through recurrence. We found drug resistance to be polyclonal in most cases, but frequently dominated by a single high-fitness and expanding clone, reducing clonal diversity in the relapsed disease state in most patients. Drug-resistant clones frequently displayed notable genomic features, including high-level amplifications of oncogenes such as CCNE1, RAB25, NOTCH3, and ERBB2. Using a population genetics Wright-Fisher model, we found evolutionary trajectories of these features were consistent with drug-induced positive selection. In select cases, these alterations impacted selection of secondary lines of therapy with positive patient outcomes. For cases with matched single-cell RNA sequencing data, pre-existing and genomically encoded phenotypic states such as upregulation of EMT and VEGF were linked to drug resistance. Together, our findings indicate that drug resistant states in HGSOC pre-exist at diagnosis and lead to dramatic clonal expansions that alter clonal composition at the time of relapse. We suggest that combining tumor single cell sequencing with cfDNA enables clonal tracking in patients and harbors potential for evolution-informed adaptive treatment decisions.

cancer biology↗

bioRxiv2024

Ongoing genome doubling promotes evolvability and immune dysregulation in ovarian cancer

Whole-genome doubling (WGD) is a critical driver of tumor development and is linked to drug resistance and metastasis in solid malignancies. Here, we demonstrate that WGD is an ongoing mutational process in tumor evolution. Using single-cell whole-genome sequencing, we measured and modeled how WGD events are distributed across cellular populations within tumors and associated WGD dynamics with properties of genome diversification and phenotypic consequences of innate immunity. We studied WGD evolution in 65 high-grade serous ovarian cancer (HGSOC) tissue samples from 40 patients, yielding 29,481 tumor cell genomes. We found near-ubiquitous evidence of WGD as an ongoing mutational process promoting cell-cell diversity, high rates of chromosomal missegregation, and consequent micronucleation. Using a novel mutation-based WGD timing method, doubleTime, we delineated specific modes by which WGD can drive tumor evolution: (i) unitary evolutionary origin followed by significant diversification, (ii) independent WGD events on a pre-existing background of copy number diversity, and (iii) evolutionarily late clonal expansions of WGD populations. Additionally, through integrated single-cell RNA sequencing and high-resolution immunofluorescence microscopy, we found that inflammatory signaling and cGAS-STING pathway activation result from ongoing chromosomal instability and are restricted to tumors that remain predominantly diploid. This contrasted with predominantly WGD tumors, which exhibited significant quiescent and immunosuppressive phenotypic states. Together, these findings establish WGD as an evolutionarily active mutational process that promotes evolvability and dysregulated immunity in late stage ovarian cancer.

cancer biology↗

bioRxiv2022

TCRi: Information theoretic metrics for single cell RNA and TCR sequencing in cancer

Single-cell T cell repertoire sequencing can pair both T cell receptor (TCR) and gene expression sequence data, providing an enriched view of T cell behavior. This powerful tool can identify and characterize specific clonotypes and phenotypes as well as track their changes in response to therapy, such as immune checkpoint blockade (ICB). We present a novel information theoretic framework called TCRi for characterizing single cell T cell repertoires by formalizing the relationship between clonotype and phenotype in a joint probability distribution. Our strategy allows for the identification of subpopulations of T cells and jointly quantifies their TCR and expression profiles in response to stimuli, in addition the framework tracks the phenotypic changes in individual T cell clones over time. We applied this framework to four datasets of T cells sequenced from cancer patients treated with anti-PD-(L)1 ICB immunotherapies and examined evolution of T cell responses pre- and post-treatment. Quantitative of phenotypic and clonotypic entropy analysis with TCRi demonstrated improvements in characterization of the transcriptional signature of clonotypes. Furthermore, TCRi highlighted the importance of phenotypic flux and specific T-cell phenotypes as determinants of therapeutic response.

bioinformatics↗

bioRxiv2022

GeneVector: Identification of transcriptional programs using dense vector representations defined by mutual information.

Deciphering individual cell phenotypes from cell-specific transcriptional processes requires high dimensional single cell RNA sequencing. However, current dimensionality reduction methods aggregate sparse gene information across cells, without directly measuring the relationships that exist between genes. By performing dimensionality reduction with respect to gene co-expression, low-dimensional features can model these gene-specific relationships and leverage shared signal to overcome sparsity. We describe GeneVector, a scalable framework for dimensionality reduction implemented as a vector space model using mutual information between gene expression. Unlike other methods, including principal component analysis and variational autoencoders, GeneVector uses latent space arithmetic in a lower dimensional gene embedding to identify transcriptional programs and classify cell types. In this work, we show in four single cell RNA-seq datasets that GeneVector was able to capture phenotypespecific pathways, perform batch effect correction, interactively annotate cell types, and identify pathway variation with treatment over time.

bioinformatics↗

bioRxiv2021

Immune and malignant cell phenotypes of ovarian cancer are determined by distinct mutational processes

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability patterned by distinct mutational processes, intratumoral heterogeneity and intraperitoneal spread. We investigated determinants of immune recognition and evasion in HGSOC to elucidate co- evolutionary processes underlying malignant progression and tumor immunity. Mutational processes and anatomic sites of tumor foci were key determinants of tumor microenvironment cellular phenotypes, inferred from whole genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumor sites from 42 treatment-naive HGSOC patients. Homologous recombination-deficient (HRD)-Dup (BRCA1 mutant-like) and HRD- Del (BRCA2 mutant-like) tumors harbored increased neoantigen burden, inflammatory signaling and ongoing immunoediting, reflected in loss of HLA diversity and tumor infiltration with highly- differentiated dysfunctional CD8+ T cells. Foldback inversion (FBI, non-HRD) tumors exhibited elevated TGF{beta} signaling and immune exclusion, with predominantly naive/stem-like and memory T cells. Our findings implicate distinct immune resistance mechanisms across HGSOC subtypes which can inform future immunotherapeutic strategies. HIGHLIGHTSO_LIMulti-region, multi-modal profiling of malignant and immune cell phenotypes in ovarian cancer C_LIO_LIAnatomic site specificity is a determinant of cancer cell and intratumoral immune phenotypes C_LIO_LITumor mutational processes impact mechanisms of immune control and immune evasion C_LIO_LISpatial topology of HR-deficient tumors is defined by immune interactions absent from immune inert HR-proficient subtypes C_LI

cancer biology↗

bioRxiv2021

Resource-efficient pooled sequencing expands translational impact in solid tumors

Intratumoral genetic heterogeneity (ITH) poses a significant challenge to utilizing sequencing for decision making in the management of cancer. Although sequencing of multiple tumor regions can address the pitfalls of ITH, it does so at a significant increase in cost and resource utilization. We propose a pooled multiregional sequencing strategy, whereby DNA aliquots from multiple tumor regions are mixed prior to sequencing, as a cost-effective strategy to boost translational value by addressing ITH while preserving valuable residual tissue for secondary analysis. Focusing on kidney cancer, we demonstrate that DNA pooling from as few as two regions significantly increases mutation detection while reducing clonality misattribution. This leads to an increased fraction of patients identified with therapeutically actionable mutations, improved patient risk stratification, and improved inference of evolutionary trajectories with an accuracy comparable to bona fide multiregional sequencing. The same approach applied to non-small-cell lung cancer data substantially improves tumor mutational burden (TMB) detection. Our findings demonstrate that pooled DNA sequencing strategies are a cost-effective alternative to address intrinsic genetic heterogeneity in clinical settings.

genomics↗

bioRxiv2021

Evolutionary tracking of cancer haplotypes at single-cell resolution

Cancer genomes exhibit extensive chromosomal copy number changes and structural variation, yet how allele specific alterations drive cancer genome evolution remains unclear. Here, through application of a new computational approach we report allele specific copy number alterations in 11,097 single cell whole genomes from genetically engineered mammary epithelial cells and 21,852 cells from high grade serous ovarian and triple negative breast cancers. Resolving single cell copy number profiles to individual alleles uncovered genomic background distributions of gains, losses and loss of heterozygosity, yielding evidence of positive selection of specific chromosomal alterations. In addition specific genomic loci in maternal and paternal alleles were commonly found to be altered in parallel with convergent phenotypic transcriptional effects. Finally we show that haplotype specific alterations trace the cyclical etiology of high level amplifications and reveal clonal haplotype decomposition of complex structures. Together, our results illuminate how allele and haplotype specific alterations, here determined across thousands of single cell cancer genomes, impact the etiology and evolution of structural variations in human tumours.

cancer biology↗

bioRxiv2021

The impact of mutational processes on structural genomic plasticity in cancer cells

Structural genome alterations are determinants of cancer ontogeny and therapeutic response. While bulk genome sequencing has enabled delineation of structural variation (SV) mutational processes which generate patterns of DNA damage, we have little understanding of how these processes lead to cell-to-cell variations which underlie selection and rates of accrual of different genomic lesions. We analysed 309 high grade serous ovarian and triple negative breast cancer genomes to determine their mutational processes, selecting 22 from which we sequenced >22,000 single cell whole genomes across a spectrum of mutational processes. We show that distinct patterns of cell-to-cell variation in aneuploidy, copy number alteration (CNA) and segment length occur in homologous recombination deficiency (HRD) and fold-back inversion (FBI) phenotypes. Widespread aneuploidy through induction of HRD through BRCA1 and BRCA2 inactivation was mirrored by continuous whole genome duplication in HRD tumours, contrasted with early ploidy fixation in FBI. FBI tumours exhibited copy number distributions skewed towards gains, widespread clone-specific variation in amplitude of high-level amplifications, often impacting oncogenes, and break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). SSVs were consistent with a CNA-based molecular clock reflecting a continual and distributed process across clones within tumours. These observations reveal previously obscured genome plasticity and evolutionary properties with implications for cancer evolution, therapeutic targeting and response.

cancer biology↗

bioRxiv2020

Single cell fitness landscapes induced by genetic and pharmacologic perturbations in cancer

Tumour fitness landscapes underpin selection in cancer, impacting etiology, evolution and response to treatment. Progress in defining fitness landscapes has been impeded by a lack of timeseries perturbation experiments over realistic intervals at single cell resolution. We studied the nature of clonal dynamics induced by genetic and pharmacologic perturbation with a quantitative fitness model developed to ascribe quantitative selective coefficients to individual cancer clones, enable prediction of clone-specific growth potential, and forecast competitive clonal dynamics over time. We applied the model to serial single cell genome (>60,000 cells) and transcriptome (>58,000 cells) experiments ranging from 10 months to 2.5 years in duration. We found that genetic perturbation of TP53 in epithelial cell lines induces multiple forms of copy number alteration that confer increased fitness to clonal populations with measurable consequences on gene expression. In patient derived xenografts, predicted selective coefficients accurately forecasted clonal competition dynamics, that were validated with timeseries sampling of experimentally engineered mixtures of low and high fitness clones. In cisplatin-treated patient derived xenografts, the fitness landscape was inverted in a time-dependent manner, whereby a drug resistant clone emerged from a phylogenetic lineage of low fitness clones, and high fitness clones were eradicated. Moreover, clonal selection mediated reversible drug response early in the selection process, whereas late dynamics in genomically fixed clones were associated with transcriptional plasticity on a fixed clonal genotype. Together, our findings outline causal mechanisms with implication for interpreting how mutations and multi-faceted drug resistance mechanisms shape the etiology and cellular fitness of human cancers.

cancer biology↗