Biology subjects

Roberts, R. D.

Publications and source records attributed to Roberts, R. D..

11 recordsLinked to original sources

bioRxiv2026

Structure-Function Analysis of an Infantile Rhabdomyosarcoma Fusion-Oncogene Reveals Domain Requirements for Tumorigenesis

Rhabdomyosarcoma is a pediatric soft-tissue sarcoma that reflects impaired skeletal muscle differentiation. Spindle cell/sclerosing rhabdomyosarcoma is a histological subtype found in children and adults. In children under two, spindle cell rhabdomyosarcoma is genetically driven by fusion-oncogenes of transcription factors or co-activators important for skeletal muscle lineage commitment and development. The most common fusion-oncogene in this group is VGLL2::NCOA2, a fusion between vestigial-like family member 2 (VGLL2) and nuclear receptor coactivator 2 (NCOA2). Children with fusion-driven rhabdomyosarcoma are treated with general chemotherapy, surgery, and radiation, which are not specific to the molecular underpinnings, and increase risk of secondary malignancies and premature death. Previously, we showed that VGLL2::NCOA2 is transforming in mouse myoblasts and transgenic zebrafish tumor models. Here, we build on this work by detailing the structure-function relationship of the retained VGLL2::NCOA2 protein domains to define domain requirements and their molecular interactions for tumorigenesis. We created deletion mutants of the VGLL2 vestigial/Tondu (TDU) domain and the NCOA2 activating domains 1 and 2 (AD1 and AD2), and a double mutant lacking the TDU+AD1 domains and assessed their function in a mouse myoblast model. We find that the TDU and AD1 domains support nuclear fusion-oncogene localization, transformation in vitro, and tumorigenesis in a mouse allograft model. Furthermore, transcriptomic analysis demonstrates that the TDU or AD1 mutant VGLL2::NCOA2 drives an upregulation of myogenesis gene sets and supports terminal differentiation. Overall, our results indicate that VGLL2::NCOA2 requires the TDU and AD1 domains for optimal transformation, and that targeting these domains, or their interacting protein partners, represents a therapeutic opportunity.

cancer biology↗

bioRxiv2026

Spatially coordinated RTK-ERK signaling dynamics shape osteosarcoma single-cell drug response in the lung microenvironment

Purpose: Lung metastases are the primary cause of death in osteosarcoma patients and treatment response often vary due to convergence of genetic and phenotypic influences in the context of a dynamic tumor-host tissue environment. The purpose of this study was to determine how dynamic tumor- and host-driven receptor tyrosine kinase (RTK)-ERK signaling interactions contribute to phenotypic drug response heterogeneity. Experimental Design: Live-cell time-lapse microscopy and data-driven dynamical modeling was used to resolve ERK activity, FOSL1/Fra-1 reporter expression, and spatiotemporal cell fate in living, biosensor-expressing osteosarcoma lung metastases ex vivo following MCL1 inhibitor exposure. RTK inhibitors were then evaluated for their ability to suppress adaptive signaling and enhance MCL1 inhibitor efficacy ex vivo and in vivo. Results: MCL1 inhibition induced extensive tumor-cell death; however, persistent ERKhigh/Fra-1high reporter cells emerged and were enriched at the tumor-lung interface, consistent with adaptive resistance. Experimental and computational approaches revealed that tumor-cell lysis amplified fibroblast growth factor receptor (FGFR)-mediated ERK signaling in neighboring cells, promoting a targetable drug-resistant state marked by adaptive ERK induction, FOSL1/Fra-1 expression, and correlated with upregulation of known drug resistance pathways Nrf2 and HMOX1. Combinatorial inhibition of FGFR with fexagratinib, or broad-spectrum RTK inhibitor, pazopanib, suppressed adaptive ERK activation and enhanced MCL1 inhibitor response resulting in greater efficacy ex vivo and in vivo. Conclusions: Tumor-host and tumor-cell-lysis-induced FGFR-ERK signaling contribute to spatiotemporally heterogeneous adaptive resistance in metastatic osteosarcoma. These data support combinatorial targeting of RTK signaling with cytotoxic or targeted agents as a strategy to block adaptive resistance and improve treatment efficacy.

cancer biology↗

bioRxiv2026

SCANBIT facilitates identification of tumor cell populations in scRNAseq data using pseudobulked SNV calls

MotivationSingle cell RNAseq (scRNAseq) is an ideal tool to characterize the heterogeneity within the tumor microenvironment, however, accurate identification of tumor cells can be a challenge. Reference-based methods can be inaccurate, if reference datasets are even available. Current purpose-built methods can be inaccurate, particularly with highly heterogeneous tumor types. Improved methods are needed. We explored the use of genetic variants to distinguish tumor from normal cells within scRNAseq data. ResultsWe characterized the limitations inherent to calling variants from scRNAseq data, quantifying how data sparsity precludes genetic distance calculation between single cells. As a novel workaround, we pooled data from transcriptionally similar cell clusters to call high quality variants and then calculated pairwise differences between cell populations and performed hierarchical clustering. We quantified confidence in genetic divergence between tumor and normal cell populations using bootstrapping. We performed extensive validation to assess accurate identification of tumor cells using ground-truth datasets. Application of our method to human scRNAseq samples highlighted the utility of our approach and revealed how mutational burden influences successful tumor cell identification. Improved cell type assignment in scRNAseq data will facilitate analysis of tumor samples and, in turn, accelerate our understanding of the mechanisms underlying tumor progression and reveal potential biological vulnerabilities that can be exploited to develop improved treatment options. Availability and implementationOur method is publicly available as an R package: SCANBIT (Single Cell Altered Nucleotide Based Inference of Tumor) https://github.com/kidcancerlab/scanBit.

bioinformatics↗

bioRxiv2026

Integrative Single-cell and Spatial Transcriptomic Analysis of Osteosarcoma Reveals Conserved and Distinct Ecosystems Across Sites and Species

Osteosarcoma is a heterogeneous malignancy, exhibiting significant variability among patients, individual cancer cells within a tumor, and the stromal cells that compose primary and metastatic lesions. To facilitate the study of this complex disease, we compiled a unique cross-species single-cell transcriptomic dataset totaling over a million cells/nuclei from human specimens, canine specimens, patient-derived xenografts/PDX, and syngeneic mouse models at both primary (bone) and metastatic (lung) sites. Using a rigorous process for multi-species alignment and annotation, we identified six conserved tumor cell transcriptional states organized along hierarchical differentiation trajectories from progenitor to differentiated phenotypes. Parallel analysis of tumor-associated cells identified conserved macrophage, fibroblast, and endothelial populations that exhibit species- and site-specific reprogramming. Validation by mapping cell types using spatial transcriptomics revealed structured neighborhood architectures that were reproduced across multiple samples. Cell-cell interaction analysis revealed similarities and differences in tumor-host networks across primary and metastatic sites and across species. This analysis enabled pathway-specific assessment of tumor-host communication fidelity across osteosarcoma model systems relative to humans, revealing canine osteosarcoma as a more faithful model. Metastatic lung lesions, counterintuitively, exhibited more intense and complex extracellular matrix (ECM) signaling than primary bone tumors. A key example was tumor-derived fibronectin (FN1), which engages integrin and syndecan receptors on lung epithelial cells, driving a pathological mesenchymal and profibrotic state that promotes fibrotic niche formation and metastatic lung colonization. Together, this cross-species resource delineates both conserved and divergent tumor microenvironment programs, demonstrates how model-aware analyses uncover previously unrecognized tumor-host interactions, and underscores the need for therapies that co-target tumor heterogeneity and its supportive metastatic niche. Statement of SignificanceWe show that the tumor microenvironment in human osteosarcoma patients has biological phenotypes that are conserved across both patients and species. This points towards underlying molecular mechanisms that could be therapeutically targeted.

cancer biology↗

bioRxiv2025

NR0B1 alters the 9-cis-retinoic acid response in Ewing Sarcoma cells

Pediatric cancers are often characterized by relatively low DNA mutation rates and are frequently driven instead by alterations in gene expression programs. In Ewing Sarcoma (EwS), these changes result from the activity of an oncogenic fusion transcription factor, EWS::FLI1, which alters the enhancer landscape and rewires transcriptional programs to promote oncogenic transformation. Among the targets directly activated by EWS::FLI1, NR0B1 has emerged as a potentially targetable transcription co-regulator and as a manifold for widespread alterations in downstream gene expression programs mediated by members of the ligand-inducible nuclear receptor superfamily of transcription factors. We have dissected the gene regulatory activity of NR0B1 in EwS models, showing its role in altering the basal gene expression programs of nuclear receptors COUP-TFII, EAR2, RXRa, and TR4. Additionally, we show that NR0B1 impacts the EwS response to retinoids, particularly 9-cis-retinoic acid signaling in part through RXRa. Our findings suggest NR0B1 silencing or inhibition offer a means of achieving a more potent response to retinoids, which may activate differentiation programs, decrease EwS gene expression signatures, and limit in vitro transformation phenotypes. Taken together, our study presents evidence of NR0B1 acting canonically as a nuclear receptor co-regulator in EwS, revealing a potential pathway to treatment of this aggressive cancer by combining NR0B1 inhibition with therapeutic nuclear receptor ligands.

cancer biology↗

bioRxiv2024

Metastasis-initiating osteosarcoma subpopulations establish paracrine interactions with both lung and tumor cells to create a metastatic niche

Osteosarcoma is an aggressive and deadly bone tumor, primarily afflicting children, adolescents, and young adults. Poor outcomes for osteosarcoma patients are intricately linked with the development of lung metastasis. While lung metastasis is responsible for nearly all deaths caused by osteosarcoma, identification of biologically defined, metastasis-targeting therapies remains elusive because the underlying cellular and molecular mechanisms that govern metastatic colonization of circulating tumor cells to the lung remains poorly understood. While thousands of tumor cells are released into circulation each day, very few can colonize the lung. Herein, using a combination of a novel organotypic metastasis in vitro model, single-cell RNA sequencing, human xenograft, and murine immunocompetent osteosarcoma models, we find that metastasis is initiated by a subpopulation of hypo-proliferative cells with the unique capacity to sustain production of metastasis promoting cytokines such as IL6 and CXCL8 in response to lung-epithelial derived IL1. Critically, genomic and pharmacologic disruption of IL1 signaling in osteosarcoma cells significantly reduces metastatic progression. Collectively, our study supports that tumor-stromal interactions are important for metastasis, and suggests that metastatic competency is driven, in part, by the tumor cells ability to respond to the metastatic niche. Our findings support that disruption of tumor-stromal signaling is a promising therapeutic approach to disrupt metastasis progression.

cancer biology↗

bioRxiv2024

Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

cancer biology↗

bioRxiv2024

Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells

PurposeLung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. The purpose of this study is to identify metastasis-specific therapeutic vulnerabilities by delineating the cellular and molecular mechanisms underlying osteosarcoma lung metastatic niche formation. Experimental designUsing single-cell transcriptomics (scRNA-seq), we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multi-parameter immunofluorescence and spatial transcriptomics. Based on these findings, we evaluated the ability of nintedanib, a kinase inhibitor used to treat patients with pulmonary fibrosis, to impair metastasis progression in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Single-nucleus and spatial transcriptomics was used to perform molecular pharmacodynamic studies that define the effects of nintedanib on tumor and non-tumor cells within the metastatic microenvironment. ResultsOsteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially differentiated epithelial intermediates and macrophages. Our data demonstrated that nintedanib prevented metastatic progression in multiple murine and human xenograft models by inhibiting osteosarcoma-induced fibrosis. ConclusionsFibrosis represents a targetable vulnerability to block the progression of osteosarcoma lung metastasis. Our data support a model wherein interactions between osteosarcoma cells and epithelial cells create a pro-metastatic niche by inducing tumor deposition of extracellular matrix proteins such as fibronectin that is disrupted by the anti-fibrotic TKI nintedanib. Our data shed light on the non-cell autonomous effects of TKIs on metastasis and provide a roadmap for using single-cell and spatial transcriptomics to define the mechanism of action of TKI on metastases in animal models. Statement of translational relevanceTherapies that block metastasis have the potential to save the majority of lives lost due to solid tumors. Disseminated tumor cells must integrate into the foreign, inhospitable microenvironments they encounter within secondary organs to facilitate metastatic colonization and progression. Our study elucidated that disseminated osteosarcoma cells survive within the lung by co-opting and amplifying the lungs endogenous wound-healing response program. This osteosarcoma-induced wound response results in fibrosis of the surrounding microenvironment. Our data implicates fibrosis and abnormal wound healing as key drivers of osteosarcoma lung metastasis that can be targeted therapeutically to disrupt metastasis progression.

cancer biology↗

bioRxiv2021

Single-cell RNA Sequencing Reveals Immunosuppressive Myeloid Cell Diversity and Restricted Cytotoxic Effector Cell Trafficking and Activation During Malignant Progression in Glioma

Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a marker of tumor associated macrophages. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II gliomas. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

cancer biology↗

bioRxiv2021

Remarkably stable copy-number profiles in osteosarcoma revealed using single-cell DNA sequencing

Osteosarcoma is an aggressive malignancy characterized by high genomic complexity. Identification of few recurrent mutations in protein coding genes suggests that somatic copy-number aberrations (SCNAs) are the genetic drivers of disease. Models around genomic instability conflict - it is unclear if osteosarcomas result from pervasive ongoing clonal evolution with continuous optimization of the fitness landscape or an early catastrophic event followed by stable maintenance of an abnormal genome. We address this question by investigating SCNAs in >12,000 tumor cells obtained from human osteosarcomas using single cell DNA sequencing, with a degree of precision and accuracy not possible when inferring single cell states using bulk sequencing. Using the CHISEL algorithm, we inferred allele- and haplotype-specific SCNAs from this whole-genome single cell DNA sequencing data. Surprisingly, despite extensive structural complexity, these tumors exhibit a high degree of cell-cell homogeneity with little sub-clonal diversification. Longitudinal analysis of patient samples obtained at distant therapeutic time points (diagnosis, relapse) demonstrated remarkable conservation of SCNA profiles over tumor evolution. Phylogenetic analysis suggests that the majority of SCNAs were acquired early in the oncogenic process, with relatively few structure-altering events arising in response to therapy or during adaptation to growth in metastatic tissues. These data further support the emerging hypothesis that early catastrophic events, rather than sustained genomic instability, give rise to structural complexity, which is then preserved over long periods of tumor developmental time. Significance StatementChromosomally complex tumors are often described as genomically unstable. However, determining whether complexity arises from remote time-limited events that give rise to structural alterations or a progressive accumulation of structural events in persistently unstable tumors has implications for diagnosis, biomarker assessment, mechanisms of treatment resistance, and represents a conceptual advance in our understanding of intra-tumoral heterogeneity and tumor evolution.

cancer biology↗

bioRxiv2020

Osteosarcoma tumors maintain intertumoral heterogeneity, even while adapting to environmental pressures that drive clonal selection

BackgroundTumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms--it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. ResultsWe investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. ConclusionsResponse to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.

cancer biology↗