Biology subjects

Kendall, G. C.

Publications and source records attributed to Kendall, G. C..

12 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

HES3-dependent regulatory functions in development and fusion-positive rhabdomyosarcoma

HES3/Her3 is a transcription factor that functions in non-canonical STAT3 signaling to promote the renewal of neural stem cells and has roles in multiple cancer contexts. To study its role in development and disease, we previously generated a CRISPR/Cas9 zebrafish knockout of her3, the ortholog to human HES3. HES3 is also a cooperating gene in fusion-positive rhabdomyosarcoma, an aggressive pediatric cancer, where HES3 prevents terminal myogenic differentiation, and high expression correlates with worse patient outcomes. Here, we utilize our her3/HES3 knockout model with chromatin and transcriptional profiling techniques to assess its role during early zebrafish gastrulation with the goal of understanding the function of this transcription factor and how these activities are co-opted in cancer. We found that the Her3/HES3 preferential binding motif is distinct from other HES-family members, including a CG-rich E-box motif, that it leverages to modulate the expression of genes involved in neurogenesis and WNT signaling. We also determined that motif preferences of Her3/HES3 altered its interactions with DNA, allowing it to function canonically as a transcriptional repressor with additional duality as an activator. In the context of PAX3::FOXO1, a monogenic driver of fusion-positive rhabdomyosarcoma, we find that Her3/HES3 plays an influential role in modulating the initial activities of this core oncogenic transcription factor. Upon expressing PAX3::FOXO1 in early developing zebrafish embryos, her3 knockout allowed for enhanced activation of neural programs, which are observed in the human disease, along with alterations to cell adhesion programs. Patient tumor samples could be clustered and stratified based on HES3 expression alone. We saw that patient PAX3::FOXO1-positive tumors with high levels of HES3 contained a more neural identity than those with low levels of HES3, altogether suggesting HES3 plays a critical role in regulating this neural signature during both the initial functions of PAX3::FOXO1 and in established tumors.

cancer biology↗

bioRxiv2026

Comparative modes of chromatin engagement by PAX::FOXO1 fusions in rhabdomyosarcoma

Fusion positive rhabdomyosarcoma (FP-RMS) is an aggressive soft-tissue sarcoma that most frequently affects children and adolescents. Treatment options and outcomes for children with this cancer remain poor, non-specific, and broadly toxic despite decades of research. The defining molecular drivers of the more aggressive fusion-positive subtype of the disease arise from chromosomal translocations that fuse PAX3 or PAX7 to FOXO1 to form PAX3::FOXO1 or PAX7::FOXO1, encoding fusion oncoprotein transcription factors. Despite their high degree of similarity, PAX3::FOXO1 correlates with worse patient overall survival than PAX7::FOXO1. Previous work from our groups and others has revealed evidence focused in chromatin accessibility contexts that PAX3::FOXO1 has key characteristics of a pioneer transcription factor, a specialized subclass of transcription factors that can bind nucleosomal DNA prior to generation of local accessibility. However, evidence at the genome scale for PAX3/7::FOXO1 direct nucleosome targeting, prior to the accessibility step in pioneering, has remained elusive and challenging to capture methodologically for RMS fusion oncoproteins. In this work, we compare the cellular functions of these PAX::FOXO1 fusions, including new approaches for identifying nucleosome targeting at the genome scale. We find that in zebrafish RMS initiation models, the fusions initially activate similar neural transcriptional programs but to different extents, and we further evaluate their mechanisms in RMS cells at the genome scale with modified MNase XChIP to detect nucleosome and subnucleosome fusion/chromatin binding. In establishing our cross-species comparative oncology approach, we report, to our knowledge, the first high resolution nucleosome positioning data in rhabdomyosarcoma. We find that both PAX::FOXO1 fusions bind nucleosomal DNA, but with varied motif preferences and histone mark co-localization patterns. Altogether, we establish the nucleosome targeting functions of PAX7::FOXO1 and PAX3::FOXO1 pioneering and uncover key mechanistic distinctions for chromatin engagement of the two most common RMS fusion oncoproteins. HIGHLIGHTSO_LIPartially overlapping gene signatures are activated by PAX3/7::FOXO1 in vivo C_LIO_LIModified MNase ChIP reveals PAX3/7::FOXO1 bind nucleosomal and subnucleosomal DNA C_LIO_LIPAX7::FOXO1 binds degenerate paired/homeobox motifs within nucleosome targets C_LIO_LIEach fusion engages distinct nucleosomal gene targets C_LI

cancer biology↗

bioRxiv2024

RNA-SeqEZPZ: A Point-and-Click Pipeline for Comprehensive Transcriptomics Analysis with Interactive Visualizations

RNA-Seq analysis has become a routine task in numerous genomic research labs, driven by the reduced cost of bulk RNA sequencing experiments. These generate billions of reads that require accurate, efficient, effective, and reproducible analysis. But the time required for comprehensive analysis remains a bottleneck. Many labs rely on in-house scripts, making standardization and reproducibility challenging. To address this, we developed RNA-SeqEZPZ, an automated pipeline with a user-friendly point-and-click interface, enabling rigorous and reproducible RNA-Seq analysis without requiring programming or bioinformatics expertise. For advanced users, the pipeline can also be executed from the command line, allowing customization of steps to suit specific requirements. This pipeline includes multiple steps from quality control, alignment, filtering, read counting to differential expression and pathway analysis. We offer two different implementations of the pipeline using either (1) bash and SLURM or (2) Nextflow. The two implementation options allow for straightforward installation, making it easy for individuals familiar with either language to modify and/or run the pipeline across various computing environments. RNA-SeqEZPZ provides an interactive visualization tool using R shiny to easily select the FASTQ files for analysis and compare differentially expressed genes and their functions across experimental conditions. The tools required by the pipeline are packaged into a Singularity image for ease of installation and to ensure replicability. Finally, the pipeline performs a thorough statistical analysis and provides an option to perform batch adjustment to minimize effects of noise due to technical variations across replicates. RNA-SeqEZPZ is freely available and can be downloaded from https://github.com/cxtaslim/RNA-SeqEZPZ.

genomics↗

bioRxiv2024

Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, functions as a chimeric gain-of-function transcription factor. An incomplete understanding of PAX3::FOXO1s in vivo epigenetic mechanisms has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. We investigated PAX3::FOXO1s recognition of chromatin and subsequent transcriptional consequences. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired-box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a new mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.

cancer biology↗

bioRxiv2024

Highly quantitative measurement of differential protein-genome binding with PerCell chromatin sequencing

We report a universal strategy for 2D chromatin sequencing, to increase uniform data analyses and sharing across labs, and to facilitate highly quantitative comparisons across experimental conditions. Within our system, we provide wetlab and drylab tools for researchers to establish and analyze protein-genome binding data with PerCell ChIP-seq. Our methodology is virtually no cost and flexible, enabling rapid, quantitative, internally normalized chromatin sequencing to catalyze project development in a variety of systems, including in vivo zebrafish epigenomics and cancer cell epigenomics. While we highlight utility in these key areas, our methodology is flexible enough such that rapid comparisons of cellular spike-in versus non spike-in are possible, and generalizability to nuclease-based 2D chromatin sequencing would also be possible within the framework of our pipeline. Through the use of well-defined cellular ratios containing orthologous species chromatin, we enable cross-species comparative epigenomics and highly quantitative low-cost chromatin sequencing with utility across a range of disciplines.

genomics↗

bioRxiv2024

Engineering an fgfr4 knockout zebrafish to study its role in development and disease

Fibroblast growth factor receptor 4 (FGFR4) has a role in many biological processes, including lipid metabolism, tissue repair, and vertebrate development. In recent years, FGFR4 overexpression and activating mutations have been associated with numerous adult and pediatric cancers. As such, FGFR4 presents an opportunity for therapeutic targeting which is being pursued in clinical trials. To understand the role of FGFR4 signaling in disease and development, we generated and characterized three alleles of fgfr4 knockout zebrafish strains using CRISPR/Cas9. To generate fgfr4 knockout crispants, we injected single-cell wildtype zebrafish embryos with fgfr4 targeting guide RNA and Cas9 proteins, identified adult founders, and outcrossed to wildtype zebrafish to create an F1 generation. The generated mutations introduce a stop codon within the second Ig-like domain of Fgfr4, resulting in a truncated 215, 223, or 228 amino acid Fgfr4 protein compared to 922 amino acids in the full-length protein. All mutant strains exhibited significantly decreased fgfr4 mRNA expression during development, providing evidence for successful knockout of fgfr4 in mutant zebrafish. We found that, consistent with other Fgfr4 knockout animal models, the fgfr4 mutant fish developed normally; however, homozygous fgfr4 mutant zebrafish were significantly smaller than wildtype fish at three months post fertilization. These fgfr4 knockout zebrafish lines are a valuable tool to study the role of FGFR4 in vertebrate development and its viability as a potential therapeutic target in pediatric and adult cancers, as well as other diseases.

developmental biology↗

bioRxiv2024

New Dual Inducible Cellular Model to Investigate Temporal Control of Oncogenic Cooperating Genes

The study of cooperating genes in cancer can lead to mechanistic understanding and identifying potential therapeutic targets. To facilitate these types of studies, we developed a new dual-inducible system utilizing the tetracycline- and cumate-inducible systems driving HES3 and the PAX3::FOXO1 fusion-oncogene, respectively, as cooperating genes from fusion-positive rhabdomyosarcoma. With this new model, we can independently induce expression of either HES3 or PAX3::FOXO1, as well as simultaneously induce expression of both genes. This new model will allow us to further investigate the cooperation between HES3 and PAX3::FOXO1 including the temporal requirements for genetic cooperation. This dual-inducible model can be adapted for any cooperating genes, allowing for independent, simultaneous, or temporally controlled gene expression.

cancer biology↗

bioRxiv2023

A robust closed-tube method for resolving tp53M214K genotypes

The tp53M214K zebrafish mutant developed by Berghmans et al 20051 is a versatile platform with which to model a diverse spectrum of human diseases. However, currently available genotyping methods for this mutant require lengthy processes such as restriction digests and outsourced Sanger sequencing. To address this deficiency, we leveraged high-resolution melting analysis (HRMA) technology in conjunction with a parallel, in-tandem wildtype spike-in approach to develop a robust genotyping protocol capable of discriminating tp53M214K zygosity. Here, we describe our method in detail. We anticipate that our genotyping protocol will benefit researchers utilizing the tp53M214K zebrafish mutant by offering reliable results with a faster turnaround time than conventional approaches.

cancer biology↗

bioRxiv2022

Zebrafish her3 knockout impacts developmental and cancer-related gene signatures

HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3/HES3s role in neurogenesis. To investigate HES3s function during development, we generated a zebrafish CRISPR/Cas9 knockout of her3, the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 knockout embryos are enriched for HOX and SOX10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3-mediated neural development and its misappropriation in cancer contexts. Summary StatementHere, we generate and characterize a zebrafish her3/HES3 knockout to elucidate the functional role of her3/HES3, a transcriptional repressor, in neural development and tumorigenic processes.

developmental biology↗

bioRxiv2021

VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

Clinical sequencing efforts are uncovering numerous fusion genes in childhood solid tumors, yet few methods exist to delineate fusion-oncogenes from structural changes of unknown significance. One such novel fusion gene is VGLL2-NCOA2, which was described by us and others in patients with congenital sarcomas but has lacked functional validation. To determine if this fusion is an oncogene, and how it is driving disease, we developed a vertebrate zebrafish model and mouse allograft model of human VGLL2-NCOA2 driven sarcomagenesis. We found that VGLL2-NCOA2 is indeed an oncogene and is sufficient to generate mesenchymal tumors that recapitulate the human disease at the histological and transcriptional level. Zebrafish VGLL2-NCOA2 tumors display features of arrested skeletal muscle development, and a subset transcriptionally cluster with somitogenesis in developing embryos. By comparing tumor and embryonic gene expression signatures, we identified developmentally regulated targets that VGLL2-NCOA2 potentially leverages for tumorigenesis. These targets highlight the core biology of the disease and could represent therapeutic opportunities. Specifically, a RAS family GTPase, arf6/ARF6, involved in actin remodeling and rapid cycling of endocytic vesicles at the plasma membrane, is highly expressed during zebrafish somitogenesis and in VGLL2-NCOA2 tumors. In zebrafish tumors, arf6 protein is highly expressed and is absent from mature skeletal muscle. In VGLL2-NCOA2 mouse allograft models and patient tumors, ARF6 mRNA is overexpressed as compared to skeletal muscle or normal controls. More broadly, ARF6 is overexpressed in adult and pediatric sarcoma subtypes as compared to mature skeletal muscle. Overall, our cross-species comparative oncology approach provides evidence that VGLL2-NCOA2 is an oncogene which leverages developmental programs for tumorigenesis, and that one of these programs, the reactivation or persistence of arf6/ARF6, could represent a therapeutic opportunity.

cancer biology↗

bioRxiv2019

CIC-DUX4 expression drives the development of small round cell sarcoma in transgenic zebrafish: a new model revealing a role for ETV4 in CIC-mediated sarcomagenesis

CIC-DUX4 sarcoma is a rare subtype of sarcoma characterized by a devastating prognosis and resistance to conventional therapeutic strategies. So far, only few models of the disease have been reported, and its biological mechanisms remain to be elucidated. We established mosaic transgenic zebrafish expressing the human CIC-DUX4 fusion under the control of the {beta}-actin promoter. CIC-DUX4 transgenic fish rapidly developed aggressive soft tissue tumors with a high penetrance. RNAseq profiling revealed that fish tumors shared major common targets with human tumors and cell lines, including the overexpression of the Pea3 transcription factors, etv4 and etv5. Tumor development was strongly impaired in etv4-deficient zebrafish, implicating Etv4 as a critical effector of CIC-DUX4-mediated oncogenesis. Altogether, we report here the first in vivo model of CIC-DUX4 sarcoma in zebrafish, which will represent a major tool for future preclinical research.

cancer biology↗