Biology subjects

Theisen, E. R.

Publications and source records attributed to Theisen, E. R..

11 recordsLinked to original sources

bioRxiv2026

Fusion Oncoprotein EWSR1::FLI1 Invades Nucleosomes at Consensus ETS Motifs and GGAA Microsatellites

Ewing sarcoma is an aggressive bone malignancy occurring in children, adolescents, and young adults. Most cases are caused by expression of the fusion oncoprotein EWSR1::FLI1, which contains the low complexity domain (LCD) of EWSR1 and the DNA-binding domain (DBD) of FLI1. Previous genomic studies indicate EWSR1::FLI1 accesses GGAA microsatellites in chromatin to function as a potent transcriptional regulator. Due to the technical challenges of purifying full-length EWSR1::FLI1, mechanistic studies biochemically characterizing its pioneer activities have been lacking. Here, we purified both full-length EWSR1::FLI1 and truncated DBD constructs to conduct biochemical and fluorescence-based experiments investigating interactions with different motifs in free DNA and nucleosomes. Both truncated and full-length EWSR1::FLI1 show efficient target binding in nucleosomes, and that the fourth alpha-helix in the FLI1 DBD enhances nucleosome-binding efficiency. Surprisingly, we also observe differences in both free DNA binding affinity and sequence preference between truncated and full-length proteins, though these changes are not apparent in nucleosome-binding assays. These findings reveal that EWSR1::FLI1 possesses a key pioneer factor property, efficiently targeting its binding site within nucleosomes, and that full-length EWSR1::FLI1 binding shifts to preferentially target GGAA repeats, even on motifs that bind a single EWSR1::FLI1 protein.

biochemistry↗

bioRxiv2025

LSD1 Performs Demethylase-Independent and Context-Specific Roles in Ewing Sarcoma

Lysine specific demethylase 1 (LSD1), encoded by the gene KDM1A, is overexpressed and correlates with poor patient prognosis in Ewing sarcoma. LSD1 and the pathognomonic fusion oncoprotein, EWSR1::FLI1, colocalize throughout the genome, suggesting LSD1 is a critical co-regulator driving the progression of Ewing sarcoma. However, therapeutic targeting of LSD1 by competitive and noncompetitive inhibitors has yielded mixed results. Irreversible, enzymatic inhibition seems ineffective, but reversible noncompetitive inhibition has predominant off target mechanisms, leaving open the question of LSD1 function in Ewing sarcoma. Here we take a robust approach through multiple methods of depletion in multiple EwS cell lines to define enzymatic and nonenzymatic contributions of LSD1 to transcriptional regulation. We define a core set of 22 genes that are commonly repressed by LSD1 in all cell lines, and that repression of these genes downregulates synapse functioning and e-cadherin target genes. Derepression of these genes with LSD1 loss is an early and sustained genotype in all cell lines tested. We further define distinct gene sets in each cell line that are regulated by enzymatic and nonenzymatic LSD1 activity and find repression of e-cadherin target genes to be nonenzymatically regulated. This finding supports the growing body of evidence that in addition to their canonical catalytic activity, chromatin regulatory enzymes serve essential noncanonical roles as well. Furthermore, we uncovered evidence through use of the irreversible inhibitor OG-L002 that 2D cytotoxicity and proliferation assays may be insufficient to determine Ewing sarcoma response to LSD1 inhibition. SIGNIFICANCEHere we address a long-standing question in the field surrounding LSD1 and define the distinct enzymatic and nonenzymatic functions of LSD1 in Ewing sarcoma. In doing so, we have created a robust data set using genetic and pharmacological techniques in multiple models to thoroughly characterize LSD1 function in Ewing sarcoma cell lines.

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↗

bioRxiv2025

N'-(1-phenylethylidene)-benzohydrazide cytotoxicity is LSD1 independent and linked to Fe-S cluster disruption in Ewing sarcoma

The noncompetitive LSD1 inhibitors SP-2509 and SP-2577 are N-(1-phenylethylidene)-benzohydrazides that display potent activity in Ewing sarcoma. They block transcriptional regulation of the causative oncogenic fusion protein, EWSR1::FLI1, and cause cell death. However, SP-2509 and SP-2577 are the only LSD1 inhibitors active in Ewing sarcoma; other LSD1 inhibitors have little effect. Studies from our group and others suggest SP-2509 activity may result from off-target activity affecting the mitochondria. Here we identified potential off-target mechanisms of N-(1-phenylethylidene)-benzohydrazides using an unbiased approach, cellular thermal shift assay coupled to mass spectrometry (CETSA-MS). Interestingly, this revealed significant destabilization of the electron transport chain complex III protein ubiquinol-cytochrome c reductase (UQCRFS1). We find that UQCRFS1 destabilization is likely linked to impaired iron-sulfur (Fe-S) cofactor binding, and that SP-2509 broadly destabilizes cellular Fe-S proteins. Using both chemical and genetic tools, we show that SP-2509 mediated cell death is LSD1 independent and instead requires a N-(2-hydroxybenzylidene)hydrazide. Our studies suggest this core moiety alters iron metabolism in the cell. Importantly, we also find that the reversal of EWSR1::FLI1 transcriptional regulation by SP-2509 is independent from LSD1 inhibition. This unique activity is instead associated with the N-(2-hydroxybenzylidene)-hydrazide core and destabilization of Fe-S proteins. These findings reveal a novel mechanism of action for this class of compounds and raise additional questions regarding how EWSR1::FLI1 transcriptional regulation is linked to Fe-S biogenesis, the precise mechanisms of cell death, the biological features of susceptible cancer cells, and strategies for clinical translation.

pharmacology and toxicology↗

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

Seclidemstat blocks the transcriptional function of multiple FET-fusion oncoproteins

Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting patients of all ages. New therapies are needed. These translocations fuse the 5 portion of the FET gene with a 3 partner gene encoding a transcription factor (TF). The resulting fusion proteins are oncogenic TFs with a FET protein low complexity domain (LCD) and a DNA binding domain. FET fusion proteins have proven stubbornly difficult to target directly and promising strategies target critical co-regulators. One candidate is lysine specific demethylase 1 (LSD1). LSD1 is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 activity and treatment with the noncompetitive inhibitor SP-2509 blocks EWSR1::FLI1 transcriptional function. A similar molecule, seclidemstat (SP-2577), is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against FET fusions has not been demonstrated. Here, we evaluate the in vitro potency of seclidemstat against multiple FET-rearranged sarcoma cell lines, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also define the transcriptomic effects of seclidemstat treatment and evaluated the activity of seclidemstat against FET fusion transcriptional regulation. Seclidemstat showed potent activity in cell viability assays across FET-rearranged sarcomas and disrupted the transcriptional function of all tested fusions. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas. SIGNIFICANCEHere, we show the noncompetitive inhibitor, seclidemstat, has in vitro activity against multiple FET fusion proteins that cause a number of rare and aggressive sarcomas. These data represent one of the largest analyses of FET fusion activity across multiple malignancies and are a valuable resource for those studying FET-rearranged sarcomas.

cancer biology↗

bioRxiv2024

DBD-α4 helix of EWS::FLI is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma

Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWSR1::FLI1. EWSR1::FLI1 is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWSR1 portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWSR1::FLI1. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI1, DBD-4 helix, is critical for the transcription function of EWSR1::FLI1. In this study, we aimed to understand the mechanism by which the DBD-4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatin marks, genome binding, and gene expression in cells expressing EWSR1::FLI1 constructs with and without the DBD-4 helix. Our studies revealed DBD-4 helix is crucial for cooperative binding of EWSR1::FLI1 at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWSR1::FLI1 such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.

cancer biology↗

bioRxiv2021

Mitochondrial Dysfunction is a Driver for SP-2509 Drug Resistance in Ewing Sarcoma

Expression of the fusion oncoprotein EWS/FLI causes Ewing sarcoma, an aggressive pediatric tumor characterized by widespread epigenetic deregulation. These epigenetic changes are targeted by novel lysine specific demethylase-1 (LSD1) inhibitors, which are currently in early phase clinical trials. Single agent targeted therapy often induces resistance, and successful clinical development requires knowledge of resistance mechanisms, enabling the design of effective combination strategies. Here, we used a genome-scale CRISPR-Cas9 loss-of-function screen to identify genes whose knockout (KO) conferred resistance to the LSD1 inhibitor SP- 2509 in Ewing sarcoma cell lines. Multiple genes required for mitochondrial electron transport chain (ETC) complexes III and IV function were hits in our screen. We validated this finding using genetic and chemical approaches including CRISPR KO, ETC inhibitors, and mitochondrial depletion. Further global transcriptional profiling revealed that altered complex III/IV function disrupted the oncogenic program mediated by EWS/FLI and LSD1 and blunted the transcriptomic response to SP-2509. These findings demonstrate that mitochondrial dysfunction modulates SP-2509 efficacy and suggest that new therapeutic strategies combining LSD1 with agents which prevent mitochondrial dysfunction may benefit patients with this aggressive malignancy.

cancer biology↗

bioRxiv2021

EWS/FLI mediated reprogramming of 3D chromatin promotes an altered transcriptional state in Ewing sarcoma

Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or highly related fusions. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. EWS/FLI binding is primarily associated with compartment activation, establishment of topologically-associated domain (TAD) boundaries, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators through three-dimensional reprogramming of chromatin.

cancer biology↗

bioRxiv2020

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma

BackgroundEwing sarcoma is an aggressive bone cancer in children and young adults that contains a pathognomonic chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the low-complexity amino-terminal portion of EWS to the carboxyl-terminus of FLI. The FLI portion contains an ETS DNA-binding domain and adjacent amino- and carboxyl-regions. Early studies using non-Ewing sarcoma cellular models provided conflicting information on the role of these adjacent regions in the oncogenic function of EWS/FLI. We therefore sought to define the specific contributions of each FLI region to EWS/FLI activity in an appropriate Ewing model, and in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. MethodsWe used a "knock-down/rescue" system to replace endogenous EWS/FLI expression with mutant forms of the protein in Ewing sarcoma cells and tested these for oncogenic transformation using soft-agar colony forming assays. These data were complemented by DNA-binding assays using fluorescence anisotropy, genomic localization assays using CUT&RUN, transcriptional regulation studies using luciferase reporter assays and RNA-sequencing, as well as chromatin accessibility assays using ATAC-sequencing. ResultsWe found that the DNA-binding domain and short flanking regions of FLI were required for oncogenic transformation, gene expression, genomic localization and chromatin accessibility when fused to the amino-terminal EWS-portion from EWS/FLI, but that the remaining regions of FLI were dispensable for these functions. Removal of a carboxyl-terminal alpha-helix from the short flanking regions of the DNA-binding domain of FLI created a hypomorphic EWS/FLI that retained normal DNA binding, genomic localization, and chromatin accessibility, but had significantly restricted transcriptional activity and a near total loss of oncogenic transformational capacity. ConclusionsThe DNA-binding domain and carboxyl-terminal short flanking region of FLI are the only portions of FLI required for EWS/FLI-mediated oncogenic transformation in a Ewing sarcoma cellular context. In addition to the well-defined DNA-binding function of FLI, this additional alpha-helix immediately downstream of the DNA-binding domain contributes a previously-undescribed function in gene regulation and oncogenic transformation. Understanding the function of this critical region could provide new therapeutic opportunities to target EWS/FLI in Ewing sarcoma.

cancer biology↗

bioRxiv2020

Chromatin profiling reveals reorganization of lysine specific demethylase 1 by an oncogenic fusion protein

Pediatric cancers commonly harbor quiet mutational landscapes and are instead characterized by single driver events such as the mutation of critical chromatin regulators, expression of oncohistones, or expression of oncogenic fusion proteins. These events ultimately promote malignancy through disruption of normal gene regulation and development. The driver protein in Ewing sarcoma, EWS/FLI, is an oncogenic fusion and transcription factor that reshapes the enhancer landscape, resulting in widespread transcriptional dysregulation. Lysine-specific demethylase 1 (LSD1) is a critical functional partner for EWS/FLI as inhibition of LSD1 reverses the transcriptional activity of EWS/FLI. However, how LSD1 participates in fusion-directed epigenomic regulation and aberrant gene activation is unknown. We now show EWS/FLI causes dynamic rearrangement of LSD1 and we uncover a role for LSD1 in gene activation through colocalization at EWS/FLI binding sites throughout the genome. LSD1 is integral to the establishment of Ewing sarcoma super-enhancers at GGAA-microsatellites, which ubiquitously overlap non-microsatellite loci bound by EWS/FLI. Together, we show that EWS/FLI induces widespread changes to LSD1 distribution in a process that impacts the enhancer landscape throughout the genome.

cancer biology↗