Biology subjects

Selich-Anderson, J.

Publications and source records attributed to Selich-Anderson, J..

7 recordsLinked to original sources

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

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↗

bioRxiv2018

Dual BRD4 and AURKA Inhibition is Synergistic against MYCN-amplified and nonamplified Neuroblastoma

A majority of cases of high-risk neuroblastoma, an embryonal childhood cancer, are driven by MYC or MYCN-driven oncogenic signaling. While considered to be directly \"undruggable\" therapeutically, MYC and MYCN can be repressed transcriptionally by inhibition of Bromodomain-containing protein 4 (BRD4) or destabilized posttranslationally by inhibition of Aurora Kinase A (AURKA). Preclinical and early-phase clinical studies of BRD4 and AURKA inhibitors, however, show limited efficacy against neuroblastoma when used alone. We report our studies on the concomitant use of the BRD4 inhibitor IBET-151 and AURKA inhibitor alisertib. We show that, in vitro, the drugs act synergistically to inhibit viability in three models of high-risk neuroblastoma. We demonstrate that this synergy is driven, in part, by the ability of IBET-151 to mitigate reflexive upregulation of AURKA, MYC, and MYCN in response to AURKA inhibition. We then demonstrate that IBET-151 and alisertib are effective in prolonging survival in three xenograft neuroblastoma models in vivo, and this efficacy is augmented by the addition of the antitubule chemotherapeutic vincristine. These data suggest that epigenetic and posttranslational inhibition of MYC/MYCN-driven pathways may have significant clinical efficacy against neuroblastoma.\n\nAbbreviationsAURKA, aurora kinase A; BRD4, Bromodomain-containing protein 4; CI, combination index; Fa, fraction affected; LOH, loss of heterozygosity; 11q, long arm of chromosome 11\n\nFunding sourcesThis work was supported by the Families for a Cure foundation (grant number 20064014) and CancerFree Kids (grant number 82178416).

cancer biology↗

bioRxiv2018

HOTAIR primes the Ewing sarcoma family of tumors for tumorigenesis via epigenetic dysregulation involving LSD1

The EWS-FLI1 fusion protein drives oncogenesis in the Ewing sarcoma family of tumors (ESFT) in humans, but its toxicity in normal cells requires additional cellular events for oncogenesis. We show that the lncRNA HOTAIR maintains cell viability in the presence of EWS-FLI1 and redirects epigenetic regulation in ESFT. HOTAIR is consistently overexpressed in ESFTs and is not driven by EWS-FLI1. Repression of HOTAIR in ESFT cell lines significantly reduces anchorage-independent colony formation in vitro and impairs tumor xenograft growth in vivo. Overexpression of HOTAIR in human mesenchymal stem cells (hMSCs), a putative cell of origin of ESFT, and IMR90 cells induces colony formation. Critically, HOTAIR-expressing hMSCs and IMR90 cells remain viable with subsequent EWS-FLI1 expression. HOTAIR induces histone modifications and gene repression through interaction with the epigenetic modifier LSD1 in ESFT cell lines and hTERT-hMSCs. Our findings suggest that HOTAIR maintains ESFT viability through epigenetic dysregulation.\n\nSignificanceWhile the EWS-FLI1 fusion gene was determined to be the oncogenic driver in the overwhelming majority of ESFT, it is toxic to cell physiology and requires one or more additional molecular events to maintain cell viability. As these tumors have surprisingly few genetic mutations at diagnosis, epigenetic changes have been considered to be such an event, but the mechanism by which these changes are driven remains unclear. Our work shows that HOTAIR is consistently expressed among ESFT and induces epigenetic and gene expression changes that cooperate in tumorigenesis. Furthermore, expression of HOTAIR allows for cell viability in the setting of subsequent EWS-FLI1 expression. Our findings elucidate new steps of malignant transformation in this cancer and identify novel therapeutic targets.

cancer biology↗