Discovery of genes required for expression or activity of fusion oncogenes

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

EWSR1's functions at the intersection of transcription and RNA processing: Proteins categorized as hnRNPs have diverse functions critical to the regulation of transcription, RNA processing and RNA transport, translation, and turnover. FUS, EWSR1, and TAF15, the FET family of proteins are multifunctional hnRNPs that function in transcriptional regulation and RNA metabolism; however, how these proteins mediate these diverse functions is still the subject of much debate. Addressing this question is essential because several disease states involve the genes encoding the FET proteins, including many fusion-driven cancers, including Ewing sarcoma (EWS), desmoplastic small round tumors, and liposarcomas and other sarcomas. We are assessing endogenous EWSR1's canonical functions, as well as determining if there are context-specific alterations in EWSR1 function in EWS cells. Employing super-resolution microscopy, including, we have demonstrated the exclusion of endogenous EWSR1 from condensed regions of DNA and its presence in euchromatic regions. At a resolution of ~120 nm, obtained using structured illumination microscopy (SIM), images show endogenous EWSR1 exists in a distributed state found throughout the nucleoplasm and as foci. EWSR1 in both states (distributed and foci) associates with nascent RNA, suggesting that EWSR1 provides a scaffold for pre-mRNA (Sundara Rajan ... Caplen, Mol. Cell Bio., 2024). We observed BRD4 and MED1 adjacent to EWSR1 foci and the significant colocalization of EWSR1 foci with a subset of phosphorylated RNA flank pol II. We are currently assessing the nuclear localization at higher resolutions using stimulated emission depletion (STED) microscopy and are compare these with other members of of the FET protein family. We are also examining whether the presence of an EWSR1-fusion oncoprotein alters EWSR1's nuclear organization and thus its functions. The processing of EWSR1-fusion transcripts: EWSR1::FLI1 is an oncogenic transcription factor expressed because of a genetic event that involves rearrangement of human chromosomes 11 and 22. This chr. 11:22 translocation is the most frequent primary genetic event observed in Ewing sarcoma (EWS), a cancer of the bone and soft tissues. We began our work to discover genes required for the expression or function of fusion oncogenes by using the results of a collaborative genome-wide RNAi screening project designed to identify genes needed for the activity of the fusion oncoprotein EWSR1::FLI1. The RNAi screen revealed that EWSR1::FLI1 fusion transcript expression is sensitive to inhibiting the activity of specific splicing factors. For example, we identified that the processing of EWSR1::FLI1 transcripts expressed in EWS cells harboring translocations where the breakpoint in chr. 22 occurs within a region of DNA (intron 8 of the EWSR1 gene) requires HNRNPH1 to generate an in-frame mRNA (Grohar, Kim ... Caplen, Cell Reports, 2016). The depletion of HNRNPH1 in EWS cells that harbor a chr. 22 breakpoint in EWSR1-intron 8 disrupts the expression and the activity of the EWSR1::FLI1 protein and reduces cell survival. A follow-up study (Neckles ... Caplen, RNA 2019), demonstrated that guanine (G)-rich sequences within EWSR1-exon 8 that can fold into RNA G-quadruplex (G4) structures influence the recruitment of HNRNPH1. Critically, we showed that EWSR1-exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-G4 binding molecule, pyridostatin (PDS), than EWSR1-exon 8 fusion-negative lines. Also, the treatment of EWSR1-exon 8 fusion-positive cells with PDS decreases EWSR1::FLI1 transcriptional activity and reverses the transcriptional deregulation driven by EWSR1::FLI1. More recently (Vo et al., Nucleic Acids Research, 2022), we have shown that though the HNRNPH1/H2 and F proteins are highly homologous, only HNRNPH1 is responsible for excluding EWSR1-exon 8. Furthermore, a series of minigenes in which we included G-A substitutions to disrupt G-tracts to which HNRNHP1 could bind, demonstrated a shift in splicing from detection of both exon inclusion and exclusion events to only exon inclusion. Furthermore, analysis of HNRNPH1's binding of RNA oligomers corresponding to G-rich regions of the EWSR1-exon 8 3' end demonstrated HNRNPH1 binds these G-rich sequences with low nM affinities irrespective of structural state - unfolded or folded as parallel G4 structures. Critically, we showed that the binding of HNRNPH1 to EWSR1-exon 8 G-rich sequences in a folded state results in a concentration-dependent change in RNA structure to that of one that more resembles an unfolded state. Our current research aims are as follows (1) Determination of cis-regulatory sequences defining EWSR1-exon 8 exclusion in the context of the EWSR1::FLI1 transcript as the basis of a strategy for inhibition of fusion oncoprotein expression in a subset of Ewing sarcomas. (2) Exploration of the adaption of mechanisms that regulate the processing of the pre-mRNAs expressed by EWSR1 or its partner genes to express the translatable product of a fusion gene. The functions of HNRNPH1: We are also evaluating other splicing events regulated by HNRNPH1 that contribute to the transcript variants expressed by cancer-associated genes. We discussed the importance of understanding the function of the HNRNPF/H proteins in further detail in a recently published review - Brownmiller and Caplen, WIREs RNA 2023 DOI: 10.1002/wrna.1788. Ewing sarcoma - the regulation of gene expression and disease progression: Our research is also interrogating mechanisms resulting from the EWSR1-fusion oncoprotein's deregulation of gene expression. Current studies focus on processes that have the potential to favor dissemination of tumor cells and thus metastasis. For example, we have initiated investigation of the transcription factor (TF) genes my group's cell-based functional studies suggest the transcriptional activity of EWSR1::FLI1 protein represses but exhibit inter-tumoral variations in expression when we compared data from EWS cell lines and tumors available to us through interrogation of published data. Studying the functional consequence of the expression of one of these TF genes, ETS1, we showed that depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of EWS cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. Notably, ETS1 regulates the movement of several early progenitor sub-types, including neural crest cells. Next, we showed that EWS cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared to control cells. Visualization of control EWS cells showed a distributed vinculin signal and a network-like organization of F-actin. In contrast, ETS1-activated EWS cells showed an accumulation of vinculin and F-actin towards the plasma membrane. Interestingly, the phenotype of ETS1-activated EWS cell lines depleted of TNS3 resembled that of the control cells. These findings have clinical relevance as TNS3 and ETS1 expression in EWS tumors positively correlate (multiple cohorts). The identification of mechanisms that promote EWS metastasis are critical to determining means of identifying individuals at an increased risk of poor survival and improving the outcome for these patients (Ebegboni ... Caplen, Mol. Cancer. Res. 2024). Our ongoing work focuses on determining how the expression in an EWS tumor cell of one or more transcriptional factors that function in early development, including ETS1 promotes cell dissemination.