Discovery of genes required for expression or activity of fusion oncogenes

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

We began our work to discover genes required for the expression or function of fusion oncogenes by using the results of a genome-wide RNAi screen designed to identify genes needed for the activity of the fusion oncoprotein EWSR1::FLI1. The development of this RNAi screen was a collaborative effort involving the Caplen laboratory and researchers from CCR's Pediatric Oncology Branch and the trans-NIH RNAi screening facility at the National Center for Advancing Translational Sciences (NCATS). EWSR::FLI1 is an oncogenic transcription factor that results from a chromosomal rearrangement involving chromosomes 11 and 22. Chromosome 11:22 translocation is the most frequent primary genetic event observed in Ewing sarcoma (EWS), a cancer of the bone and soft tissues. The genome-wide RNAi screen revealed that EWSR1::FLI1 fusion transcript expression is sensitive to inhibiting the activity of specific splicing factors. We identified that expression of the EWSR1::FLI1 transcripts expressed in EWS cells harboring translocations where the breakpoint in chromosome 22 occurs within a region of DNA (intron 8 of the EWSR1 gene) requires the splicing factor HNRNPH1 to generate an in-frame mRNA (Grohar, Kim ... Caplen, Cell Reports, 2016). The depletion of HNRNPH1 in EWS cells that harbor a chromosome 22 breakpoint in EWSR1-intron 8 disrupts the expression and the activity of the EWSR::FLI1 protein and reduces cell survival. A follow-up study (Neckles ... Caplen, RNA 2019), demonstrated that guanine-rich sequences within EWSR1-exon 8 that can fold into RNA G-quadruplex 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-quadruplex binding molecule, pyridostatin (PDS), than EWSR1-exon 8 fusion-negative lines. Also, the treatment of EWSR1-exon 8 fusion-positive cells with PDS decreases EWSR::FLI1 transcriptional activity and reverses the transcriptional deregulation driven by EWSR1::FLI1. Most recently (Vo et al., Nucleic Acids Research, 2022), we performed a meta-analysis of studies reporting Chr. 22 breakpoints and determined that about 35% of EWSR::FLI1-positive Ewing sarcomas harbor EWSR1-intron 8 breakpoints. Using long-read sequencing, we have determined that the exclusion of EWSR1-exon 8 is the dominant HNRNPH1-dependent event observed in the processing of EWSR1::FLI1 in EWS cell lines harboring EWSR1-intron 8 breakpoints. Though the HNRNPH1/H2 and F proteins are highly homologous (90 and 70%, respectively), minigenes focused on EWSR1-exon 8, confirmed our previous findings that 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. Analysis of HNRNPH1's binding of RNA oligomers corresponding to G-rich regions of the EWSR1-exon 8 3' end using Bio-layer Interferometry demonstrated its binding of these G-rich sequences. HNRNPH1 binds these G-rich sequences with low nM affinities irrespective of structural state - unfolded or folded as parallel G4 structures but exhibits faster association and dissociation rates when these RNAs are in a folded state. Critically, using spectroscopic assays 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 results indicate that HNRNPH1's binding of G-rich sequences can destabilize rG4 structures in a non-catalytic fashion. In summary, our results provide the first evidence that HNRNPH1's binding of G-rich sequences in a quadruplex state can alter this structural conformation, provides insights into the function of HNRNPH1 as a regulator of alternative splicing, and suggest means for inhibiting EWSR1::FLI1 expression through disruption of EWSR1::FLI1 mRNA biogenesis. 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. (3) The evaluation of splicing regulation by HNRNPH1 to define its functions further and inform the study of disease states associated with mutations in HNRNPF/H genes or sequences they bind. 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. Our follow-up studies of the genome-wide RNAi screen of EWSR1::FLI1 activity also identified SF3B1, the catalytic component of the spliceosome, a protein complex required for splicing, as a protein that EWS cells are dependent upon for cell survival. We discovered that the biogenesis of the EWSR1::FLI1 mRNA is sensitive to disruption of spliceosome function and that canonical splicing is a potential vulnerability in EWS. In 2020, we published an opinion piece that discussed the rationale for studying the biology of cancer-associated fusion transcripts in further detail, which included discussion of several other fusion driven cancers, including subtypes of leukemia, lung and prostate cancer, and NUT-midline carcinoma (Neckles, Sundara Rajan, Caplen, Wiley Interdiscip Rev RNA, 2020). Building on these previous studies, we have recently initiated studies that use multi-dimensional assays (quantitative and image-based) to identify additional proteins that regulate the expression or activity of EWSR1 and/or the fusion oncoprotein EWSR1::FLI1 and the respective functions of these proteins. Our research also involves evaluating the hypothesis that mechanisms contributing to and resulting from the EWSR1-fusion oncoprotein's deregulation of gene expression represent potential tumor-specific dependencies with therapeutic potential. We are investigating how gene expression regulatory mechanisms contribute to the pathology of an EWSR1-fusion driven cancer with an initial focus on EWSR1 and the study of pathways EWSR1::FLI1 deregulates. For example, we have demonstrated that EWSR1::FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine (Sen .. Caplen, Molecular Carcinogenesis, 2018). Specifically, we show that EWSR1::FLI1 can alter the expression of PHGDH, PSAT1, PSPH, and SHMT2, genes encoding enzymes required for serine-glycine biosynthesis. Using cell-based studies, we also established that EWS cells are dependent on glutamine for cell survival and that EWSR1::FLI1 positively regulates the expression of the glutamine transporter, SLC1A5, and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state, leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that most of the genes identified by our cell-based studies also exhibit increased expression compared with non-diseased tissues, including PHGDH and SHMT2. Current studies aim to examine other cellular processes EWSR1::FLI1 regulates to promote EWS tumorigenesis, in particular processes that have the potential to favor dissemination of tumor cells and thus metastasis.