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 EWS-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). EWS-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 EWS-FLI1 fusion transcript expression is sensitive to inhibiting the activity of specific splicing factors. We identified that expression of the EWS-FLI1 transcripts expressed in ES 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 EWS-FLI1 protein and reduces cell survival. A follow-up study, published in December 2019, showed that the processing of EWS-FLI1 pre-mRNAs containing EWSR1 exon 8 by HNRNPH1, but not other homologous family members, resembles the alternative splicing of transcript variants of EWSR1 (Neckles .. Caplen, RNA 2019). We also 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 EWS-FLI1 transcriptional activity and reverses the transcriptional deregulation driven by EWS-FLI1. Our findings illustrated that modulation of the alternative splicing of EWS-FLI1 pre-mRNA as a strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma. Our ongoing studies aim to determine how HNRNPH1 facilitates the splicing of EWS-FLI1 in those EWS cells that depend upon its function, with the long-term aim of identifying a selective means of inhibiting this event. Our specific objectives include identifying the sequence or sequences recognized in the EWS-FLI1 pre-mRNA by HNRNPH1 that mediates the exclusion of EWSR1 exon 8. We will use this information to test different approaches for blocking the interaction of HNRNPH1 with the biologically relevant sequence or sequences. Follow-up studies of the genome-wide RNAi screen of EWS-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 EWS-FLI1 mRNA is sensitive to disruption of spliceosome function and that canonical-splicing is a potential vulnerability in EWS. We have recently initiated studies that will extend our analysis of the biogenesis of the EWS-FLI1 pre-mRNA using complementary sequencing approaches. 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). We have also recently initiated gene-editing-based studies that use multi-dimensional assays to identify additional proteins that regulate the expression or activity of the fusion oncoprotein EWS-FLI1. The inhibition of transcription factor function using small molecules has proven challenging. A potential alternative strategy is the targeting of post-translational modifications of proteins. Previous studies have reported post-translational modifications of the EWS-FLI1 protein, but we know little about the proteins that catalyze these changes. We are thus using functional genetic approaches coupled with biochemical methods to identify candidate proteins required for the post-translational modification of the EWS-FLI1 protein, particularly proteins that may regulate protein stability. In related, collaborative work, we contributed to a recent publication that highlighted RNA polymerase II processivity as an approach to improving the activity and limiting the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for the treatment of Ewing sarcoma (Flores ..Caplen, Helman, Grohar, Molecular Cancer Therapeutics, 2020). Our research also includes investigating the reprogramming of the transcriptome and signaling networks by the EWS-FLI1 fusion oncoprotein that suppresses the differentiation of the progenitor cells in which EWS arises and promotes tumor growth. For example, we demonstrated recently that EWS-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 EWS-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 EWS-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 EWS-FLI1 regulates to favor EWS tumorigenesis, including those that support cell proliferation or metastasis.