Hayriye V. Erkizan

A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma

Many sarcomas and leukemias carry nonrandom chromosomal translocations encoding tumor-specific mutant fusion transcription factors that are essential to their molecular pathogenesis. Ewings sarcoma family tumors (ESFTs) contain a characteristic t(11;22) translocation leading to expression of the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disordered protein that precludes standard structure-based small-molecule inhibitor design. EWS-FLI1 binding to RNA helicase A (RHA) is important for its oncogenic function. We therefore used surface plasmon resonance screening to identify compounds that bind EWS-FLI1 and might block its interaction with RHA. YK-4-279, a derivative of the lead compound from the screen, blocks RHA binding to EWS-FLI1, induces apoptosis in ESFT cells and reduces the growth of ESFT orthotopic xenografts. These findings provide proof of principle that inhibiting the interaction of mutant cancer-specific transcription factors with the normal cellular binding partners required for their oncogenic activity provides a promising strategy for the development of uniquely effective, tumor-specific anticancer agents.

Oncogenic Partnerships: EWS-FLI1 Protein Interactions Initiate Key Pathways of Ewing's Sarcoma

Targeted therapy for cancer, which is specifically directed toward the cancer without any potential for effects outside of controlling the tumor, is a gold standard for treatment. Ewings sarcoma contains the potential target EWS-FLI1, as a result of a pathognomonic chromosomal translocation. The EWS-FLI1 fusion protein includes the EWS domain, a potent transcriptional activator alongside the highly conserved FLI1 ets DNA-binding domain. Because of the combination of these domains, the EWS-FLI1 fusion protein acts as an aberrant transcription factor whose expression results in cellular transformation. EWS-FLI1 functions by binding to normal cellular protein partners in transcription and splicing, similar to how a virus would corrupt normal cellular machinery for virion production. Therefore, understanding the protein-protein interactions of EWS-FLI1 and the pathways that are regulated by these partnerships will inform both oncogenesis and therapeutics. This review describes the known protein partners and transcriptional targets of EWS-FLI1, while proposing strategies for exploiting these partnerships with targeted therapy. Clin Cancer Res; 16(16); 4077–83. ©2010 AACR.

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

Significance Alternative splicing of RNA allows a limited number of coding regions in the human genome to produce proteins with diverse functionality. Alternative splicing has also been implicated as an oncogenic process. Identifying aspects of cancer cells that differentiate them from noncancer cells remains an ongoing challenge, and our research suggests that alternatively spliced mRNA and subsequent protein isoforms will provide new anticancer targets. We determined that the key oncoprotein of Ewing sarcoma (ES), EWS-FLI1, regulates alternative splicing in multiple cell line models. These experiments establish oncogenic aspects of splicing that are specific to cancer cells and thereby illuminate potentially oncogenic splicing shifts as well as provide a useful stratification mechanism for ES patients. The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4–279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

Combining PARP-1 Inhibition and Radiation in Ewing Sarcoma Results in Lethal DNA Damage

Ewing sarcomas (ES) harbor a chromosomal translocation that fuses the EWS gene to an ETS transcription factor, most commonly Friend leukemia integration 1 (FLI1). The EWS-FLI1 fusion protein acts in a positive feedback loop to maintain the expression of PARP-1, which is involved in repair of DNA damage. Here, we examine the effects of PARP-1 inhibition and radiation therapy on Ewing sarcomas. In proliferation assays, the Ewing sarcoma cell lines RD-ES and SK-N-MC were much more sensitive than non-Ewing sarcoma cell lines to the PARP-1 inhibitor olaparib (Ola; IC50 0.5–1 μmol/L vs. >5 μmol/L) and to radiation (IC50 2–4 Gy vs. >6 Gy). PARP-1 inhibition with short hairpin RNA (shRNA) or Ola sensitized Ewing sarcoma cells, but not non-Ewing sarcoma cells, to radiation therapy in both proliferation and colony formation assays. Using the Comet assay, radiation of Ewing sarcoma cells with Ola, compared to without Ola, resulted in more DNA damage at 1 hour (mean tail moment 36–54 vs. 26–28) and sustained DNA damage at 24 hours (24–29 vs. 6–8). This DNA damage led to a 2.9- to 4.0-fold increase in apoptosis and a 1.6- to 2.4-fold increase in cell death. The effect of PARP-1 inhibition and radiation therapy on Ewing sarcoma cells was lost when EWS-FLI1 was silenced by shRNA. A small dose of radiation therapy (4 Gy), when combined with PARP-1 inhibition, stopped the growth of SK-N-MC flank tumors xenografts. In conclusion, PARP-1 inhibition in Ewing sarcomas amplifies the level and duration of DNA damage caused by radiation therapy, leading to synergistic increases in apoptosis and cell death in a EWS-FLI1–dependent manner. Mol Cancer Ther; 12(11); 2591–600. ©2013 AACR.

Novel peptide binds EWS-FLI1 and reduces the oncogenic potential in Ewing tumors

Ewing tumor is driven by the oncogenic EWS-FLI1 fusion protein that functions as an aberrant transcription factor. The identification of EWS-FLI1 protein partners is essential to enhance its vulnerability as a therapeutic target. We utilized phage display library screening against recombinant EWS-FLI1 protein. We identified 27 unique Ewing sarcoma binding peptides. The cytotoxicity evaluation of these peptides with in EWS-FLI1 containing cell lines yielded one potent peptide called ESAP1 (TMRGKKKRTRAN). ESAP1 binds EWS-FLI1 with 0.202 micromolar affinity as measured in surface plasmon resonance. The minimal interaction region of ESAP1 is characterized and found that the lysine residues are critical for cellular cytotoxicity. ESAP1 reduces the transcriptional activity of EWS-FLI1 as well as disrupts cell cycle kinetics in Ewing Tumor cells. These findings provide both a novel experimental probe and a potential therapeutic scaffold for Ewing Tumor.

YK-4-279 effectively antagonizes EWS-FLI1 induced leukemia in a transgenic mouse model

Ewing sarcoma is an aggressive tumor of bone and soft tissue affecting predominantly children and young adults. Tumor-specific chromosomal translocations create EWS-FLI1 and similar aberrant ETS fusion proteins that drive sarcoma development in patients. ETS family fusion proteins and over-expressed ETS proteins are also found in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Transgenic expression of EWS-FLI1 in mice promotes high penetrance erythroid leukemia with dense hepatic and splenic infiltrations. We identified a small molecule, YK-4-279, that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing sarcoma cell lines and xenograft mouse models. Herein, we tested in vivo therapeutic efficacy and potential side effects of YK-4-279 in the transgenic mouse model with EWS-FLI1 induced leukemia. A two-week course of treatment with YK-4-279 significantly reduced white blood cell count, nucleated erythroblasts in the peripheral blood, splenomegaly, and hepatomegaly of erythroleukemic mice. YK-4-279 inhibited EWS-FLI1 target gene expression in neoplastic cells. Treated animals showed significantly better overall survival compared to control mice that rapidly succumbed to leukemia. YK-4-279 treated mice did not show overt toxicity in liver, spleen, or bone marrow. In conclusion, this in vivo study highlights the efficacy of YK-4-279 to treat EWS-FLI1 expressing neoplasms and support its therapeutic potential for patients with Ewing sarcoma and other ETS-driven malignancies.

RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1

RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.

Acetylation increases EWS-FLI1 DNA binding and transcriptional activity

Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level.

ABSTRACT Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.

A new sub‐pathway of long‐patch base excision repair involving 5′ gap formation

Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub‐pathway of conventional long‐patch BER that involves formation of a 9‐nucleotide gap 5′ to the lesion. This new sub‐pathway is mediated by RECQ1 DNA helicase and ERCC1‐XPF endonuclease in cooperation with PARP1 poly(ADP‐ribose) polymerase and RPA. The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto‐(ADP‐ribosyl)ation and the choice between long‐patch and single‐nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long‐patch BER and a new key regulation point for pathway choice in BER.