181 research outputs found
EWS-FLI1 as a molecular target: Small molecule inhibitors for a disordered protein
No full textPh.D.Ewing sarcoma family of tumors (ESFT) consists of highly malignant tumors of the bone and soft tissue. Ninety-five percent of cases contain a balanced t(11;22) or t(21;22) rearrangement, combining the amino-terminus of EWS to the carboxy-terminus of FLI1 or ERG, which contain the highly conserved ets DNA binding domain. As the EWS-FLI1 protein is found only in ESFT cells and its expression is required for the oncogenic phenotype, it presents a promising molecular target for anti-cancer therapies. EWS-FLI1 is a hydrophobic disordered protein with unknown three-dimensional structure, precluding standard structure-based drug design. RNA Helicase A (RHA) enhances EWS-FLI1 driven oncogenesis and interruption of this protein-protein complex validates this interaction as a unique therapeutic target. Surface plasmon resonance screening identified compounds that bind to EWS-FLI1, including a lead compound that induces apoptosis in ESFT cells and reduces the growth of xenografts. Our compound, YK-4-279, has a chiral center and can be separated into enantiomers, only one of which is able to specifically target the protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein-protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be highly specific. Given the challenges of drug design targeted to EWS-FLI1, we proposed that characterization of the physical interaction points between EWS-FLI1 and RHA would allow us to better alter the lead compound to block this protein-protein interaction. While full length EWS-FLI1 is able to pull down RHA, fragments of the protein are not. Although we can successfully crosslink EWS-FLI1 and RHA, we have yet to identify what region of EWS-FLI1 is involved. We are able to show specific regions of order and disorder of EWS-FLI1, which may lead to the identification of the binding site for YK-4-279. The development of higher-throughput methods for testing small molecules that bind to or inhibit EWS-FLI1 function will allow us to further investigate protein structure and function. These data are a contribution to the future development of small molecules in an era where novel approaches to cancer therapy are critical for improving patient care
Cassette Exon RNA Splicing Altered in Ewing Sarcoma Is Characterized by Higher Guanine-Cytosine Content and Lower Splice Site Strength Indicative of Intron-Definition Splicing
No full textPh.D.Ewing sarcoma (EwS) is a pediatric cancer that typically presents at a mean of 15 years of age and is the second most common bone tumor in children and adolescents. While there have been substantial investigations into the mechanism of EwS oncogenesis, few of these findings have translated to improved therapeutics, and treatment of EwS has largely remained static for the last 30 years. Patients that present with localized EwS have an approximately 70% 5-year survival rate whereas patients that present with metastatic EwS have only a 30% 5-year survival rate. New approaches to understanding the oncogenic process and potential vulnerabilities behind EwS are badly needed, as targeted therapeutics for EwS patients have the opportunity to drastically improve long-term outcomes and reduce systemic effects. In this dissertation I present first the creation of a model to study EwS using bone marrow-derived human mesenchymal stem cells (hMSC-BM), as the lack of patient samples presents a substantial barrier to EwS research. I then describe my efforts to create a reference transcriptome using long-read sequencing that is more comprehensive than commonly used reference transcriptomes. I use this transcriptome to quantify mRNA splicing in tumor samples, followed by a detailed analysis of sequence features associated with altered RNA splicing in EwS
Abstract 694: TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of Ewing Sarcoma
No full textAbstract
One of the most significant challenges in creating more potent, less toxic treatments for patients is to identify new cancer therapeutic targets that distinguish the malignant from normal cells. EWS-FLI1 is a well-established Ewing sarcoma (ES) oncogene that has the potential to be an ideal therapeutic target by directly impacting malignant cells. We have previously reported the discovery and characterization of YK-4-279, an enantiomer-specific inhibitor of EWS-FLI1, which has been demonstrated to induce apoptosis, inhibit EWS-FLI1 transcription, block RNA helicase A co-immunoprecipitation with EWS-FLI1, and result in alternative splicing to mimic EWS-FLI1 knockdown. Continuous efforts in structure-guided medicinal chemistry has yielded TK-216, an analog of YK-4-279 inhibitor of EWS-FLI1, which is 3-4 fold more potent with excellent drug-like properties. TK-216 potently inhibits the proliferation of ES cells. Induces apoptosis in a dose -dependent manner as measured by caspase-3 activity in multiple ES cell lines with distinct translocation variants. The effects of TK-216 on alternative splicing (AS) were further validated using genes including ARID1A, CLK1, CASP3, PPFIBP1 and RUNX2. The splicing pattern was similar between TK-216 and YK-4-279. In addition to the in vitro activity of TK-216 , we show that TK-216 displays anti-tumor activity in a number of ES xenograft models. In summary, TK-216, a novel, first-in-class therapeutic which directly inhibits EWS-FLI1, offers a promising approach for the treatment of Ewing Sarcoma and is currently in Phase 1 clinical trials in patients with relapsed or refractory Ewing Sarcoma (clinicaltrials.gov - NCT02657005).
Citation Format: Saravana P. Selvanathan, Eric Moseley, Garrett T. Graham, Katti Jessen, Brian Lannutti, Aykut Üren, Jeffrey A. Toretsky. TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2017-694</jats:p
An Intrinsically Disordered Region of the Acetyltransferase p300 with Similarity to Prion-Like Domains Plays a Role in Aggregation
No full textSeveral human diseases including neurodegenerative disorders and cancer are associated with abnormal accumulation and aggregation of misfolded proteins. Proteins with high tendency to aggregate include the p53 gene product, TAU and alpha synuclein. The potential toxicity of aberrantly folded proteins is limited via their transport into intracellular sub-compartments, the aggresomes, where misfolded proteins are stored or cleared via autophagy. We have identified a region of the acetyltransferase p300 that is highly disordered and displays similarities with prion-like domains. We show that this region is encoded as an alternative spliced variant independently of the acetyltransferase domain, and provides an interaction interface for various misfolded proteins, promoting their aggregation. p300 enhances aggregation of TAU and of p53 and is a component of cellular aggregates in both tissue culture cells and in alpha-synuclein positive Lewy bodies of patients affected by Parkinson disease. Down-regulation of p300 impairs aggresome formation and enhances cytotoxicity induced by misfolded protein stress. These data unravel a novel activity of p300, offer new insights into the function of disordered domains and implicate p300 in pathological aggregation that occurs in neurodegeneration and cancer. © 2012 Kirilyuk et al
- …
