Small Molecule Therapeutics An Oral Formulation of YK-4-279: Preclinical Efficacy and Acquired Resistance Patterns in Ewing Sarcoma Salah-Eddine Lamhamedi-Cherradi 1 , Brian A. Menegaz 1 , Vandhana Ramamoorthy 1 , Ramani A. Aiyer 2 , Rebecca L. Maywald 3 , Adrianna S. Buford 1 , Dannette K. Doolittle 4 , Kirk S. Culotta 4 , James E. O'Dorisio 5 , and Joseph A. Ludwig 1 Abstract Ewing sarcoma is a transcription factor–mediated pediatric bone tumor caused by a chromosomal translocation of the EWSR1 gene and one of several genes in the ETS family of transcription factors, typically FLI1 or ERG. Full activity of the resulting oncogenic fusion protein occurs only after binding RNA helicase A (RHA), and novel biologically targeted small molecules designed to interfere with that interaction have shown early promise in the preclinical setting. Herein, we demonstrate marked preclinical antineoplastic activity of an orally bioavailable for- mulation of YK-4-279 and identify mechanisms of acquired chemotherapy resistance that may be exploited to induce collat- eral sensitivity. Daily enteral administration of YK-4-279 led to significant delay in Ewing sarcoma tumor growth within a murine model. In advance of anticipated early-phase human clinical trials, we investigated both de novo and acquired mechanism(s) by which Ewing sarcoma cells evade YK-4-279–mediated cell death. Drug-resistant clones, formed by chronic in vitro exposure to steadily increased levels of YK-4-279, overexpressed c-Kit, cyclin D1, pStat3(Y705), and PKC isoforms. Interestingly, cross-resis- tance to imatinib and enzastaurin (selective inhibitors of c-Kit and PKC-b, respectively), was observed and the use of YK-4-279 with enzastaurin in vitro led to marked drug synergy, suggesting a potential role for combination therapies in the future. By advanc- ing an oral formulation of YK-4-279 and identifying prominent mechanisms of resistance, this preclinical research takes us one step closer to a shared goal of curing adolescents and young adults afflicted by Ewing sarcoma. Mol Cancer Ther; 14(7); 1591–604. Ó2015 AACR. Introduction Ewing sarcoma family of tumors, which encompass traditional Ewing sarcoma of bone (1), primitive neuroectodermal tumors (PNET; refs. 2), and Askin tumor of chest wall (3–5), have for more than a two decades been considered to represent clinical variants of the same molecularly distinct sarcoma subtype that bears a pathognomonic EWS–ETS oncogenic fusion protein (6). Heretofore, simply referred to as Ewing sarcoma, this sarcoma subtype is a highly aggressive malignancy of adolescents and young adults (7) that is rapidly fatal without effective multi- modality treatment that includes surgery and/or radiation and extensive use of systemic chemotherapy. Although polyche- motherapy has led to marked improvement in 5-year survival for patients diagnosed with localized Ewing sarcoma—approach- ing 80% with current generation clinical trials—those with met- astatic disease or rapid tumor recurrence fare poorly and often quickly succumb to their disease (8–11). Although cytotoxic chemotherapy will remain the mainstay for treating Ewing sarcoma in the near future given its well- trodden track record of antineoplastic activity, high-throughput "-omic" technologies and paired biomarkers are enabling an era of precision medicine that, as has occurred for more common carcinomas such as breast or lung cancer, will increas- ingly target the unique molecular complexity linked to each patient's respective tumor. Therapies targeting the insulin-like growth factor 1 receptor (IGFIR), alone or in combination with inhibitors of mammalian target of rapamycin (mTOR), provide just one example of selective biologically targeted therapies capable of inducing striking tumor regression among a subset of Ewing sarcoma patients (12, 13). Although such therapies show tremendous potential for the treatment of Ewing sarcoma, and are likely to be even more effective once tissue-based biomarkers reliably predict a priori who will respond, recent evidence suggests that no single therapy will be universally effective. Rather, antineoplastic effects will rely upon a number of secondary mutation/amplification events or perturbed signaling cascades that maintain an aberrant cancer phenotype. Fortunately, though most nonleukemic malignancies lack a consistent primary oncogenic driver protein, Ewing sarco- ma and nearly one third of the approximately 50 sarcoma 1 Department of Sarcoma Medical Oncology, MD Anderson Cancer Center, Houston, Texas. 2 Shasta BioVentures, San Jose, California. 3 Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas. 4 Laboratory of Experimental Therapeutics, MD Anderson Can- cer Center, Houston, Texas. 5 Star Biotechnology, LLC, San Francisco, California. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Joseph Ludwig, Department of Sarcoma Medical Oncol- ogy, MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77005. Phone: 713-792-3626; Fax: 713-563-1185; E-mail: jaludwig@mdanderson.org doi: 10.1158/1535-7163.MCT-14-0334 Ó2015 American Association for Cancer Research. 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