RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma

Molecular Cancer RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma Shilpi Arora 0 2 Irma M Gonzales 0 1 R Tanner Hagelstrom 0 1 Christian Beaudry 0 Ashish Choudhary 0 Chao Sima 3 Raoul Tibes 1 Spyro Mousses 0 David O Azorsa 0 1 0 Pharmaceutical Genomic Division, Translational Genomics Research Institute , Scottsdale, AZ, 85259 , USA 1 Clinical Translational Research Division, The Translational Genomics Research Institute , Scottsdale, Arizona 85259 , USA 2 Cancer and Cell Biology Division, Translational Genomics Research Institute , Phoenix, AZ, 85004 , USA 3 Computational Biology Division, Translational Genomics Research Institute , Phoenix, AZ, 85004 , USA Background: Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Results: Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. Conclusion: In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma. - Introduction Ewings sarcoma represents approximately three percent of pediatric cancers and is the second most common bone malignancy in children and adolescents [1,2]. It is an aggressive cancer with a tendency to recur following resection and it metastasizes to the lung, bone and bone marrow. Ewings sarcomas harbor unique chromosomal translocations that give rise to fusion genes that act as oncoproteins [3]. Rearrangement of the EWS gene on chromosome 22q12 with an ETS gene family member is the underlying molecular genetic abnormality for Ewings sarcoma. The most common translocation involves the genes EWS and Friend Leukemia Integration Site 1 (FLI1). This translocation can be further subdivided into two separate types, Type I and Type II, with Type I resulting from the translocation fusing EWS exon 7 to FLI-1 exon 6 and Type II resulting from the fusion of EWS exon 7 to FLI1 exon 5. The newly formed EWS-FLI1 fusion protein is a transcription factor that can then lead to aberrant transcription [4]. Morphologically, Ewings sarcoma is composed of small round cells with high nuclear to cytoplasmic ratio and cells from more than 90% of patients express the adhesion receptor CD99 [5,6]. Disease management for patients with localized disease has substantially improved but the prognosis for those with metastatic or recurrent disease has changed very little over the past three decades. Currently, Ewings sarcoma patients are treated with a combination of surgery, radiation and chemotherapy [7]. Five-year event free survival for patients with metastatic disease is only 20% and curative therapy does not exist for patients whose disease recurs rapidly following therapy for localized disease [7,8]. Recently, expression of several individual genes has been linked to the development and progression of the disease, but so far there has been no comprehensive systematic study undertaken to identify functionally relevant genes in Ewings sarcoma [9-13]. The genomic translocations in Ewings sarcoma provide a valuable tool for accurate diagnosis. In addition, these common genetic abnormalities could serve in identifying specific genetic vulnerabilities, which would be useful in development of targeted therapeutics for this disease. In order to identify novel therapeutic targets for Ewings sarcoma, we employed a functional genomics approach based on high-throughput RNA interference (HT-RNAi), which is also known as loss-of-function screening. The basis of this technology is RNA interference (RNAi), a robust method of post-transcriptional silencing of genes using double-stranded RNA in the form of either siRNA (short interfering RNA) or shRNA (short hairpin RNA) with sequence homology driven specificity [14]. Largescale libraries of siRNA and shRNA have been used to identify genes involved in many biological functions [15-19]. We utilized a siRNA library targeting human kinases to identify single siRNA kinase targets for Ewings sarcoma cells. The availability of four Ewings sarcoma cell lines that transfect well and are amenable to high throughput screening enables us to identify essential kinase that regulate growth of Ewings sarcoma cells. Numerous small molecule kinase inhibitors to various different targets are fairly well developed and rapid translation of our results into the clinic is a real prospect from such screens. Results from HT-RNAi screening of kinases identified seventeen specific siRNAs (corresponding to sixteen genes) that lead to reduced growth and proliferation of Ewings sarcoma cells. We showed that two kinases, STK10 and TNK2, are important in survival of Ewings sarcoma cells and represent potential therapeutic targets for future drug development in this disease. Materials and methods Cell Culture The human Ewings sarcoma cell lines TC-32 and TC71 were a kind gift from Dr. Javed Khan (Pediatric Oncology Branch, National Cancer Institute, Gaithersburg, MD). The Ewings sarcoma cell lines RD-ES and SK-ES-1 were obtained from ATCC (Manassas, VA). The human normal fibroblast cell line GM05659 was obtained from the Coriell Institute (Camden, NJ). TC32, TC-71, and RD-ES cell lines were grown in RPMI, supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin G, and 100 g/ml streptomycin. SK-ES1 cells were grown in McCoys 5A media supplemented with 15% FBS, 2 mM L-glutamine, 100 IU/ml penicillin G, and 100 g/ml streptomycin. The normal human fibroblast cell line GM05659 was grown in Minimum Essentials Media (MEM) with 10% FBS and 2 mM Lglutamine, 100 IU/ml penicillin G, and 100 g/ml streptomycin. All media reagents were obtained from Invitrogen (Carlsbad, CA). The cell lines were routinely maintained at 37C in a humidified 5% CO2 atmosphere. Reagents The validated kinase siRNA library version 1.0 was obtained from Qiagen (Valencia, CA). Short interfering RNAs targeting TNK2, STK10, PLK1 and non-silencing control were (...truncated)