Work over the past year has focused on 3 major areas, IGFIR signaling, the identification of oncogene addiction pathways in rhabdomyosarcomas (RMS) using shRNA screening techniques, and screening drug and natural product libraries to identify inhibitors of the EWS-FLI-1 fusion protein in Ewings sarcoma. We have continued to study IGF signaling in pediatric sarcomas now focusing on trying to identify relevant biomarkers that would allow us to enrich patients entered onto clinical studies using IGFIR Abs. We have shown that RMS tumor specimens as well as cell lines have variable IGFIR levels, and that very low levels predict lack or response to IGFIR blockade, as expected. We are now working with the Childrens Oncology Group to develop quantitative IGFIR assays that might be used in clinical studies for enrichment strategies in clinical studies of IGFIR blockade. However, we still have not identified markers predictive of response, and work is ongoing in this area. Currently, in collaboration with Dr. Paul Meltzer and SARC, we are evaluating tumor specimens obtained for Ewings sarcoma patients who responded to IGFIR Ab therapy and specimens from those who did not respond. We are sequencing target genes thought to play a role in IGFIR signaling, as well as some selected tumor suppressor pathway genes. In preclinical studies, in collaboration with Dr. Liang Cao we have found that there may be some RMS tumor cells that use the IGFIR pathway for both proliferation as well as anti-apoptotic signaling, and these tumors are particularly sensitive to IGFIR Ab treatment. Ongoing studies in xenografts are attempting to model optimal timing and combinations of combination therapy of IGFIR Ab and mTOR inhibitors to pick an optimal way to test these combinations in the clinic. We have continued to mine our data from our high throughput shRNA screening to identify critical pathways for survival of human RMS cell lines. Using an inducible shRNA library containing specific barcodes for clone identification in collaboration with Dr. Lou Staudt, we screened an alveolar and an embryonal RMS cell line to identify specific RNAs that when knocked-down with shRNA would lead to growth arrest. We have identified a number of candidate genes that appear to be critical for survival of these tumor cells. The first gene we have just completed our analysis of confirms that CrkL is required for RMS survival and tumor growth both in vitro and in vivo. We have most recently demonstrated that CrkL signaling in RMS is independent of PI3K-Akt signaling but appears to be via Src family kinase signaling, thus identifying a new potential critical signaling pathway for RMS. We are currently preparing a manuscript describing this finding. We have also identified Bub1b, a spindle assembly checkpoint gene, as critical for growth of RMS genes. We are currently studying this pathway in RMS to understand its importance in growth of these tumors. We have also used this screen to identify genes whose expression is necessary for survival only in the presence of the Pax-3-Foxo1 gene fusion found in alveolar RMS. We have identified TNK2, a cytoplasmic tyrosine kinase as necessary for survival of alveolar RMS and work is ongoing to study the mechanism of activity. An additional list of 12 other genes has been identified and we are confirming these with follow-up screens. We have developed and utilized a high-throughput screen to evaluate more than 50,000 compounds for inhibition of EWS-FLI1 activity in collaboration with Drs. Woldemichael and McMahon at the Molecular Targets laboratory. We used a cell based luciferase reporter screen utilizing the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen employing a novel list of more than 10 downstream targets to sort and prioritize the compounds. We identified a lead compound that appears to have specific activity against the EWS-FLI-1 transcription factor, have confirmed its activity in mouse models and hope to test it in the clinic within the next year.
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