Work over the past year has continued to focus on 3 major areas, IGFIR signaling, the role of ezrin in metastatic behavior of pediatric sarcomas, and the identification of oncogene addiction pathways in rhabdomyosarcomas using shRNA screening techniques. We have continued to study IGF signaling in pediatric sarcomas and this is becoming increasingly more clinically relevant as we now have onging clinical studies using IGFIR Ab treatment in the clinic. Since our previous finding correlating IGFIR levels with sensitivity to IGFIR Ab treatment, we now have data to suggest that while low levels of IGFIR will predict lack of response, high IGFIR levels are not sufficient to predict reponse. In collaboration with Dr. Liang Cao we have also 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 tumor are particularly sensitive to IGFIR Ab treatment. We have also continued to analyze the potential interactions between IGFIR blockade and mTOR inhibition. We have shown and published data demonstrating that although IGFIR Ab treatment of RMS xenografts leads to a reduction of tumor growth, invariably tumors begin to re-grow after long-term exposure to Ab. Furthermore, we have shown that this re-growth is associated with a reactivation of Akt signaling in spite of continued suppression of IGFIR levels. 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 study the molecular mechanisms of ezrin signaling in pediatric sarcomas. We have now published our studies linking beta-4 integrin to metastatic behavior in osteosarcoma models. Furthermore, we find high beta-4 integrin expression in human osteosarcoma tumor samples. Finally we demonstrated that ezrin interacts with beta-4 integrin and this interaction is required for continued expression of beta-4 integrin in tumors thus linking ezrin, previously shown to be important for metastatic behavior of osteosarcoma, and beta-4 integrin, now shown to be critical for metastatic behavior. We have completed 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 kinase signaling, thus identifying a new potential critical signaling pathway for RMS. We are currently preparing a manuscript describing this finding. We are also beginning to evaluate additional hits from this shRNA screen. We are also using the same inducible shRNA approach to specifically regulate the expression of the PAX3-FKHR fusion gene in alveolar RMS. We have prepared the construct and plan to use this regulation to identify the critical downstream targets of this fusion transcription factor by expression profiling of alveolar RMS under native conditions and under conditions of PAX3-FKHR supressison via the shRNA.
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