Work over the past year has focused 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 clinical relevant as we now have onging clinical studies using IGFIR Ab treatment in the clinic. We have now clearly demonstrated and published data correlating responsiveness of rhabdomyosarcoma (RMS) cell lines and tumor xenografts to IGFIR levels and to the ability of the IGFIR Ab to rapidly decrease phospho-Akt levels. We found that there is a wide range of IGFIR density on cell lines and human tumors ranging from 3000 receptors/cell to over 40,000 receptors/cell. We believe that ability to prospectively measure these levels in the clinic may help select patients more likely to benefit from IGFIR Ab treatment. Most recently 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. This Akt reactivation is somewhat mitigated by simultaneous treatment with the IGFIR Ab and the mTOR inhibitor, rapamycin. These data were also recently published and suggest potential synergy. We plan to explore this in a clinical study currently being written. The rationale for mTOR inhibition of these tumors as a therapeutic approach has been strengthened by our recent analysis of primary RMS tumors using reverse-phase proteomics demonstrating that activation of the mTOR signaling pathway in these tumors is a poor prognostic factor. We are also performing additional xenograft studies trying to model optimal timing of combination mTOR/IGFIR inhibition and to identify mechanisms responsible for reactivation of Akt signaling despite continued suppression of IGFIR levels. We have continued to study the molecular mechanisms of ezrin signaling in pediatric sarcomas. We have previously linked ezrin to mTOR signaling, and current work has suggested that beta-4 integrin signaling is also required for metastatic functioning in a human osteosarcoma xenograft model. Furthermore, we find high beta-4 integrin expression in human osteosarcoma tumor samples. We have now 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. Finally, we have begun to use 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 completed our first screen and analysis and 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. Since CrkL is an adapter signaling molecule responsible for integrating various kinase signaling pathways, we are now working to identify the mechanisms whereby loss of CrkL leads to loss of tumor cell survival. We are currently preparing a manuscript describing this initial observation and plan to continue to mine this data in the future.
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