Combination therapy targeting the EWS/FLI-1 oncogene with antisense oligonucleotides and rapamycin induces apoptosis of Ewing’s sarcoma cells in vitro and inhibits tumor growth in vivo

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Tumors of the Ewing’s sarcoma family (ESFT) are common bone and soft-tissue malignancies in adolescents and young adults. Chromosomal translocation t(11;22)(q24;q12), which is detected in aproximately 90% of ESFT, generates the EWS/FLI-1 chimeric gene fusion that encodes EWS/FLI-1, a fusion protein acting as an aberrant transcriptional activator. Despite aggressive treatment strategies, such as high-dose chemotherapy in combination with surgical an/or extended radiotherapy, the prognosis for ESFT patients with large primary tumors or metastatic disease remains quite poor. Therefore, targeting of the EWS/FLI-1 protein may represent an efficient approach for the treatment of ESFT. We previously demonstrated that the mammalian Target of Rapamycin (mTOR) signaling pathway plays a central role in EWS cell pathobiology. We demonstrated that rapamycin, a mTOR inhibitor, efficiently blocked the proliferation of EWS cell lines carrying EWS/FLI-1, by promoting G₁ cell cycle arrest. The use of antisense oligodeoxynucleotides (AS-ODN) to selectively modulate the expression of individual genes, has become a promising area for cancer therapeutics. Previously, we described that administration of an AS-ODN designed against the breakpoint of EWS/FLI-1 together with the mTOR inhibitor rapamycin induced apoptotic cell death of EWS cells in vitro. In the present study we demonstrate the molecular mechanisms underlying this apoptotic process. We observed that, although the levels of Bax and Bcl-2 were not altered, the Bcl-xl levels were downregulated as a consequence of the combined treatment. The levels of the Inhibitors of Apoptosis (IAP) XIAP and Survivin were also downregulated. Executioner caspase-7 was shown to be involved. Moreover, in vivo experiments demonstrated complete inhibition of tumor xenograft growth in mice. These data provide proof of principle for the inclusion of this combined therapy as a novel strategy for the treatment of ESFT.