Background: Despite multi-modality therapy, the majority of patients with metastatic or recurrent Ewing sarcoma (ES), the second most common pediatric bone malignancy, will die of their disease. ES tumors express aberrantly activated ETS transcription factors through translocations that fuse the EWS gene to ETS family genes FLI1 or ERG. The aberrant activation of ETS transcription factors promotes malignant transformation and proliferation. While, FLI1 or ERG cannot be readily targeted, there is an opportunity to deploy functional genomics screens, to develop novel therapeutic approaches by identifying targetable liabilities in EWS/FLI1 dependent tumors. Materials and Methods: We performed a near whole-genome pooled shRNA screen in a panel of five EWS/FLI1 dependent Ewing sarcoma cell lines and one EWS/ERG cell line to identify essential genes. Essential genes were defined as those genes whose loss resulted in reduced viability selectively in ES cells compared to non-Ewing cancer cell lines. Essential hits were subsequently validated with genomic knockdown and chemical inhibition in vitro, followed by validation of the on-target effect of chemical inhibition. Next, we determined the in vivo effects of small-molecule inhibition on survival and tumor growth in NOD scid gamma (NSG) mice with established subcutaneous ES xenografts. Results: Top hits in our screen that could be readily targeted by small-molecule inhibitors, and thus have potential for rapid clinical validation, were selected for further investigation. These hits included IKBKE, CCND1 and CDK4. IKBKΕ, a non-canonical IKK with an oncogenic role in breast cancer, was one of the top kinase hits in the screen. IKBKΕ shares significant homology to TBK1, another non-canonical IKK that is essential in k-RAS dependent lung cancer. We validated IKBKE through small-molecule inhibition of IKBKE/TBK1 and shRNA based knockdown. Ewing sarcoma cell lines are sensitive to low micromolar concentrations of two IKBKE/TBK1 inhibitors (CYT387 and MRT67307). Additionally, in a panel of ES cell lines, knockdown of IKBKE resulted in decreased growth and impaired colony formation. These observations, paired with impairment of NF-κB nuclear localization following CYT387 treatment suggests that non-canonical IKK mediated signaling may be essential in Ewing sarcoma. We further validated these results through inhibition of IKBKE/TBK1 in in vivo xenograft models treated with 100 mg/kg/day of CYT387. Treatment over the course of twenty-nine days resulted in a significant increase in survival (p-value = 0.0231) and a significant decrease (p-value = 0.036) in tumor size after fifteen days of treatment. CDK4 and CCND1 are highly expressed in Ewing sarcoma as compared to other tumor types. shRNA mediated knockdown of CDK4 and CCND1 resulted in impaired viability and anchorage independent growth. Furthermore, treatment of Ewing sarcoma cell lines with a highly selective CDK4/6 inhibitor, LEE011, resulted in decreased viability (IC50 range of 0.26-18.06 μM), potent G1 arrest in six of eight EWS/FLI1 containing Ewing sarcoma lines tested and apoptosis in a panel of four highly sensitive lines. Administration of 75 mg/kg/day and 250 mg/kg/day of LEE011 in NSG mice with Ewing xenografts resulted in significant impairment of tumor growth, (p-value <0.001 for both treatment arms), as compared to vehicle control. Conclusions: These studies suggest a role for the targeting of IKBKE and CDK 4/6 in Ewing sarcoma, findings with immediate clinical relevance for patients with this malignancy, because small-molecule inhibitors of these proteins have already entered clinical trial for other disease indications.

Plusieurs molécules nouvelles ont été obtenues à partir du Gossypol, extrait des graines de cotonnier. Dans la première partie, de nouvelles bases de Schiff du gossypol et de la gossypolone ont été synthétisées, les énantiomères du gossypol et leurs bases de Schiff sont optiquement stables, tandis que, les énantiomères de la gossypolone ne sont pas stables à température ambiante, mais il est possible de les observer vers 0°C. La cytotoxicité de ces bases de Schiff a été évaluée principalement sur des cellules KB, la méthylimine et l'éthylimine de la gossypolone sont les plus toxiques (IC50= 0.8 et 1.2 µM). La toxicité du gossypol et de la gossypolone augmente quand les tests sont effectués en absence de sérum et elle diminue en présence de catalase ou de mannitol dans le milieu de culture. L'énantiomère (-)-gossypol est plus toxique que le (+)-gossypol, ceci est aussi valable pour les bases de Schiff des énantiomères du gossypol. Dans la deuxième partie, une nouvelle classe de dérivés du gossypol et de la gossypolone, les dithianes et les dithiolanes, a été développée. Les dithianes/dithiolanes du gossypol et de la gossypolone ont été synthétisés par action de dithiols en présence d'éthérate de trifluoroborate. La même réaction effectuée avec les monothiols, conduit à des mélanges complexes. L'action du propanedithiol ou de l'ethanedithiol sur le tetraméthyle ou l'hexaméthyle éthers du gossypol ne conduit pas aux dithianes ou dithiolanes attendus mais à de nouveaux dérivés cycliques. La toxicité de ces nouveaux thio-dérivés sur les cellules KB est assez modeste, mais sous l'action de NO ou d'un sel de nitrosonium, ces dérivés se transforment en dérivés plus toxiques dans le cas du gossypol ou régénèrent la molécule de départ dans le cas de la gossypolone. Nous formulons l'hypothèse que dithianes et dithiolanes du gossypol et de la gossypolone pourraient être des modèles de prodrogues ciblées sur les cellules exprimant de fortes concentrations d'oxyde nitrique.