Background: Ewing sarcoma (EwS) is an aggressive pediatric tumor. The 5-year survival rate for localized disease has increased to 70-75%, attributed to an intensified use of multi-agent cytotoxic chemotherapies. However, severe toxicity and high rate of life-threatening late events are associated with such intensive chemotherapy. In addition, therapy amelioration in patients with metastatic or relapsed disease, whose overall survival is only 20-30%, is an unmet medical need. Metformin has long been perceived to be an attractive therapeutic option for EwS, but hypoxia limits its efficacy as EwS cells were reported to adapt to hypoxia by redefining their transcriptome and acquiring a distinct hypoxic phenotype. We hypothesize that another drug can be combined with metformin to reduce viability of EwS cells and bring forth in vivo efficacy of metformin. Methods: We assembled a pool of fifty-seven anti-EwS drugs reported in the literature and Clinicaltrial.gov and composed a customized drug library in a 96-well plate for drug screening to evaluate synergistic combinations under hypoxia. RNA-seq and reverse-phase protein array were generated and analyzed to delineate the underlying signaling pathways for synergistic combinations founded, followed by xenograft experiments to determine in vivo efficacy and mechanism. Results: Our drug combination screening results indicated the anti-proliferation effect of metformin was significantly enhanced by seven drugs. In particular, imatinib stands out as most potent and has a strong synergy with metformin in inhibiting cell growth on several EwS cell lines, as well as on clinically relevant plasma concentrations. Furthermore, the combination significantly enhances in vitro cell inhibition efficacy of two standard chemotherapeutics drugs, cyclophosphamide and ifosfamide, indicating a potential benefit of lower dosage and better outcomes. In vivo studies on two EwS xenograft mouse models confirmed superior anti-tumor efficacy of the metformin-imatinib combination. Functional evaluation revealed imatinib strikingly reversed the hypoxia-specific transcriptional signature in EwS cells while the drug combination notably induced cell cycle arrest and apoptosis. The drug combination regulated a network of sixty-one signaling molecules and inhibited the activity of PI3K-AKT-mTOR signaling pathway. These genes regulated by drug combination highly correlates with poor clinical outcome of reported clinical data of EwS and sarcoma. Conclusions: The novel drug combination identified revives the potential of metformin in treating EwS by using another known drug, imatinib, to attenuate tumor hypoxic response through a systematic integration of drug combination screening, bioinformatics studies, functional and in vivo validations, and clinical outcome correlation. The combination may also apply to localized disease to reduce adverse side effects of intensive cytotoxic chemotherapy.
Citation Format: Xiang Nan, Jiang Wang, Jianting Sheng, Ching C. Lau, Jason T. Yustein, Hong Zhao, Stephen Wong. Imatinib revives the therapeutics potential of metformin in Ewing sarcoma by attenuating tumor hypoxic response and inhibiting convergent signaling pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A140. doi:10.1158/1535-7163.TARG-19-A140