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. 2017 Jan 17;8(3):4079-4095.
doi: 10.18632/oncotarget.13385.

In silico and in vitro drug screening identifies new therapeutic approaches for Ewing sarcoma

Ziyan Y Pessetto  1 , Bin Chen  2 , Hani Alturkmani  1 , Stephen Hyter  1 , Colleen A Flynn  3 , Michael Baltezor  3 , Yan Ma  1 , Howard G Rosenthal  4 , Kathleen A Neville  5 , Scott J Weir  4   6   7 , Atul J Butte  2 , Andrew K Godwin  1   4
Affiliations

In silico and in vitro drug screening identifies new therapeutic approaches for Ewing sarcoma

Ziyan Y Pessetto et al. Oncotarget. .

Abstract

The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease.

Keywords: Ewing sarcoma; auranofin; drug repurposing; ganetespib; high-throughput screening.

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Conflict of interest statement

CONFLICTS OF INTEREST

All authors claim there is no conflicts of interest.

Figures

Figure 1
Figure 1. In silico prediction of drugs for EWS using three approaches
(A) Identify drugs that are likely to reverse disease gene expression (disease-based approach). A disease gene expression signature was created from the results of two meta-analysis studies [75, 76], where disease tissue samples and normal tissue samples were compared. (B) Identify drugs that mediate gene expression similar to siRNA EWS/FLI1 (siEWS/FLI1) (siRNA-based approach). The siEWS/FLI signature was taken from the previous study [77]. (C) Identify drugs that are likely to sensitize drug resistance expression (resistance-based approach). A drug resistance expression signature was computed by comparing the pre-treatment samples of patients who responded to chemotherapy versus those who did not respond to chemotherapy [78]. Drug gene expression databases were built from the CMap 2.0 and part of the LINCS. In the plot, each dot represents the score of one drug instance. One drug may have multiple instances due to multiple experiments. False discovery rate (FDR) value < 0.05 was used to select drug hits and only the top 20 drug hits for each approach were selected. In the heat map, the first column represents the disease gene expression ranked by fold change and the remaining columns represent the gene expression of drug hits. Red shows up-regulated genes and green shows down-regulated genes. All the drug hits from the three approaches were merged and manually evaluated. The drugs colored by black were selected for further experimental validation.
Figure 2
Figure 2. Combination analysis for auranofin and ganetespib in six EWS cell lines and three control (Hs 822.T., Hs 863.T., and Hs 919.T.) cell lines
Inhibitory concentration values for individual auranofin or ganetespib as well as drug combinations are shown. (A) Color scale for drug inhibition values. (B–J) Synergy between auranofin and ganetespib was tested by CellTiter-Glo assay at 64 different drug combinations for each cell line. (K) Color scale for Combination Index (CI) values. (L–Q) CI values for auranofin and ganetespib combination treatment for six EWS cell lines. CI value of > 1 indicates antagonism effects; CI = 1 indicates additive effects; CI value of < 0.9 indicates synergy effects; and CI value of < 0.5 indicates strong synergy effects. (Negative inhibition values cannot be used to calculate CI values).
Figure 3
Figure 3. The sensitivity of EWS cells to auranofin is dependent on expression of the EWS-FLI 1 oncogene
(A) Representative western blot data shows the significant decrease in the expression of EWS-FLI1 in A673 cells 48h post-transfection. (B) Cell viability was assessed by the CellTiter Blue assay in A673 cells treated by siControl, siFLI1-#3 or siBPEF1 for 48 h then followed by the incubation of auranofin ranging from 7.8 nmol/L to 1 μmol/L for 72h. Data are presented as the mean ± SD of three independent experiments performed in triplicate. (C) In vivo studies of auranofin, ganetespib and the combination treatment in an EWS xenograft mice model (n = 12 per group). The percentage of survival rate in control, auranofin treated, ganetespib treated, and auranofin in combination with ganetespib treated groups.
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