Abstract
Simple SummaryEwing sarcoma is a cancer of the bone and soft tissues that affects children and adolescents. Unfortunately, only 20–30% of patients with metastatic Ewing sarcoma survive, necessitating the need to identify new, more effective therapies. We screened natural product extracts from plants and fungal cultures to identify compounds with selective cytotoxic activity against Ewing sarcoma cells, which led to the identification of altertoxin II as a compound with highly selective activity against Ewing sarcoma cells. Mechanism of action studies showed that altertoxin II selectively induces DNA damage in Ewing sarcoma cells, but does not bind to DNA. Additionally, we found that altertoxin II has antitumor activity in a mouse model of Ewing sarcoma, suggesting it will be useful as a lead compound to help identify new molecular targets for the development of new Ewing-sarcoma-specific therapies.A screening program designed to identify natural products with selective cytotoxic effects against cell lines representing different types of pediatric solid tumors led to the identification of altertoxin II as a highly potent and selective cytotoxin against Ewing sarcoma cell lines. Altertoxin II, but not the related compounds altertoxin I and alteichin, was highly effective against every Ewing sarcoma cell line tested, with an average 25-fold selectivity for these cells as compared to cells representing other pediatric and adult cancers. Mechanism of action studies revealed that altertoxin II causes DNA double-strand breaks, a rapid DNA damage response, and cell cycle accumulation in the S phase. Our studies also demonstrate that the potent effects of altertoxin II are partially dependent on the progression through the cell cycle, because the G1 arrest initiated by a CDK4/6 inhibitor decreased antiproliferative potency more than 10 times. Importantly, the cell-type-selective DNA-damaging effects of altertoxin II in Ewing sarcoma cells occur independently of its ability to bind directly to DNA. Ultimately, we found that altertoxin II has a dose-dependent in vivo antitumor efficacy against a Ewing sarcoma xenograft, suggesting that it has potential as a therapeutic drug lead and will be useful to identify novel targets for Ewing-sarcoma-specific therapies.
Highlights
Ewing sarcoma (ES) is an aggressive bone and soft tissue cancer affecting children, adolescents, and young adults
Concurrent with the bioassay-guided fractionation, LC-MS analysis was performed on the biologically active fraction, alerting us to the presence of two co-eluting metabolites. Based on their estimated molecular weights and photodiode array data, the metabolites were suspected of being putative analogues of altertoxin II (ATXII)
We evaluated the ES-selectivity of a panel of other DNA damage-inducing agents. including gemcitabine, etoposide, SN38, melphalan, and the PARP1 inhibitor olaparib, but none of these agents show the degree of selectivity for ES cells that we see with ATXII (Supplementary Figure S1)
Summary
Ewing sarcoma (ES) is an aggressive bone and soft tissue cancer affecting children, adolescents, and young adults. This disease is most often caused by a chromosomal translocation leading to the expression of an abnormal fusion protein that is commonly designated as EWS-FLI1 [1,2]. Based on the knowledge that ES tumors are caused by the expression of the EWS-FLI1 fusion protein, the possibility exists for the identification of targeted fusionprotein-dependent therapies that could achieve high levels of efficacy against ES tumors while avoiding toxicity to other tissues that do not express the abnormal fusion protein. The identification of a selective inhibitor of EWS-FLI1 has not yet been successful, in part because EWS-FLI1 is a highly disordered protein [4]
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