Mechanistic studies of altertoxin II: A fungal metabolite with selective activity against Ewing sarcoma

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are driven by chromosomal translocations that most frequently result in expression of the abnormal EWSR1-FLI1 fusion protein, a transcriptional activator and modulator of RNA splicing, or similar fusion proteins. Despite significant advances in therapies for pediatric hematological malignancies over the past 30 years, there has been less progress in the development of curative therapies for advanced or metastatic pediatric solid tumors. Approximately 35% of ES are resistant to standard-of-care chemotherapeutic agents, and nearly 70% of ES patients with metastatic disease at the time of diagnosis die within 5 years. Therefore, there is a need to develop new, targeted therapies for ES that improve patient survival and decrease toxicity. We performed high-content phenotypic screens of natural product libraries to identify compounds with selective actions against pediatric solid tumor types and found that the fungal metabolite altertoxin II (ATXII) has highly selective cytotoxic activity against 6 ES cells lines compared to a panel of other pediatric and adult cancer cell lines. On average, the IC50 of ATXII is 94-fold higher (range 16- to 400-fold) in rhabdomyosarcoma (RMS) cells than in ES cells, indicating high selectivity for ES. Mechanistic studies were conducted to determine this compound’s mechanisms of actions in ES cells, with the goal of identifying new molecular targets for the treatment of ES. We found that 100 nM ATXII, a concentration that has no effect on RMS cell viability, induces cell cycle accumulation in ES cells in the S and G2/M phases, and rapid phosphorylation of histone H2A.X (gamma-H2A.X) and checkpoint kinase 1, suggesting ATXII selectively induces cell cycle arrest and DNA damage in ES cells. To determine if ES sensitivity to ATXII is a dominant or recessive phenotype, we hybridized sensitive EW8 cells (ES) with resistant Rh30 cells (RMS) and determined the sensitivity of these hybrids to ATXII. Five single-cell subclones derived from these hybrids were resistant to ATXII, suggesting that sensitivity to ATXII is a recessive phenotype in ES cells. Ongoing studies are aimed at determining the cellular uptake and binding kinetics of radiolabeled ATXII and identifying genes that mediate sensitivity and resistance to this compound by performing a genome-wide CRISPR/Cas9 knockout screen. Citation Format: Andrew J. Robles, Wentao Dai, Robert H. Cichewicz, Susan L. Mooberry, Peter J. Houghton. Mechanistic studies of altertoxin II: A fungal metabolite with selective activity against Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2141.