Deciphering the Mechanisms of Action of Altertoxin II in Ewing Sarcoma

Abstract

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are characterized by chromosomal translocations that result in expression of an oncogenic fusion protein (typically EWSR1-FLI1). Despite advances in cure rates for many childhood malignancies, advanced, metastatic or relapsed ES remain a challenge. We identified the fungal metabolite altertoxin II (ATXII) as having highly selective activity against six ES cell lines compared to a panel of pediatric and adult cancer cells. On average, ATXII is 94-fold (range 16- to 400-fold) more potent against ES cells compared to rhabdomyosarcoma (RMS) cells, indicating high selectivity for ES. Mechanism of action studies indicated that ATXII selectively induces DNA damage in ES cells. However, the high degree of selectivity for ES suggests other mechanisms may be involved. 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. Based on these data, we further investigated the mechanism of action of ATXII to identify which cellular pathways confer resistance. A genome-wide CRISPR-Cas9 knockout screen was conducted in resistant Rh30 cells using the GeCKOv2 library. Cells were treated for three weeks with ATXII or vehicle and genomic DNA was isolated every 4 days. Next-generation sequencing and bioinformatic analysis identified multiple differentially expressed sgRNAs in ATXII-treated Rh30 cells compared to vehicle-treated cells, suggesting loss of these genes influences growth in the presence of ATXII. Ongoing studies are aimed at confirming the influence of these genes on resistance to ATXII with additional gene knockdown experiments. Additionally, mass spectrometry-based thermal proteome profiling was conducted on ATXII-treated ES cell lysates and identified potential protein binding partners of ATXII. Overall, these studies will identify mechanisms of resistance to ATXII and ultimately identify new molecular targets for the development of ES therapies.