Deciphering the Mechanisms of Action of Altertoxin II with Genome‐Wide CRISPR/Cas9 Knockout Screening

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. After selection with puromycin, the transduced cells were treated for three weeks with ATXII or vehicle and genomic DNA was isolated every 4 days. These samples were subsequently prepared for next‐generation sequencing to identify sgRNAs that are depleted in the ATXII‐treated samples compared to vehicle‐treated control samples. Sequencing of these samples has recently been completed and initial bioinformatic analysis was conducted. Preliminary results have identified multiple differentially expressed sgRNAs in ATII‐treated Rh30 cells compared to vehicle‐treated cells, suggesting loss of these genes influences growth in the presence of ATXII. Overall, these studies have the potential to identify new molecular targets for the development of ES therapies.Support or Funding InformationFunded by P01CA165995 (PJH), R01GM107490 (RHC, SLM), T32CA148724 (Cancer Biology Training Program) and RP170345 (CPRIT Training Program)