Cellular Binding Kinetics of Altertoxin II: A Fungal Metabolite with Highly Selective Activity Against 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 fuse EWSR1 with FLI1 (~85% of ES) or another ETS transcription factor gene, resulting in expression of an oncogenic fusion protein (typically EWSR1‐FLI1). Despite significant advances over the past 40 years in therapy for pediatric hematological malignancies, less progress has been made with therapies for ES and other childhood solid tumors. Thus, there is a need to develop new and more efficacious therapies for ES that improve patient survival. We recently 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. To characterize the cellular uptake of ATXII and identify potential molecular binding partners, a tritiated sample of the compound was produced and evaluated in cellular assays. We observed significantly different kinetics for 3H‐ATXII accumulation between sensitive EW8 and resistant Rh30 cells. In EW8 cells, maximum 3H‐ATXII accumulation was observed five minutes after treatment, with cellular levels slowly decreasing over the next hour. In contrast, cellular abundance of 3H‐ATXII steadily increased in Rh30 cells for one hour following treatment, with final levels approximately 1.6‐fold higher than those in EW8 cells. These data suggest that ATXII rapidly binds and saturates its binding partner(s) in EW8, but not Rh30 cells. Additionally, these data indicate that the increased sensitivity of EW8 cells to ATXII is not simply due to greater drug accumulation. Differences in uptake kinetics were also observed between EW8 and Rh30 cells when cellular uptake was measured at 4°C. While we measured 2‐fold less uptake in Rh30 cells at 4°C compared to 37°C, uptake increased nearly 5‐fold in EW8 cells at 4°C. To determine whether uptake of ATXII is ATP dependent, we evaluated the uptake of 3H‐ATXII following treatment with the uncoupling agent carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP). While pretreatment with FCCP reduced 3H‐ATXII accumulation in Rh30 cells, we observed an increase in 3H‐ATXII accumulation in EW8 cells. Together, these data suggest that EW8 cells actively export ATXII, while uptake is a passive process. Ongoing studies are aimed at identifying the molecular binding partners of ATXII, and identifying genes that mediate resistance in RMS cells by performing a genome‐wide CRISPR/Cas9 knockout screen. Overall, these studies have the potential to identify new molecular targets for the development of ES therapies.Support or Funding InformationNCI R01 GM107490, PO1 CA165995This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.