Abstract 1950: The eIF4A inhibitors didesmethylrocaglamide and rocaglamide as effective treatments for pediatric bone and soft-tissue sarcomas
Authors: Chang Long-Sheng , Oblinger Janet L. , Agarwal Garima , Wilson Tyler A. , Roberts Ryan , Fuchs James , O'Keefe Barry R. , Kinghorn A. Douglas , Collins Jerry M. 
Published: September 2020

Abstract
Background: Development of an effective medical therapy for osteosarcoma and Ewing sarcoma, the two most common aggressive forms of pediatric bone and soft-tissue sarcomas, is urgently needed as current treatments are inadequate particularly for those with recurrent or metastatic diseases. To achieve this goal, we have identified two natural eIF4A inhibitors, rocaglamide (Roc) and didesmethylrocaglamide (DDR), which potently inhibit proliferation of a series of commonly-used osteosarcoma and Ewing sarcoma cell lines. We also demonstrated that Roc effectively suppresses tumor growth in patient-derived xenograft (PDX) models of osteosarcoma and Ewing sarcoma. Both Roc- and DDR-treated sarcoma cells show decreased levels of multiple oncogenic kinases, including insulin-like growth factor-1 receptor (IGF-1R). Importantly, these rocaglamides are not sensitive to multidrug resistance 1 (MDR1) efflux. In addition, Roc exhibits 50% oral bioavailability, is well-tolerated in mice, and does not induce pulmonary toxicity in dogs as found with another eIF4A inhibitor silvestrol (Chang et al. Mol Cancer Ther. 2019; In Revision).
Methods: Various osteosarcoma and Ewing sarcoma cell lines were treated with DDR and Roc, followed by cell proliferation assay, flow cytometry, and immunoblotting. Mesenchymal stem cells (MSCs) were used as a control for comparing the expression levels of the eIF4F components important for translation initiation. RNA interference (RNAi) and CRISPR/Cas9 techniques were used to verify eIF4A dependency. An orthotopic cell line-derived xenograft (CDX), a metastatic xenograft, and a PDX models for osteosarcoma and immunohistochemistry were used to assess antitumor efficacy.
Results: We hypothesize that osteosarcoma and Ewing sarcoma cells are highly addicted to active protein synthesis and that Roc and DDR, by acting as potent eIF4A inhibitors with an ability to induce apoptosis and the DNA damage response, effectively eliminate these malignant sarcomas. We found that various osteosarcoma and Ewing sarcoma cell lines expressed higher levels of eIF4A2, but not eIF4A1, eIF4E, and eIF4G, than MSCs, suggesting a role of eIF4A2 in enhancing protein translation in these sarcoma cells. RNAi knockdown and CRISPR/Cas9 knockout experiments are being evaluated to verify this finding. Both DDR and Roc effectively blocked tumor growth in an orthotopic CDX model. As found with Roc previously, DDR also potently inhibited the growth of an osteosarcoma PDX model. Both rocaglamides were well tolerated at 3 mg/kg by IP every other day and did not cause any significant changes in body weight, compared with vehicle-treated controls. We are presently determining the effects of Roc and DDR on osteosarcoma metastasis.
Conclusions: The potent anti-tumor activity and favorable toxicity profile of Roc and DDR suggest that these rocaglamides should be further evaluated as potential treatments for osteosarcomas and Ewing sarcomas.
Citation Format: Long-Sheng Chang, Janet L. Oblinger, Garima Agarwal, Tyler A. Wilson, Ryan Roberts, James Fuchs, Barry R. O'Keefe, A. Douglas Kinghorn, Jerry M. Collins. The eIF4A inhibitors didesmethylrocaglamide and rocaglamide as effective treatments for pediatric bone and soft-tissue sarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1950.