Abstract PR002: Identification of metabolic adaptation mechanisms that drive anoikis suppression and metastasis in Ewing sarcoma
Authors: Zhang Hai-Feng , Hughes Christopher S. , Delaidelli Alberto , Huang Yue Zhou , Shyp Taras , Yang Xiaqiu , Sorensen Poul H. 
Published: January 2023

Metastasizing cancer cells must overcome anoikis (detachment-induced death) prior to colonization in distant organs. Ewing sarcoma (EwS) is a highly aggressive bone and soft tissue cancer that mainly occurs in children, adolescents, and young adults. EwS patients with metastatic disease have a 5-year survival rate of only 15-20%, which has not changed for decades. Our recent study identified that EwS cells are highly dependent on augmented cysteine metabolism and glutathione biogenesis in anoikis suppression and metastasis through the IL1RAP protein. Mechanistically, IL1RAP binds the cell surface system Xc- transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and glutathione antioxidant pools. Moreover, under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Furthermore, we show that inhibitors of Glutaminase (GLS) and Glutamate cysteine ligase (GCL), two critical enzymes for glutamate and glutathione synthesis, synergistically induce massive anoikis and ferroptosis in EwS cells. However, as a single agent, GLSi or GCLi only had moderate effects on EwS survival. Our global proteomic analysis identified dramatic adaptive changes in response to GLSi and GCLi, which may facilitate EwS cell survival and metastasis upon blockade of these metabolic processes. To further pinpoint critical regulators of anoikis and metastasis, we found that anokis suppression is governed by EWS-FLI1, a driver oncogene in EwS, and depletion of EWS-FLI1 triggered anoikis and led to cell death of EwS tumor spheroids formed in 3D cultures, whereas only a mild cytostatic effect was induced in 2D cultures. Given the predominant role of oncogenic EWS-FLI1 in anoikis suppression, we performed proteomic analysis in EwS cells +/- EWS-FLI1 depletion or gene rescue cultured under either 2D or 3D conditions prior to anoikis onset. We found that the 2D-to-3D transition induced marked global proteomic changes, suggesting a molecular reprogramming in response to 3D-induced anoikis stress that might be crucial for anoikis suppression. Moreover, a significant subset of these adaptive proteomic changes was driven by EWS-FLI1. Among these proteins, we validated that CDH11 is highly expressed in EwS compared with &gt;1000 other human cancer cell lines, and localized on the EwS cell surface, which might be targeted via immunotherapeutic strategies. Thus, these studies provide insights into adaptive mechanisms upon perturbation of glutathione metabolism and oncogenes that drive anoikis suppression and metastasis in EwS.

Citation Format: Hai-Feng Zhang, Christopher S. Hughes, Alberto Delaidelli, Yue Zhou Huang, Taras Shyp, Xiaqiu Yang, Poul H. Sorensen. Identification of metabolic adaptation mechanisms that drive anoikis suppression and metastasis in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR002.