Abstract 4973: MYCN mediates cysteine addiction and sensitizes to ferroptosis in cancer cells
Authors: Westermann Frank , Alborzinia Hamed , Gogolin Sina , Flórez Andrés F. , Brückner Lena M. , Gartlgruber Moritz , Hartlieb Sabine , Dreidax Daniel , Nadler-Holly Michal , Ziehm Matthias , Shao Chunxuan , Selbach Matthias , Stresemann Carlo , Poschet Gernot , Nicke Barbara , Wölfl Stefan , Henrich Kai O. , Höfer Thomas 
Published: August 2018

Abstract
Aberrant expression of MYC family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. The purpose of the study was to analyze the interplay of oncogenic MYCN or c-MYC, referred to here as MYC(N), activity with cysteine metabolism and ferroptosis, an oxidative, non-apoptotic, and iron dependent form of regulated cell death caused by ROS-mediated massive lipid peroxidation (L-ROS), using MYC(N)-driven childhood neuroblastoma as a model.The intracellular amino acid levels at MYC(N)-high and MYC(N)-low cellular states were analyzed by HPLC. Effects on cell viability upon depletion of individual amino acids from the growth medium was tested in various cancer cell lines with regulable MYC(N). An unbiased high-throughput MYCN synthetic lethal siRNA screen was used to identify genes preferentially acting in the 'MYC(N)-high' state and protecting cells from ROS accumulation and ferroptosis. The capacity of cyst(e)ine uptake, intracellular cysteine synthesis via transsulfuration and glutathione biosynthesis was assessed in various neuroblastoma cell lines and tissues using metabolome, RNAseq, ChiP-seq and global proteome analyses. To investigate L-ROS formation at various conditions cells were stained with the lipid peroxidation sensor, C11-BODIPY, and flow cytometrically analyzed. Ferroptosis inducers (FINs) and inhibitors of transsulfuration were used to test their activity in various MYC(N)-dependent neuroblastoma cell lines and in vivo xenografts.We found that intracellular cysteine depletion in a 'MYC(N)-high' context induces cell death by ferroptosis and identified multiple points in glutathione synthesis and metabolism, particularly detoxification of L-ROS, that are vulnerable in the 'MYC(N)-high' state as compared to the 'MYC(N)-low' context. We could show that ferroptosis was dependent on MYC(N) expression and was enhanced by iron. We further demonstrated that both cystine import and intracellular cysteine synthesis via transulfuration achieved the intracellular state supportive of oncogenic MYC(N)-driven growth without endangering the cell to ferroptosis. We demonstrated the MYC(N) drives increased transsulfuration activity, rather than cysteine import, in tumor cells to maintain the cellular cysteine supply for glutathione synthesis. Our findings together with new descriptions of the ferroptotic process establish a novel functional link between oncogenic MYC(N) and ferroptosis, and imply regulation by cysteine-dependent glutathione availability. In MYCN-amplified childhood neuroblastoma, MYCN mediates resistance to ferroptosis by activating transsulfuration of methionine to cysteine. We identified enzymes and antiporter proteins crucial to ferroptotic escape, providing multiple previously unknown sites that may be acted on therapeutically.
Citation Format: Frank Westermann, Hamed Alborzinia, Sina Gogolin, Andrés F. Flórez, Lena M. Brückner, Moritz Gartlgruber, Sabine Hartlieb, Daniel Dreidax, Michal Nadler-Holly, Matthias Ziehm, Chunxuan Shao, Matthias Selbach, Carlo Stresemann, Gernot Poschet, Barbara Nicke, Stefan Wölfl, Kai O. Henrich, Thomas Höfer. MYCN mediates cysteine addiction and sensitizes to ferroptosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4973.