Genome-wide CRISPR screens reveal APR-246 (Eprenetapopt) triggers ferroptosis and inhibits iron-sulfur cluster biogenesis

• Published in: bioRxiv
• Main Authors: Fujihara, Kenji M, Zhang, Bonnie, Jackson, Thomas D, Nijiagel, Brunda, Ching-Seng Ang, Nikolic, Iva, Sutton, Viv, Trapani, Joe, Simpson, Kaylene J, Stojanovski, Diana, Leimuehler, Silke, Haupt, Sue, Phillips, Wayne A, Clemons, Nicholas J
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 29.11.2020
• Subjects: Antioxidants; Apoptosis; Biosynthesis; Cancer; Cell death; CRISPR; Cysteine; Ferroptosis; Genomes; Glycine; Iron; Metabolism; Metabolites; Mitochondria; Mutants; NADH; Oxidative stress; p53 Protein; Proteomics; Sulfur; Tumors
• DOI: 10.1101/2020.11.29.398867

ABSTRACT The mechanisms by which cells respond and adapt to oxidative stress are largely unknown but are key to developing a rationale for cancer therapies that target antioxidant pathways. APR-246 is a mutant-p53 targeted therapeutic currently under clinical investigation in myeloid dysplastic syndrome (MDS) and acute myeloid leukemia1. Whilst the mechanism of action of APR-246 is thought to be reactivation of wild-type p53 activity through covalent modification of cysteine residues in the core domain of mutant-p53 protein2,3, here we report that the anti-neoplastic capacity of APR-246 lies predominantly in the conjugation of free cysteine. Genome-wide CRISPR perturbation screening, metabolite profiling and proteomics in response to APR-246 treatment in mutant-p53 cancer cells highlighted the role of GSH and mitochondrial metabolism in determining APR-246 efficacy. APR-246 sensitivity was increased through loss of key enzymes in mitochondrial one-carbon metabolism, SHMT2 and MTHFD1L, due to diminished glycine supply for de novo GSH synthesis. Critically, we show that APR-246 induces iron-dependent, apoptotic machinery-independent cell death, ferroptosis. Whole-cell proteomics analyses indicated an upregulation of proteins involved in iron-sulfur cluster biogenesis (eg. FDX1). GSH, acetyl-CoA and NADH levels were also depleted in APR-246 treated cells. Importantly, we found that APR-246 inhibits iron-sulfur cluster biogenesis in the mitochondria of cancer cells through cysteine conjugation. This work not only details novel determinants of APR-246 activity in cancer cells, but also provides a clinical roadmap for targeting antioxidant pathways in tumours - beyond targeting mutant-p53 tumours. Figure1 Figure1 * Download figure * Open in new tab Competing Interest Statement The authors have declared no competing interest. Footnotes * Declaration: The authors disclose that there are no conflicts of interest in the work that contributed towards this manuscript.