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

Fujihara, K. M.; Zhang, B. Z.; Jackson, T. D.; Nijagal, B.; Ang, C.-S. M.; Nikolic, I.; Sutton, V.; Trapani, J.; Simpson, K. J.; Stojanovski, D.; Leimkuehler, S.; Haupt, S.; Phillips, W. A.; Clemons, N. J.

doi:10.1101/2020.11.29.398867

bioRxiv · 10.1101/2020.11.29.398867

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

Fujihara, K. M.·Zhang, B. Z.·Jackson, T. D.·Nijagal, B.·Ang, C.-S. M.·Nikolic, I.·Sutton, V.·Trapani, J.·Simpson, K. J.·Stojanovski, D.·Leimkuehler, S.·Haupt, S.·Phillips, W. A.·Clemons, N. J.

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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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/398867v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@11155f8org.highwire.dtl.DTLVardef@1231cb7org.highwire.dtl.DTLVardef@324881org.highwire.dtl.DTLVardef@136146f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Fujihara, K. M., Zhang, B. Z., Jackson, T. D., Nijagal, B., Ang, C.-S. M., Nikolic, I., Sutton, V., Trapani, J., Simpson, K. J., Stojanovski, D., Leimkuehler, S., Haupt, S., Phillips, W. A., Clemons, N. J.. 2020-11-29. Genome-wide CRISPR screens reveal APR-246 (Eprenetapopt) triggers ferroptosis and inhibits iron-sulfur cluster biogenesis. https://doi.org/10.1101/2020.11.29.398867

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