The cysteine-reactive covalent RNF4 ligand CCW16 induces ferroptosis in AML cells by activation of ROS signaling

The SUMO-targeted E3 ubiquitin ligase RNF4 plays an important role in safeguarding genome and proteome integrity. RNF4 recognizes polySUMO modified proteins and induces their proteolytic or non-proteolytic ubiquitylation. Given the key function of RNF4 in maintaining proteome and genome stability, we explored its role in cancer cell survival and as a potential cancer drug target. We found that RNF4 is overexpressed in acute myeloid leukemia and high expression levels correlate with poor survival of patients. Depletion of RNF4 exerts antiproliferative effects in AML cells and increased their sensitivity towards antileukemic drugs thus validating RNF4 as a potential vulnerability in AML. To develop PROTACs for targeted degradation of RNF4, we aimed to exploit the recently described cysteine-reactive RNF4 ligand CCW16, and synthesized a set of CCW16-derived degrader molecules using established VHL- and CRBN-E3 ligands. We validated covalent binding of CCW16 and CCW16-derived PROTACs to cysteine residues outside the RING domain of RNF4 using recombinant protein. However, none of the synthesized PROTACs were active in degrading RNF4 in a panel of different cell lines. Consistent with the high reactivity of the chosen electrophile, we detected in the cellular context covalent attachment of CCW16 to accessible cysteines in a large number of proteins including covalent linkage with the catalytic residues in members of the peroxiredoxin family. Furthermore, exposure of cells to CCW16-derived PROTACs induced upregulation of heme oxygenase 1, a ferroptosis marker and impaired cell viability in a distinct, RNF4-independent, ferroptotic cell death pathway.