# Cysteine-reactive covalent chloro-N-acetamide ligands induce ferroptosis mediated cell death

## TL;DR

A covalent ligand targeting RNF4 in AML cells unexpectedly causes ferroptosis, a type of cell death, rather than degrading the intended protein.

## Contribution

The discovery that chloro-N-acetamide ligands induce ferroptosis, revealing unintended toxicity in drug development.

## Key findings

- CCW16, a covalent RNF4 ligand, forms bonds with multiple proteins including peroxiredoxins.
- CCW16 induces ferroptosis and oxidative stress in an RNF4-independent manner.
- Chloro-N-acetamide ligands like EN219 also trigger ferroptosis, indicating a broader off-target effect.

## Abstract

Covalent inhibitors are an attractive targeting strategy that has expanded the development of degraders to target poorly druggable proteins including the E3 ligase RNF4. We show that RNF4 is a potential vulnerability of AML. High RNF4 expression levels correlate with poor patient survival and depletion of RNF4 results in increased sensitivity of AML cells to antileukemic drugs. Therefore, we aimed to develop chemical degraders (PROTACs) of RNF4 using a known covalent RNF4 ligand (CCW16), containing a chloro-N-acetamide group, as well as established E3 ligands targeting CRBN or VHL. However, while CCW16 and CCW16-derived PROTACs react potently with cysteines in recombinant RNF4, in cells, CCW16 forms covalent bonds with a large number of proteins, including peroxiredoxins. Consequently, CCW16 based PROTACs do not trigger degradation of RNF4, but induce the ferroptosis marker heme oxygenase-1 and impair cell viability in a distinct, RNF4-independent, ferroptotic cell death pathway. We hypothesize that other chloro-N-acetamide-containing E3 ligase ligands would also induce ferroptosis. Indeed, the RNF114 ligand EN219 also strongly induces ferroptosis, suggesting that ligands harboring this electrophile induce undesired off-target toxicity.

The E3 ubiquitin ligase RNF4 represents a dependency factor in AML cells. Development of an RNF4 degrader reveals that the published covalent ligand CCW16 binds to the unstructured region in RNF4, induces oxidative stress and RNF4-independent ferroptotic cell death.

RNF4 represents a vulnerability in AML cells.The covalent RNF4 ligand CCW16 broadly targets cysteine residues.CCW16 induces ferroptotic cell death in an RNF4-independent pathway.Chloro-N-acetamide containing covalent ligands induce ferroptosis.

RNF4 represents a vulnerability in AML cells.

The covalent RNF4 ligand CCW16 broadly targets cysteine residues.

CCW16 induces ferroptotic cell death in an RNF4-independent pathway.

Chloro-N-acetamide containing covalent ligands induce ferroptosis.

The E3 ubiquitin ligase RNF4 represents a dependency factor in AML cells. Development of an RNF4 degrader reveals that the published covalent ligand CCW16 binds to the unstructured region in RNF4, induces oxidative stress and RNF4-independent ferroptotic cell death.