Abstract 379: Cysteine-driven drug inactivation undermines covalent drug efficacy and drives resistance.

Abstract Background: Constitutive activation of ATF4 drives metabolic rewiring in cancer, enhancing tumorigenesis and therapy resistance. A key consequence is upregulation of the cystine/glutamate antiporter SLC7A11, leading to increased cystine import and excessive intracellular cysteine. Cyclin-dependent kinase 7 (CDK7) inhibitors, such as YKL-5-124, are emerging anticancer agents that target proteins essential for cell proliferation. These inhibitors can be subdivided into covalent and noncovalent classes. Covalent drugs, such as YKL-5-124, act via electrophilic warheads that irreversibly react with nucleophilic amino acids, whereas noncovalent drugs, such as SY-5609, inhibit through intramolecular interactions at the protein’s active site. We hypothesized that excess intracellular nucleophiles, particularly cysteine, can sequester electrophilic drugs, reducing target engagement, and promoting resistance. Methods: We quantified the efficacy of CDK7 inhibitors in pancreatic ductal adenocarcinoma (PDAC) cell lines upon treatment conditions that alter cysteine availability. Cell growth was quantified using Sulforhodamine B (SRB) assay following drug exposure. For studies of drug-thiol reactivity, both CDK7 inhibitors were preincubated with freshly prepared cysteine (10:1 ratio, thiol:drug) in degassed PBS on ice for 1 hour, then applied to cells. For LC-MS analysis, polar metabolites were extracted from cells with 80% methanol normalized to total cell volume. Drug metabolites were measured by untargeted LC-MS using a Q-Exactive HF-X Orbitrap. Results: Pancreatic ductal adenocarcinoma (PDAC) cell lines with high SLC7A11 expression were resistant to the covalent CDK7 inhibitor YKL-5-124 compared to their low-SLC7A11 counterparts. Pharmacological inhibition of SLC7A11 with erastin sensitized high-SLC7A11 cells to YKL-5-124, whereas cotreatment of low-SLC7A11 cells with N-acetylcysteine, a cysteine prodrug, induced resistance. Sensitivity to SY-5609, a noncovalent CDK7 inhibitor, was unaffected by cysteine availability. These results suggested that cysteine may be directly acting upon covalent CDK7 inhibitors to prevent their efficacy. Indeed, pre-incubation with cysteine drastically decreased the efficacy of YKL-5-124, while SY-5609 was unaffected. LC-MS analysis detected YKL-cysteine conjugates in vitro and in cells grown under high-cystine conditions, supporting direct nucleophile-drug adduct formation. Conclusion: Elevated intracellular cysteine can chemically sequester electrophilic drugs and drive resistance. Ongoing studies aim to determine if cysteine accumulation alone suffices to confer resistance to covalent CDK7 inhibition. These results highlight cysteine metabolism as a therapeutically actionable vulnerability, potentially guiding drug selection and combination regimens for patients receiving covalent inhibitors. Citation Format: Maximilian Kobiesa, Jennifer A. Brain, Kelli J. Che, Leah G. Rector, Abby C. Jurasin, Lucas B. Sullivan. Cysteine-driven drug inactivation undermines covalent drug efficacy and drives resistance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 379.