Abstract 7113: Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents

Abstract

Abstract Bifunctional alkylating agents, such as nitrosoureas, platinum-based therapeutics, and chloroethylhydrazines, have demonstrated clinical successes against cancer by inducing cytotoxic interstrand or intrastrand DNA crosslinks. However, the less well-characterized capacity of these agents to couple DNA to chromosomally-associated proteins may also contribute to these compounds’ observed cytotoxicities. Bulky DNA-protein crosslinks (DPCs) could obstruct the DNA metabolism critical to cancer proliferation, thus contribute to genomic instability and cell death. To endogenously repair DPCs, human cells often utilize a DPC-specific proteolytic mechanism dependent on the metalloprotease SPRTN. Complementary approaches for quantifying DPCs, generated ex vivo, to assess the capacity of several bifunctional alkylating agents to induce these lesions in cultured human leukemia cells are presented here. In the first approach, proteins were isolated from lysates of drug-treated cells and protein-bound DNA was quantified as a ratio to the total DNA present in the lysates. In the complementary approach, DNA was extracted from lysates of drug-treated cells using a modified ‘STAR’ assay in which genomic DNA is isolated and the DNA-bound proteins were quantified by fluorescein isothiocyanate labeling. Micromolar concentrations of the nitrogen mustard mechlorethamine and nitrosourea lomustine induced significant DPC formation while more modest DPC formation was observed using similar concentrations of the chloroethylating agents carmustine and laromustine, as well as cisplatin. Bifunctional alkylating agents dependent on epoxide functionality, including dianhydrogalacticol and diepoxybutane, required drug concentrations of at least one order of magnitude larger to produce similar DPC yields measurable by these techniques. A strategy to interfere with the DPC repair pathway via SPRTN knockdown and assess increased sensitivity of cancer cells to bifunctional alkylating agents is also presented. Chemotherapeutic sensitization by targeting SPRTN could suggest a therapeutic strategy to improve effectiveness of several DNA-targeted anticancer agents, especially if extended to other therapeutics that trap DPCs such as inhibitors of topoisomerase and poly(ADP-ribose) polymerase. Further, identifying structure-function relationships with respect to bifunctional alkylating agents and DPC formation could inform the future development and clinical utilization of these chemotherapies. Citation Format: Cole Turner, Hailey Cerrato, Victoria Melehov, Charlotte Healy, Kevin P. Rice. Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7113.