Abstract 5206: Quantifying DNA-protein crosslinks induced by bifunctional alkylating agents in cultured human leukemia cells

Abstract

Abstract Bifunctional alkylating agents, such as cisplatin and several nitrosoureas, have clinical utility as antineoplastic drugs largely due to their DNA alkylating and crosslinking activity. Other drug candidates such as laromustine (VNP-40101M) and dianhydrogalacticol (VAL-083) have similar DNA-alkylating activities and have shown success in clinical trials against hard-to-treat cancers such as acute myelogenous leukemia and glioblastoma multiforme. While these agents' interstrand and instrastrand DNA crosslinking activities are widely cited as the critical cytotoxic event, their capacity to crosslink DNA to chromosomally-affiliated proteins may also contribute to their anticancer profile. Indeed, DNA-protein crosslinks (DPCs) are highly toxic phenomena that are difficult for cells to repair and severely compromise gene transcription and DNA replication. Herein, we assess concentration-dependent DPC formation by a wide variety of bifunctional alkylating agents in HL-60 human leukemia cells using two parallel approaches. In one approach, protein-bound DNA was quantified using PicoGreen after precipitating proteins from lysates of drug-treated cells and compared to total DNA concentration. In another, chromosomal DNA was extracted from lysates of drug-treated cells then treated with fluorescein isothiocyanate, which modifies primary amines on proteins. Protein concentration was measured by fluorescence and compared to that of the extracted DNA. The nitrogen mustard mechlorethamine and the chloroethyl nitrosoureas carmustine and lomustine were the most potent agents, yielding significant DPC formation at clinically relevant doses (10−5 M). Cisplatin and laromustine yielded DPCs at similar concentrations, but to a lesser extent. Dianyhydrogalacticol, epichlorohydrin, and diepoxybutane, which all rely on epoxide functional groups for attachment to cellular nucleophiles, required at least an order of magnitude higher drug concentrations for significant DPC observation. These data suggest bifunctional alkylating agents featuring halide leaving groups are most apt to crosslink DNA to nuclear proteins and may offer insights into patterns of cytotoxicity and clinical utility for this important class of chemotherapeuric agents. Citation Format: Hailey L. Cerrato, Sam W. Marchant, Allie H. Naccara, Thomas J. LaJoie, Shawnya Michaels, Dennis Brown, Kevin P. Rice. Quantifying DNA-protein crosslinks induced by bifunctional alkylating agents in cultured human leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5206.