Abstract LB180: Non-cardiotoxic properties of annamycin, a clinically evaluated anthracycline and potent topoisomerase 2β poison

Abstract

Abstract Introduction. Cardiotoxicity is an important side effect limiting the clinical use of all anthracyclines, including doxorubicin (DOX). DOX targets topoisomerase II alpha (Top2α) and beta (Top2β), two enzymes that regulate DNA topology. Top2β is present in cardiomyocytes and has been proposed as a major contributor to DOX-induced cardiotoxicity. Annamycin (ANN), a novel clinically evaluated DOX analog, displays suppressed or no cardiotoxic properties in preclinical in vivo experiments. The objective of this study was to directly assess and compare the potency of DOX and ANN against Top2α and Top2β and determine their impact on physiology of human cardiomyocytes and mice hearts following chronic exposure. Our initial data are clearly aligned with lack of drug-related cardiotoxic events in ANN-treated patients in ongoing clinical trials. Methods. The potency of ANN and DOX to inhibit Top2 was tested in DNA relaxation assays using recombinant Top2α and Top2β with kinetoplast as the DNA substrate. ANN and DOX cytotoxicity was assessed in a panel of cancer cell lines cardiomyocytes (murine and human). In addition, the effects on cardiomyocyte physiology (beating rate, contraction, electric potential,) were assessed in human cardiomyocytes using the xCELLigence RTCA CardioECR. The pathophysiology of the heart after chronic exposure to the drugs was also evaluated in mice models. Cardiotoxicity of liposome formulated ANN (L-ANN) is currently being examined in ongoing clinical studies of subjects with acute myeloid leukemia (NCT05319587) or soft tissue sarcoma with metastases to the lungs (NCT04887298). Results. The nanomolar range potency of ANN in tested cancer cell lines was 2-10 times greater than that of DOX. The DNA relaxation assay demonstrated high potency of ANN against recombinant Top2α and Topβ with IC50 values 0.23±0.06 and 0.22± 0.06 µM, respectively, whereas the IC50 for DOX was 0.65± 0.22 and 0.34± 0.31µM, respectively. Importantly, ANN did not significantly affect electric potential, contractility, and viability of human cardiomyocytes up to highest tested dose of 1,500 nM (72h exposure). In contrast, DOX caused a dramatic change in heart beating rate and decreased in contractility and field potential amplitude. In rat cardiomyocyte culture (H92C), ANN was 14-fold less cytotoxic than DOX (IC50 1,264 nM for ANN vs. 88 nM for DOX). No cardiotoxicity of L-ANN was detected in myocardium of nude mice exposed weekly for 12 weeks to therapeutic doses of L-ANN (4 mg/kg, 40% of LD10). Finally, data from ongoing clinical trials revealed no drug related cardiac events in patients receiving L-ANN (confirmed by an independent review of the Duke’s ECHO core laboratory, USA). Conclusions. Our preclinical studies demonstrate non-cardiotoxic properties of ANN. These data are clearly aligned with lack of drug-related cardiotoxic events in ANN-treated patients in ongoing clinical trials. Interestingly, ANN appeared to be a significantly more potent inhibitor of Top2β than DOX, suggesting that Top2β may not be a universal mediator of cardiotoxicity of anthracyclines. Additional studies are needed to reevaluate the role of Top2β in anthracycline-induced cardiotoxicity. Citation Format: Rafal J. Zielinski, Krzysztof Grela, Roberto Cardenas-Zuniga, Stanislaw Skora, Izabela Fokt, Mihai Gagea, Wolfram Dempke, Waldemar Priebe. Non-cardiotoxic properties of annamycin, a clinically evaluated anthracycline and potent topoisomerase 2β poison [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB180.