Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIβ has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIβ selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and β) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.

Biosciences Institute Faculty of Medical Sciences Newcastle University Newcastle upon Tyne NE2 4HH UK

Department of Biochemical Sciences Faculty of Pharmacy in Hradec Kralove Charles University Hradec Kralove 500 05 Czech Republic

Department of Organic and Bioorganic chemistry Faculty of Pharmacy in Hradec Kralove Charles University Hradec Kralove 500 05 Czech Republic

Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Faculty of Pharmacy in Hradec Kralove Charles University Hradec Kralove 500 05 Czech Republic

Department of Pharmacology Faculty of Medicine in Hradec Kralove Charles University Hradec Kralove 500 03 Czech Republic

National Center of Hematology Mustansiriyah University Baghdad Baghdad Governorate 79R2 RXM Iraq

See more in PubMed

Alousi A. M., Boinpally R., Wiegand R., Parchment R., Gadgeel S., Heilbrun L. K., Wozniak A. J., DeLuca P., LoRusso P. M. (2007). A phase 1 trial of XK469: Toxicity profile of a selective topoisomerase II beta inhibitor. Invest. New Drugs. 25, 147–154. PubMed

Austin C. A., Cowell I. G., Khazeem M. M., Lok D., Ng H. T. (2021). TOP2B’s contributions to transcription. Biochem. Soc. Trans. 49, 2483–2493. PubMed

Azarova A. M., Lin R. K., Tsai Y. C., Liu L. F., Lin C. P., Lyu Y. L. (2010). Genistein induces topoisomerase IIbeta- and proteasome-mediated DNA sequence rearrangements: Implications in infant leukemia. Biochem. Biophys. Res. Commun. 399, 66–71. PubMed PMC

Castrogiovanni C., Waterschoot B., De Backer O., Dumont P. (2018). Serine 392 phosphorylation modulates p53 mitochondrial translocation and transcription-independent apoptosis. Cell Death Differ. 25, 190–203. PubMed PMC

Chou T. C., Talalay P. (1984). Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27–55. PubMed

Corremans R., Adao R., De Keulenaer G. W., Leite-Moreira A. F., Bras-Silva C. (2019). Update on pathophysiology and preventive strategies of anthracycline-induced cardiotoxicity. Clin. Exp. Pharmacol. Physiol. 46, 204–215. PubMed

Cowell I. G., Tilby M. J., Austin C. A. (2011). An overview of the visualisation and quantitation of low and high MW DNA adducts using the trapped in agarose DNA immunostaining (TARDIS) assay. Mutagenesis  26, 253–260. PubMed

Deng S., Yan T., Nikolova T., Fuhrmann D., Nemecek A., Godtel-Armbrust U., Kaina B., Wojnowski L. (2015). The catalytic topoisomerase II inhibitor dexrazoxane induces DNA breaks, ATF3 and the DNA damage response in cancer cells. Br. J. Pharmacol. 172, 2246–2257. PubMed PMC

Dewilde S., Carroll K., Nivelle E., Sawyer J. (2020). Evaluation of the cost-effectiveness of dexrazoxane for the prevention of anthracycline-related cardiotoxicity in children with sarcoma and haematologic malignancies: A European perspective. Cost Eff. Resour. Alloc. 18, 7. PubMed PMC

Earhart R. H., Tutsch K. D., Koeller J. M., Rodriguez R., Robins H. I., Vogel C. L., Davis H. L., Tormey D. C. (1982). Pharmacokinetics of (+)-1,2-di(3,5-dioxopiperazin-1-yl)propane intravenous infusions in adult cancer patients. Cancer Res. 42, 5255–5261. PubMed

European Medicines Agency. (2017). Questions and Answers on Cardioxane (Dexrazoxane, Powder for Solution for Injection, 500 mg). Available at: https://www.ema.europa.eu/en/medicines/human/referrals/dexrazoxane. Accessed January 30, 2024.

Gao H., Huang K. C., Yamasaki E. F., Chan K. K., Chohan L., Snapka R. M. (1999). XK469, a selective topoisomerase IIbeta poison. Proc. Nat. Acad. Sci. U.S.A. 96, 12168–12173. PubMed PMC

Getz K. D., Sung L., Alonzo T. A., Leger K. J., Gerbing R. B., Pollard J. A., Cooper T., Kolb E. A., Gamis A. S., Ky B., et al. (2020). Effect of dexrazoxane on left ventricular systolic function and treatment outcomes in patients with acute myeloid leukemia: A report from the children’s oncology group. J. Clin. Oncol. 38, 2398–2406. PubMed PMC

Gewirtz D. A. (1999). A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol. 57, 727–741. PubMed

Hasinoff B. B. (2002). Dexrazoxane (ICRF-187) protects cardiac myocytes against hypoxia-reoxygenation damage. Cardiovasc. Toxicol. 2, 111–118. PubMed

Hazeldine S. T., Polin L., Kushner J., Paluch J., White K., Edelstein M., Palomino E., Corbett T. H., Horwitz J. P. (2001). Design, synthesis, and biological evaluation of analogues of the antitumor agent, 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469). J. Med. Chem. 44, 1758–1776. PubMed

Hazeldine S. T., Polin L., Kushner J., White K., Bouregeois N. M., Crantz B., Palomino E., Corbett T. H., Horwitz J. P. (2002). Synthesis and biological evaluation of some bioisosteres and congeners of the antitumor agent, 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469). J. Med. Chem. 45, 3130–3137. PubMed

Henriksen P. A. (2018). Anthracycline cardiotoxicity: An update on mechanisms, monitoring and prevention. Heart  104, 971–977. PubMed

Herman E. H., Hasinoff B. B., Steiner R., Lipshultz S. E. (2014). A review of the preclinical development of dexrazoxane. Prog. Pediat. Cardiol. 36, 33–38.

Jasra S., Anampa J. (2018). Anthracycline use for early stage breast cancer in the modern era: A review. Curr. Treat. Options Oncol. 19, 30. PubMed

Jirkovská-Vávrová A., Roh J., Lenčová-Popelová O., Jirkovský E., Hrušková K., Potůčková-Macková E., Jansová H., Hašková P., Martinková P., Eisner T., et al. (2015). Synthesis and analysis of novel analogues of dexrazoxane and its open-ring hydrolysis product for protection against anthracycline cardiotoxicity in vitro and in vivo. Toxicol. Res. 4, 1098–1114.

Jirkovska A., Karabanovich G., Kubes J., Skalicka V., Melnikova I., Korabecny J., Kucera T., Jirkovsky E., Novakova L., Piskackova H. B., et al. (2021). Structure-activity relationship study of dexrazoxane analogues reveals ICRF-193 as the most potent bisdioxopiperazine against anthracycline toxicity to cardiomyocytes due to its strong topoisomerase II beta interactions. J. Med. Chem. 64, 3997–4019. PubMed

Jirkovsky E., Jirkovska A., Bavlovic-Piskackova H., Skalicka V., Pokorna Z., Karabanovich G., Kollarova-Brazdova P., Kubes J., Lencova-Popelova O., Mazurova Y., et al. (2021). Clinically translatable prevention of anthracycline cardiotoxicity by dexrazoxane is mediated by topoisomerase II beta and not metal chelation. Circ. Heart Fail. 14, e008209. PubMed

Jirkovský E., Lenčová-Popelová O., Hroch M., Adamcová M., Mazurová Y., Vávrová J., Mičuda S., Šimůnek T., Geršl V., Štěrba M. (2013). Early and delayed cardioprotective intervention with dexrazoxane each show different potential for prevention of chronic anthracycline cardiotoxicity in rabbits. Toxicology  311, 191–204. PubMed

Khazeem M. M., Casement J. W., Schlossmacher G., Kenneth N. S., Sumbung N. K., Chan J. Y. T., McGow J. F., Cowell I. G., Austin C. A. (2022). TOP2B is required to maintain the adrenergic neural phenotype and for ATRA-induced differentiation of SH-SY5Y neuroblastoma cells. Mol. Neurobiol. 59, 5987–6008. PubMed PMC

Khazeem M. M., Cowell I. G., Harkin L. F., Casement J. W., Austin C. A. (2020). Transcription of carbonyl reductase 1 is regulated by DNA topoisomerase II beta. FEBS Lett. 594, 3395–3405. PubMed

Kim H., Kang H. J., Park K. D., Koh K.-N., Im H. J., Seo J. J., Lee J. W., Chung N.-G., Cho B., Kim H. K., et al. (2019). Risk factor analysis for secondary malignancy in dexrazoxane-treated pediatric cancer patients. Cancer Res. Treat. 51, 357–367. PubMed PMC

Kollarova-Brazdova P., Jirkovska A., Karabanovich G., Pokorna Z., Piskackova H. B., Jirkovsky E., Kubes J., Lencova-Popelova O., Mazurova Y., Adamcova M., et al. (2020). Investigation of structure-activity relationships of dexrazoxane analogs reveals topoisomerase II beta interaction as a prerequisite for effective protection against anthracycline cardiotoxicity. J. Pharmacol. Exp. Ther. 373, 402–415. PubMed

Kollarova-Brazdova P., Lencova-Popelova O., Karabanovich G., Kocurova-Lengvarska J., Kubes J., Vanova N., Mazurova Y., Adamcova M., Jirkovska A., Holeckova M., et al. (2021). Prodrug of ICRF-193 provides promising protective effects against chronic anthracycline cardiotoxicity in a rabbit model in vivo. Clin. Sci. (Lond)  135, 1897–1914. PubMed

Leger K., Slone T., Lemler M., Leonard D., Cochran C., Bowman W. P., Bashore L., Winick N. (2015). Subclinical cardiotoxicity in childhood cancer survivors exposed to very low dose anthracycline therapy. Pediatr. Blood Cancer. 62, 123–127. PubMed

Lu Y., Xu D., Zhou J., Ma Y., Jiang Y., Zeng W., Dai W. (2013). Differential responses to genotoxic agents between induced pluripotent stem cells and tumor cell lines. J. Hematol. Oncol. 6, 71. PubMed PMC

Lyu Y. L., Kerrigan J. E., Lin C. P., Azarova A. M., Tsai Y. C., Ban Y., Liu L. F. (2007). Topoisomerase IIbeta mediated DNA double-strand breaks: Implications in doxorubicin cardiotoxicity and prevention by dexrazoxane. Cancer Res. 67, 8839–8846. PubMed

Macleod K. F., Sherry N., Hannon G., Beach D., Tokino T., Kinzler K., Vogelstein B., Jacks T. (1995). P53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 9, 935–944. PubMed

Mensah-Osman E. J., Al-Katib A. M., Dandashi M. H., Mohammad R. M. (2002). 2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469) inhibition of topoisomerase II beta is not sufficient for therapeutic response in human Waldenstrom’s macroglobulinemia xenograft model. Mol. Cancer Ther. 1, 1315–1320. PubMed

Millan-Zambrano G., Burton A., Bannister A. J., Schneider R. (2022). Histone post-translational modifications—Cause and consequence of genome function. Nat. Rev. Genet. 23, 563–580. PubMed

Nitiss J. L. (2009). Targeting DNA topoisomerase II in cancer chemotherapy. Nat. Rev. Cancer  9, 338–350. PubMed PMC

Olive P. L., Banath J. P. (2006). The comet assay: A method to measure DNA damage in individual cells. Nat. Protoc. 1, 23–29. PubMed

Pommier Y., Nussenzweig A., Takeda S., Austin C. (2022). Human topoisomerases and their roles in genome stability and organization. Nat. Rev. Mol. Cell Biol. 23, 407–427. PubMed PMC

Reichardt P., Tabone M. D., Mora J., Morland B., Jones R. L. (2018). Risk-benefit of dexrazoxane for preventing anthracycline-related cardiotoxicity: Re-evaluating the European labeling. Future Oncol. 14, 2663–2676. PubMed

Roca J., Ishida R., Berger J. M., Andoh T., Wang J. C. (1994). Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp. Proc. Nat. Acad. Sci. U.S.A. 91, 1781–1785. PubMed PMC

Shapiro T. A., Klein V. A., Englund P. T. (1999). Isolation of kinetoplast DNA. Methods Mol. Biol. 94, 61–67. PubMed

Shatzkes K., Teferedegne B., Murata H. (2014). A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR. Sci. Rep. 4, 4659. PubMed PMC

Schroeder P. E., Jensen P. B., Sehested M., Hofland K. F., Langer S. W., Hasinoff B. B. (2003). Metabolism of dexrazoxane (ICRF-187) used as a rescue agent in cancer patients treated with high-dose etoposide. Cancer Chemother. Pharmacol. 52, 167–174. PubMed

Simůnek T., Klimtová I., Kaplanová J., Mazurová Y., Adamcová M., Sterba M., Hrdina R., Gersl V. (2004). Rabbit model for in vivo study of anthracycline-induced heart failure and for the evaluation of protective agents. Eur. J. Heart Fail. 6, 377–387. PubMed

Snapka R. M., Gao H., Grabowski D. R., Brill D., Chan K. K., Li L., Li G. C., Ganapathi R. (2001). Cytotoxic mechanism of XK469: Resistance of topoisomerase IIbeta knockout cells and inhibition of topoisomerase I. Biochem. Biophys. Res. Commun. 280, 1155–1160. PubMed

Stock W., Undevia S. D., Bivins C., Ravandi F., Odenike O., Faderl S., Rich E., Borthakur G., Godley L., Verstovsek S., et al. (2008). A phase I and pharmacokinetic study of XK469R (NSC 698215), a quinoxaline phenoxypropionic acid derivative, in patients with refractory acute leukemia. Invest. New Drugs. 26, 331–338. PubMed PMC

Subramanian B., Nakeff A., Media J., Wentland M., Valeriote F. (2002). Cellular drug action profile paradigm applied to XK469. J. Exp. Ther. Oncol. 2, 253–263. PubMed

Sung H., Ferlay J., Siegel R. L., Laversanne M., Soerjomataram I., Jemal A., Bray F. (2021). Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 71, 209–249. PubMed

Tebbi C. K., London W. B., Friedman D., Villaluna D., De Alarcon P. A., Constine L. S., Mendenhall N. P., Sposto R., Chauvenet A., Schwartz C. L. (2007). Dexrazoxane-associated risk for acute myeloid leukemia/myelodysplastic syndrome and other secondary malignancies in pediatric Hodgkin’s disease. J. Clin. Oncol. 25, 493–500. PubMed

Teuffel O., Leibundgut K., Lehrnbecher T., Alonzo T. A., Beyene J., Sung L. (2013). Anthracyclines during induction therapy in acute myeloid leukaemia: A systematic review and meta-analysis. Br. J. Haematol. 161, 192–203. PubMed

Uhlen M., Fagerberg L., Hallstrom B. M., Lindskog C., Oksvold P., Mardinoglu A., Sivertsson A., Kampf C., Sjostedt E., Asplund A., et al. (2015). Proteomics. Tissue-based map of the human proteome. Science  347, 1260419. PubMed

Undevia S. D., Innocenti F., Ramirez J., House L., Desai A. A., Skoog L. A., Singh D. A., Karrison T., Kindler H. L., Ratain M. J. (2008). A phase I and pharmacokinetic study of the quinoxaline antitumour agent R(+)XK469 in patients with advanced solid tumours. Eur. J. Cancer  44, 1684–1692. PubMed PMC

Vogel C. L., Gorowski E., Davila E., Eisenberger M., Kosinski J., Agarwal R. P., Savaraj N. (1987). Phase I clinical trial and pharmacokinetics of weekly ICRF-187 (NSC 169780) infusion in patients with solid tumors. Invest. New Drugs. 5, 187–198. PubMed

Wolf D., Rotter V. (1985). Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proc. Natl. Acad. Sci. U.S.A. 82, 790–794. PubMed PMC

Zhang A., Lyu Y. L., Lin C. P., Zhou N., Azarova A. M., Wood L. M., Liu L. F. (2006). A protease pathway for the repair of topoisomerase II-DNA covalent complexes. J. Biol. Chem. 281, 35997–36003. PubMed

Zhang S., Liu X., Bawa-Khalfe T., Lu L. S., Lyu Y. L., Liu L. F., Yeh E. T. (2012). Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat. Med. 18, 1639–1642. PubMed

Topobexin targets the Topoisomerase II ATPase domain for beta isoform-selective inhibition and anthracycline cardioprotection