update on cancer therapeutics 3 (2008) 13–26 available at www.sciencedirect.com journal homepage: www.updateoncancer.com Topoisomerase II inhibitors Kenneth R. Hande ∗ 777 Preston Research Building, Vanderbilt University Medical Center, Nashville, TN 37232, United States article info abstract Keywords: Topoisomerase II is an enzyme essential for DNA replication, chromosome condensation Topoisomerase II and chromosome segregation. Inhibitors of topoisomerase II are important drugs used Chemotherapy in the therapy of many neoplasms including breast cancer, lung cancer, testicular can- Etoposide cer, lymphomas and sarcomas. This paper reviews the mechanism of action, toxicities, Anthracyclines pharmacology and clinical use of topoisomerase II inhibitors including etoposide, tenipo- Pharmacology side, doxorubicin, daunorubicin, epirubicin, idarubicin and mitoxantrone. New information Chemotherapy regarding these agents and on topoisomerase II inhibitors under development is highlighted. Published by Elsevier Ltd. 1. Introduction in DNA to modulate DNA topology [4]. After strand passage, the DNA backbone is religated and DNA structure restored. DNA topoisomerases are nuclear enzymes that make tran- Etoposide prevents topoisomerase II from religating cleaved sient strand breaks in DNA to allow a cell to manipulate its DNA [5]. Etoposide thus converts topoisomerase II into a poi- topology [1,2]. Topoisomerase I makes single-strand breaks. son that introduces high levels of transient protein-associated Topoisomerase II makes double-strand breaks and passes breaks in the genome of treated cells. double-stranded DNA through the nick to allow relaxation Topoisomerase II exists as two highly homologous iso- of over-coiled DNA [3]. Topoisomerases are highly conserved forms, alpha and beta, which differ in their production during enzymes essential for the survival of all eukaryotic organisms. the cell cycle. The alpha isoform concentration increases 2–3- There is little sequence homology between topoisomerase I fold during G2/M, and orders of magnitude is higher in rapidly and II (Table 1). Topoisomerases function in DNA replication, proliferating cells than in quiescent cell populations. The chromosome condensation, and chromosome segregation. alpha isoform appears to be the target of etoposide [6]. The Several currently approved chemotherapeutic drugs interfere beta enzyme does not change significantly during the cell with the action of topoisomerases. Currently available topoi- cycle and could potentially be a target in slow growing can- somerase I inhibitors are irinotecan (CPT-11) and topotecan. cers. Two scissile bonds are formed per every topoisomerase FDA-approved topoisomerase II inhibitors are etoposide, teni- II-mediated double-stranded DNA break. Results of DNA cleav- poside, doxorubicin, idarubicin, epirubicin, and mitoxantrone. age and ligation assay studies indicate a two-site model for This chapter will review critical concepts and update new the action of etoposide against human topoisomerase II alpha. information regarding topoisomerase II inhibitors. This model suggests that drug interactions at both scissile bonds are required in order to increase enzyme-mediated 2. Epipodophyllotoxins double-stranded DNA breaks [7]. There does not appear to be a single DNA binding site 2.1. Etoposide for etoposide-topoisomerase II targeted breaks. However, selected hot spots may be present for DNA binding (see 2.1.1. Mechanism of action sections on drug toxicity). The cell-signaling pathways that Topoisomerase II is a multi-subunit enzyme which uses ATP to lead to apoptosis following topoisomerase-induced DNA pass an intact helix through a transient double-stranded break damage are not completely understood. Current research ∗ Tel.: +1 615 322 4967; fax: +1 615 343 7602. E-mail address: [email protected]. 1872-115X/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.uct.2008.02.001 14 update on cancer therapeutics 3 (2008) 13–26 the anal canal have been associated with etoposide use Table1–DNAtopoisomerases [22]. Topoisomerase I Topoisomerase II Hypersensitivity reactions, including vasomotor changes 100 kDa 170 kDa; 180 kDa in the pulmonary and gastrointestinal systems, may also Makes single-strand Makes double-strand DNA occur following etoposide (or teniposide) use. These reactions DNA breaks breaks may result from the Tween 80 needed to solubilize etopo- ATP independent ATP dependent side. These reactions can usually be ameliorated with steroids, Genes located on Gene located on chromosomes histamine blockade, and/or using a slower infusion rate [23]. chromosome 20q12 17q21 and 3p24 Two types, alpha and beta Etoposide phosphate, a water-soluble pro-drug that is rapidly converted to etoposide by endogenous phosphatases [24],may reduce the risk of a hypersensitivity reaction since no solubi- is attempting to elucidate the mechanisms involved [8]. lizer is required. Etoposide phosphate has been safely used in Caspases are a group of cysteine proteases that orchestrate patients who have had a hypersensitivity reaction to etoposide apoptosis. Robertson et al. have identified caspase 2 as an [25]. Etoposide phosphate appears to have kinetics similar to important link between etoposide-induced DNA damage and etoposide, even in the transplant setting [26]. the engagement of the mitochondrial apoptotic pathway The most serious adverse event associated with etopo- [9]. Caspase 2 activates caspase 8 resulting in mitochondrial side is the development of acute myelogenous leukemia damage and subsequent downstream caspase 9 and 3 activa- [27,28]. On the basis of current clinical evidence, the World tion [10]. Caspase 3 appears critical for apoptosis-associated Health Organization has identified etoposide as carcinogenic chromatin margination, DNA fragmentation, and nuclear to humans [29]. Therapy-related acute myelogenous leukemia collapse. Cells lacking caspase 3 are resistant to etoposide also occurs with other topoisomerase II inhibitors [30,31]. Vari- [11]. Caspase 10 appears to trigger a feedback amplification ous studies have shown that topoisomerase II inhibitors target loop that amplifies caspases 9 and 3 [12]. Tumor necrosis selected binding sites at translocation breakpoints leading factor-related apoptosis inducing ligand (TRAIL) augments to MLL, AML1-ETO, PML-RARA and NUP98 rearrangements the expression of caspases induced by etoposide [13,14]. [32,33]. Etoposide-related leukemia develops relatively early Other cell-cycle control proteins are also important medi- after therapy (2–3 years). Most often (70% of cases), etoposide- ators in etoposide-induced apoptosis. P53, c-Myc and BAFF related leukemia is distinguished by a balanced translocation have been identified as pathways utilized to arrest cell cycle involving the mixed-lineage leukemia (MLL) gene on chromo- progression and induce apoptosis in certain cell lines exposed some 11. In vitro exposure of mouse embryonic stem cells to to etoposide [15]. Etoposide activates two pathways which etoposide results in MLL fusions [34]. The MLL gene is more lead to G2M arrest, one which depends on the presence of sensitive to topoisomerase II induced cleavage than other P53 while the other is P53 independent [16]. The presence genes such as RUNX1 and MLLT3 [35,36]. Lovett et al. has found of bcr-abl, which prolongs G2M arrest and allows for DNA that not only etoposide, but also its metabolites (etoposide repair mechanisms, decreases the cytotoxicity of etoposide. quinone and etoposide catechol) enhance DNA topoisomerase Cells with dysfunctional early G2/M checkpoint control (such II cleavage near the MLL translocation breakpoints [37]. MLL as ataxia-telangiectasia mutated deficient fibroblasts) have rearrangements occur through cleavage events in MLL and the increased chromosomal abnormalities following etoposide translocated gene in which both breaks become stable, DNA exposure [17]. ends are processed and then undergo ligation [38]. The double- Resistance to etoposide arises through multiple mecha- stranded DNA breaks in the MLL gene may not be directly nisms. Mutations at ser-1106 in the topoisomerase II molecule linked to topoisomerase II exposure. Hars et al. [39] suggest abrogate phosphorylation of the enzyme and reduce sensi- that the DNA breaks generated in the MLL locus are the result tivity to etoposide [18]. Rapid repair of DNA breaks caused of caspase activation of DNase by etoposide. MLL breakpoints by etoposide can also lead to drug resistance. Repair occurs can occur in stem cells found in cord blood and in fetal hepatic through the single-strand invasion pathway of homologous hematopoietic stem cells, potentially explaining the develop- recombination or by non-homologous DNA end-joining [19]. ment of infant leukemia resulting from in utero exposure to The repair of potentially lethal DNA damage by etoposide topoisomerase II inhibitors [40]. appears to be dependent on functioning FLT3 [20]. Resis- The incidence of secondary leukemia from etoposide use tance to etoposide is noted in cells that have the multidrug and the factors increasing the risk of leukemia have varied resistance (MDR) phenotype. By modifying the structure of from study to study. With rare exceptions, the risk for devel- etoposide to make it a prodrug, etoposide analogs have been opment of acute myeloid leukemia (AML) does not exceed 5% developed which are active, in vitro, against MDR resistant in patients treated for solid tumors, even with high cumula- tumors [21]. tive doses of topoisomerase