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Posts Tagged ‘Hypoxia-inducible factor 1 (HIF-1)’


Topoisomerase 1 & II inhibitors and cancer therapy

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Topoisomerase 1 & II Inhibitors and Cancer Therapy

Julia MoukharskayaClaire Verschraegen,

Hematology/Oncology Clinics of North America  June 2012; Volume 26, Issue 3:507–525     doi:10.1016/j.hoc.2012.03.002

KEY POINTS

Topoisomerase 1 inhibitors cure human cancer xenografts in animal models, more so than most other chemotherapy agents.

However, their activity in patients with cancer is modest.

Ongoing research is studying the optimal analogs that could reproduce animal data in the cancer population.

This article analyzes the clinical research with topoisomerase 1 inhibitors in ovarian cancer.

The first type I topoisomerase (Top1) inhibitors were found in the wood bark of Camptotheca acuminata, an oriental tree, the powder or injectable extracts of which have been used in traditional Chinese medicine.1 The class was named camptothecin (CPT) for the basic CPT compound (Fig. 1). Clinical studies of this group of drugs were initiated in the 1970s, and in the 1980s the Top1 enzyme was identified as the cellular target of CPT.2,3 Topoisomerases relax the DNA supercoiling and perform catalytic functions during replication and transcription.4 There are two classes of topoisomerases. Type I enzymes cleave one strand of DNA and type II cleave both strands. Six topoisomerase genes have been identified in mammalian somatic cells within these two classes. Type IA enzymes consist of Top3 a and Top3 b; type IB consist of Top1 and Top1mt (mitochondrial); and type IIA consist of Top2 a and Top2 b. CPT is an inhibitor of Top1. Top1 cleaves the DNA phosphodiester backbone, nicking one strand of the DNA duplex and forming a Top1-DNA reversible cleavage complex by covalent bonding of a tyrosine residue. Single-strand breaks induced by Top1 help untangle excessive DNA supercoils during DNA replication and transcription (Fig. 2).5,6 Top1 is essential for survival.

 

Catalytic topoisomerase II inhibitors in cancer therapy

AK Larsen, AE Escargueil (Pierre and Marie Curie University – Paris), A Skladanowski

Pharmacology & Therapeutics  Aug 2003; 99(2):167-181      http://dx.doi.org:/10.1016/S0163-7258(03)00058-5

The nuclear enzyme DNA topoisomerase II is a major target for antineoplastic agents. All topoisomerase II-directed agents are able to interfere with at least one step of the catalytic cycle. Agents able to stabilize the covalent DNA topoisomerase II complex (also known as the cleavable complex) are traditionally called topoisomerase II poisons, while agents acting on any of the other steps in the catalytic cycle are called catalytic inhibitors. Thus, catalytic topoisomerase II inhibitors are a heterogeneous group of compounds that might interfere with the binding between DNA and topoisomerase II (aclarubicin and suramin), stabilize noncovalent DNA topoisomerase II complexes (merbarone, ICRF-187, and structurally related bisdioxopiperazine derivatives), or inhibit ATP binding (novobiocin). Some, such as fostriecin, may also have alternative biological targets. Whereas topoisomerase II poisons are used solely for their antitumor activities, catalytic inhibitors are utilized for a variety of reasons, including their activity as antineoplastic agents (aclarubicin and MST-16), cardioprotectors (ICRF-187), or modulators in order to increase the efficacy of other agents (suramin and novobiocin). In this review, the mechanism and biological activity of different catalytic inhibitors is described, with emphasis on therapeutically used compounds. We will then discuss future development and applications of this interesting class of compounds.

Fig. 1. The catalytic cycle of DNA topoisomerase II. The ATPase domains of topoisomerase II are shown in light blue, the core domain in dark blue, and the active site tyrosine residue in red. The C-terminal domain of the enzyme is not included in the diagram since its orientation, with respect to the rest of the molecule, is not known. The catalytic cycle is initiated by enzyme binding to two double-stranded DNA segments called the G segment (in red) and the T segment (in green) (Step 1). Next, two ATP molecules are bound, which is associated with dimerization of the ATPase domains (Step 2). The G segment is cleaved (Step 3) and the T segment is transported through the break in the G segment, which is accompanied by the hydrolysis of one ATP molecule (Step 4). The G segment is then religated and the remaining ATP molecule is hydrolyzed (Step 5). Upon dissociation of the two ADP molecules, the T segment is transported through the opening in the C-terminal part of the enzyme (Step 6) followed by closing of this gate. Finally, the N-terminal ATPase domains reopen, allowing the enzyme to dissociate from DNA (Step 7). Data from Berger et al. (1996), Baird et al. (1999), Brino et al. (2000), and Hu et al. (2002).     https://www.researchgate.net/profile/Alexandre_Escargueil3/publication/10638629/figure/fig1/AS:281505667534857@1444127589543/Fig-1-The-catalytic-cycle-of-DNA-topoisomerase-II-The-ATPase-domains-of-topoisomerase.png

 

Targeting HIF-1 for cancer therapy

Gregg L. Semenza

Nature Reviews Cancer 3, 721-732 (October 2003) |      http://dx.doi.org:/10.1038/nrc1187   http://www.nature.com/nrc/journal/v3/n10/full/nrc1187.html

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that are involved in crucial aspects of cancer biology, including angiogenesis, cell survival, glucose metabolism and invasion. Intratumoral hypoxia and genetic alterations can lead to HIF-1α overexpression, which has been associated with increased patient mortality in several cancer types. In preclinical studies, inhibition of HIF-1 activity has marked effects on tumour growth. Efforts are underway to identify inhibitors of HIF-1 and to test their efficacy as anticancer therapeutics.

 

Targeting DNA topoisomerase II in cancer chemotherapy

Recent molecular studies have greatly expanded the biological contexts where Top2 plays critical roles, including DNA replication, transcription and chromosome segregation. Although the biological functions of Top2 are important for insuring genomic integrity, the ability to interfere with Top2 and generate enzyme mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed understanding the biological functions of Top2 are also being applied to understanding the details of drug action. These studies promise a more refined ability to target Top2 as an effective anti-cancer strategy.

An important reason why Top2 has held the interest of researchers studying cancer was the discovery that active anti-cancer drugs, notably etoposide and doxorubicin target Top21. These studies showed that most clinically active drugs that target Top2 generate enzyme mediated DNA damage24. Since etoposide and doxorubicin are highly active anti-cancer agents in many different settings, an identification of a critical target of these drugs was a major landmark in the pharmacology of anti-cancer drugs.

Recent work has shown that there may be contexts where the level of Top2 protein predicts clinical activity (as well as many contexts where it does not). With the understanding of mechanisms of drug action and improved patient survival rates has come the appreciation that clinical treatment with drugs targeting Top2 can lead to the dire consequence of secondary malignancies. An important goal of present and future work is to maximize therapeutic efficacy of therapy using Top2 targeting agents while minimizing the risks of secondary malignancy and other toxicities. This review highlights recent work that is relevant to maximizing the potential of Top2 as an anti-cancer drug target.

Inhibition of Top2 activity by anti-cancer agents

Drugs targeting Top2 are divided into two broad classes. The first class, which includes most of the clinically active agents including etoposide, doxorubicin, and mitoxantrone, lead to increases in the levels of Top2:DNA covalent complexes. Because these agents generate a “lesion” that includes DNA strand breaks and protein covalently bound to DNA, these agents have been termed Top2 poisons. A second class of compounds inhibits Top2 catalytic activity, but do not generate increases in the levels of Top2 covalent complexes. This second class of agents is thought to kill cells through elimination of the essential enzymatic activity of Top2 and is therefore termed catalytic inhibitors (Fig. 1).

Figure 1   Mechanisms of inhibiting of Top2
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Mechanisms of inhibiting of Top2

Top2 can be inhibited at several different points in the enzyme reaction cycle, which can generate different biochemical and cellular consequences. One simple mode of inhibition is to inhibit a step early in the enzyme reaction cycle. For example, competitive inhibitors of ATP binding prevent strand passage, and do not generate enzyme mediated DNA damage. While agents such as novobiocin and coumermycin (not shown on the figure) inhibit both prokaryotic and eukaryotic Top2s, they are either less potent as well as relatively nonspecific (e.g., novobiocin) or are poorly taken up by mammalian cells (e.g., coumermycin). Similar effects would occur with inhibitors that prevent the binding of Top2 to DNA such as aclarubicin. Agents that prevent DNA cleavage by Top2, such as merbarone would also be expected to act as simple catalytic inhibitors. While merbarone clearly prevents DNA cleavage by Top2126, merbarone clearly affects other targets besides Top2. A second mode of inhibition is blocking the catalytic cycle after DNA is cleaved but prior to DNA religation. This mode of inhibition occurs for most currently used Top2 targeting agents including anthracyclines and epipodophyllotoxins, as well as for agents that target prokaryotic type II topoisomerases. These agents prevent enzyme turnover, and therefore greatly inhibit the enzyme catalytic activity, however, the clearest effect is the generation of high levels of Top2:DNA covalent complexes. Therefore, these inhibitors generate DNA damage, and interfere with many DNA metabolic events such as transcription and replication. Since agents of this class convert Top2 into an agent that induces cellular damage, they have been termed topoisomerase poisons. Top2 can be inhibited after strand passage is completed, but prior to ATP hydrolysis and dissociation of N-terminal dimerization. Bisdioxopiperazines such as dexrazoxane (ICRF-187) inhibit both ATP hydrolysis and maintain Top2 as a closed clamp 74. As is the case with Top2 poisons, bisdioxopiperazines inhibit Top2 catalytic activity mainly by blocking enzyme turnover. Although these agents are frequently termed catalytic inhibitors, they leave Top2 trapped on DNA, and may interfere with DNA metabolism in ways distinct from the inhibitors described in pathway (A). Nonetheless, since bisdioxopiperazines are relatively specific for Top2, they are the most commonly used catalytic inhibitors of Top2 in mammalian cells 143.

There are several lines of evidence indicating the importance of the distinction between Top2 poisons and Top2 catalytic inhibitors. Studies in yeast and mammalian cells demonstrated that resistance to Top2 poisons is recessive, i.e., presence of a drug resistant Top2 in the presence of a drug sensitive allele results in cells that are drug sensitive (reviewed in 5,6). The importance of enzyme mediated DNA damage is also demonstrated by observations that Top2 poisons rapidly elicit DNA damage responses such as ATM phosphorylation and activation of downstream damage responses79. Resistance to Top2 targeting drugs in mammalian cells is frequently associated with reduced expression of Top2 isoforms6, suggesting that resistance is mediated through a reduction in enzyme mediated DNA damage, rather than through enhancing available enzyme activity (where resistance would arise from increased expression of Top2 isoforms).

The generation of high levels of Top2 DNA covalent complexes has profound effects on cell physiology. Top2 poisons effectively block transcription and replication. DNA strand breaks are rapidly detected following treatment with Top2 poisons, and most of the strand breaks are protein linked, as expected10,11. Cells subsequently commit to apoptosis, in fact etoposide is a very commonly used agent to study apoptotic processes12.

The pattern of responses observed with catalytic inhibitors of Top2 differ from that observed with Top2 poisons, albeit with several important complications. Most catalytic inhibitors of Top2 are not specific for Top2 inhibition (see Box 1) with the exception of bisdioxopiperazines. While bisdioxopiperazines generate DNA damage responses following long exposure13, they do not produce a DNA damage response following short term exposure1417. Importantly, in cell culture experiments, catalytic inhibitors of Top2 antagonize the toxicity of Top2 poisons18, indicating that the agents act by separable mechanisms. An important and still unanswered question is whether Top2 inhibitors that are not poisons might be active anti-cancer agents. This issue is addressed in the concluding sections of this review.

Box 1. Many different classes of compounds target Topoisomerase II

Drugs targeting topoisomerase II fall into two categories, Top2 poisons and Top2 catalytic inhibitors. Many Top2 poisons have demonstrated anti-cancer activity. Top2 poisons can be further sub-divided into intercalating and non-intercalating poisons. The intercalators are chemically diverse, and include doxorubicin and other anthracyclines, mitoxantrone, mAMSA, and a variety of other compounds that are not currently in clinical use such as amonafide and ellipticine5. Other than their ability to intercalate in DNA, there is no obvious chemical similarity that could explain the ability of these compounds to trap Top2. Importantly, some compounds, such as oAMSA and ethidium bromide have little ability to poison Top2, suggesting that intercalation of a small molecule is insufficient to trap Top2 as a covalent complex on DNA1,110. Some of the intercalating Top2 targeting drugs, notably the anthracyclines, produce a variety of effects on cells, including many effects that are independent of their action against Top2. For example, doxorubicin is known to produce free radicals, to cause membrane damage, and to induce protein:DNA crosslinks. Whether Top2 is the most important target of anthracyclines remains a controversial issue, (reviewed in 111), although some of the results presented in the text support the hypothesis that Top2 is the most relevant target for both clinical response and cardiotoxicity. For alternate hypotheses, see 112114.

Several classes of compounds have been described that inhibit Top2 activity but do not increase DNA cleavage. Most prominent are the bisdioxopiperazines, which inhibit the enzyme ATPase activity non-competitively and trap Top2 as a closed clamp74,117,118. ICRF-187, a bisdioxopiperazine, is used as a cardioprotectant in some patients treated with anthracyclines. Other Top2 catalytic inhibitors include novobiocin119121, merbarone122, and the anthracycline aclarubicin123. All three compounds have significant targets besides Top2121,124,125; therefore these compounds have not been useful in assessing the feasibility of using catalytic inhibitors of Top2 as an anti-cancer therapy. Merbarone has attracted interest because it is the only agent that has been found to inhibit Top2 cleavage of DNA but not affect protein:DNA binding126. QAP1 is a newly described purine analog that was rationally designed to target the Top2 ATPase activity127. This compound may be particularly useful in assessing the effects of catalytic inhibition of Top2. Several other catalytic inhibitors have been described, however, their detailed mechanism of action has not been explored.

The future of Top2 as a drug target

Is there a need for new and different Top2 drugs? The first answer to this question is a resounding yes, since Top2 targeting is clearly successful in a wide variety of contexts. It is clear from broiad clinical experience that Top2 targeting drugs can be safely and effectively combined with many other agents. The Top2 targeting drugs in clinical use were identified not based on their activity against Top2, but mainly on empirical anti-tumor activity. Therefore, it would be expected that rational screening would lead to potent and specific Top2 poisons. It would be very desirable to know if greater potency and specificity would enhance clinical response.

At the time etoposide and doxorubicin were approved for use, we did not know of the existence of Top2β. The results reviewed in this article suggest that the targeting of Top2β leads to several undesirable consequences and little clear benefit. The negative effects of targeting Top2β include the induction of cardiotoxicity, and potentially a major role in secondary malignancies. On the other hand, there are potential benefits of targeting Top2β, especially the ability to kill non-proliferating cells. While targeting Top2β may contribute to toxicity, it may also be important for eliminating cancer cells that function as cancer stem cells.

An important question is whether isotype specific Top2 poisons can be identified, since the two enzymes share catalytic mechanisms, and a great deal of amino acid homology in their catalytic domains. It has been previously suggested that the intercalators mAMSA and mitoxantrone confer cytotoxicity mainly due to targeting Top2β106. More recently, a novel intercalator NK314 has been reported to be highly specific for Top2α107,108. Toyoda and colleagues also suggested that etoposide and doxorubicin generate greater cytotoxicity by targeting Top2α. Taken together, these results suggest that agents specific for Top2α may possible, and may be useful for having both greater anti-tumor activity, and reduced toxicitiy.

The search for improved Top2 targeting drugs will require further advances in both the biochemistry and structural biology of drug action. While the structures that have already been determined have provided important insights into the biochemistry of Top2, the only structure of Top2 bound to a drug that has been determined is the ATPase domain of Top2 bound to ICRF-187109. The grail for understanding the biochemistry of a drug like etoposide is the determination of a ternary complex between drug, protein, and DNA. Hopefully, the structures of the breakage/reunion domains of Top2α and Top2β, especially their DNA bound forms, will be solved soon.

An interesting question related to drug development is whether catalytic inhibitors of Top2 might be active anti-cancer agents. Much of the literature on the action of Top2 poisons implicitly assumes that they inhibit Top2 activity. Compared to many other enzyme inhibitors, any of the currently described Top2 targeting agents has relatively poor potency (for example, the Ki of etoposide for Top2 is in the 5-20 μM range, the Ki for ICRF-193 is in the 1-2 μM range). The availability of crystal structures provides the tools for addressing whether Top2 inhibition will be a valuable strategy (and will provide tools needed to answer many important biological questions).

The recent biological insights in transcription, replication and checkpoint control also offer ways to better understand drug action and resistance. Since cancer cells can clearly present with altered topoisomerase levels, whether by amplification or changes in gene regulation, these alterations provide an opportunity for enhanced therapeutic index. Finally, active anti-cancer therapy requires an understanding of how cancer cells ‘make a living’, and topoisomerases clearly are central to many of these core biological functions.

At a glance

  • Top2 is the target of several important classes of anti-cancer drugs, including the epipodophyllotoxin etoposide, and the anthracycline doxorubicin.
  • Most clinically active drugs that target Top2 kill cells by trapping an enzyme intermediate termed the covalent complex. Therefore, the principal action of Top2 targeting drugs currently used are to generate enzyme mediated DNA damage.
  • A recent structure of the breakage reunion domain of Top2 bound to DNA has been determined. This structure is likely to be of great use in understanding the protein determinants of the action of drugs targeting Top2. A drug:protein:DNA ternary complex would be extremely valuable, but has not yet been determined.
  • Top2 mediated DNA damage is repaired by multiple pathways. The DNA damage includes DNA strand breaks and proteins covalently bound to DNA. Repair of Top2 damage requires double strand break repair pathways, and other pathways specific for the removal of protein:DNA adducts.
  • Sensitivity to Top2 targeting drugs depends in part on levels of Top2 protein. Cells overexpressing Top2 are hypersensitive to Top2 poisons while cells expressing low levels of Top2 are relatively drug resistant. Top2α is frequently co-amplified with ERBB2. This can lead to some tumors with elevated levels of Top2α.
  • An important side effect of targeting Top2 with Top2 poisons are secondary malignancies arising from drug induced translocations. Top2β may be the Top2 isoform that is most responsible for secondary malignancies caused by Top2 targeting drugs.
  • Anthracycline use is limited by cardiotoxicity. Although the mechanism of the cardiotoxicity is poorly understood, recent results suggest that anthracyclines acting against Top2β may contribute significantly to cardiotoxicity. There may be considerable benefit to developing Top2 targeting drugs specific for the Top2α isoform.
  • Catalytic inhibition of Top2 may also be a useful anti-cancer strategy. New compounds are being developed to test this possibility.

 

Anticancer Chemotherapy – Topoisomerase Inhibitors Part 1 …

Early effects of topoisomerase I inhibition on RNA polymerase II along transcribed genes in human cells.

Khobta A, Ferri F, Lotito L, Montecucco A, Rossi R, Capranico G.

J Mol Biol. 2006 Mar 17;357(1):127-38. Epub 2006 Jan 6.

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RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

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. Topoisomerase I deficiency causes RNA polymerase II accumulation and increases AID abundance in immunoglobulin variable genes.

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Disifin (sodium tosylchloramide) and Toll-like receptors (TLRs): evolving importance in health and diseases.

Ofodile ON.

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Genome-wide analysis of novel splice variants induced by topoisomerase I poisoning shows preferential occurrence in genes encoding splicing factors.

Solier S, Barb J, Zeeberg BR, Varma S, Ryan MC, Kohn KW, Weinstein JN, Munson PJ, Pommier Y.

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Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

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Patterns of oligonucleotide sequences in viral and host cell RNA identify mediators of the host innate immune system.

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Gemcitabine (2′,2′-difluoro-2′-deoxycytidine), an antimetabolite that poisons topoisomerase I.

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. Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability.

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