Aging (Albany NY). 2017 Jan;9(1):68-87 

Effects of an unusual poison identify a lifespan role for Topoisomerase 2 in Saccharomyces cerevisiae

 

Gregory Tombline1*, Jonathan I. Millen1*, Bogdan Polevoda1, Matan Rapaport1, Bonnie Baxter1, Krister Wennerberg2, Joe Madrey2, Michael Van Meter1, Matthew Gilbertson3, John L. Nitiss3, Gary Piazza2 and David S. Goldfarb1 

1Biology Department, University of Rochester, Rochester, NY 14627, USA

2Southern Research Institute, Birmingham AL, 35205, USA.

3Department of Biopharmaceutical Sciences, UIC College of Pharmacy at Rockford, Rockford, IL 61107, USA

*Equal contribution

 

Correspondence: David S. Goldfarb, Biology Department, University of Rochester, Rochester, NY 14627, USA. Tel: 585-275-3890; e-mail: david.goldfarb@rochester.edu

 

Abstract

A progressive loss of genome maintenance has been implicated as both a cause and consequence of aging. Here we present evidence supporting the hypothesis that an age-associated decay in genome maintenance promotes aging in Saccharomyces cerevisiae (yeast) due to an inability to sense or repair DNA damage by topoisomerase 2 (yTop2). We describe the characterization of LS1, identified in a high throughput screen for small molecules that shorten the replicative lifespan of yeast. LS1 accelerates aging without affecting proliferative growth or viability. Genetic and biochemical criteria reveal LS1 to be a weak TOP2 poison. TOP2 poisons induce the accumulation of covalent TOP2-linked DNA double strand breaks that, if left unrepaired, lead to genome instability and death. LS1 is toxic to cells deficient in homologous recombination, suggesting that the damage it induces is normally mitigated by genome maintenance systems. The essential roles of yTop2 in proliferating cells may come with a fitness trade-off in older cells that are less able to sense or repair yTop2-mediated DNA damage. Consistent with this idea, cells live longer when yTop2 expression levels are reduced. These results identify intrinsic yTop2-mediated DNA damage as a potentially manageable cause of aging.

Keywords: replicative lifespan, aging, topoisomerase 2 poison, DNA damage, antagonistic pleiotropy

doi: 10.18632/aging.101114

 

Supplement:

The lifespan of a eukaryote, and its antecedent aging, is determined by the confluence of genetic, biochemical and environmental factors. The priority of many investigators is to discover interventions or small molecules that slow the appearance of aging phenotypes, mitigate age-associated diseases and, for some, to extend maximum lifespan. To this end the search for small molecules that extend lifespan focuses on known longevity targets, such as mTORC1 and certain sirtuins. An alternative approach is to seek small molecules that shorten lifespan. Our primary aim was to elucidate basic biology by identifying modulators, usually inhibitors, of conserved factors that are required for longevity. These novel targets may or may not be amenable as drug targets. In this study, we describe LS1, an unusual catalytic poison of Topoisomerase 2 (Top2), and define Top2 as a novel, druggable longevity factor, but with a twist. LS1 causes the (transient) arrest of Top2 covalently attached to the ends of a DNA double strand break (DSB). This alone cannot explain its lifespan shortening activity, since LS1, unlike most Top2 poisons, is not toxic to young proliferating cells, and regardless, why would a Top2 poison act selectively in older cells? Our current hypothesis is that Top2 exhibits pleiotropic antagonism, that is, it promotes proliferation in young cells, but is deleterious in older cells. We hypothesize that older cells lose the capacity to efficiently recognize and repair gratuitous Top2-arrested DSBs that occur naturally at low levels, and which are slightly stabilized by LS1. As proof of this concept, we showed that a moderate reduction in the expression of TOP2, to levels above that which are required for housekeeping functions and proliferation, extend both mean and maximum replicative lifespan. These results identify Top2 as a potential therapeutic target for certain age-associated diseases.

 

Death of Daughters (DeaD): A facile high throughput assay for the discovery of small molecules that affect replicative lifespan in yeast.

The replicative lifespan (RLS) of Saccharomyces cerevisiae is the number of daughter cells a mother cell can produce before it senesces and dies. Mammalian lifespan and yeast RLS are sensitive to many of the same environmental and genetic interventions; hence, yeast RLS has served well to discover and elucidate longevity genes and environmental conditions with relevance to humans. The standard yeast RLS assay involves microdissecting aging mother cells away from their daughter cells for typically 40 generations, depending on the strain, and thus is slow, tedious and unsuited to high throughput studies. We report improvements to a RLS proxy (Jarolim et al., 2004), that uses a genetically engineered yeast strain (Death of Daughters: DeaD) whose growth rate in glucose-containing medium is, in some but not all circumstances, directly proportional to RLS. For example, the DeaD assay recapitulates the classic lifespan effects of increasing and reducing the expression of the sirtuin SIR2, as well as to the deletion of DNA repair genes. A caveat, likely stemming from the design of the assay, is that it is largely unresponsive to caloric restriction or nutrient signaling by regulators such as Tor kinase. However, when applicable, the DeaD assay offers a uniquely facile, very high throughput option for the study of RLS.

With regard to the search for lifespan shortening molecules or conditions, the DeaD assay includes a permissive mode that allows for the screening out of toxic molecules or conditions that reduce proliferation or lower viability. Positive hits using the DeaD assay are always validated using the standard RLS microdissection assay. We employed the DeaD assay to screen a large chemical library for small molecules that shorten RLS, with the expectation that bona fide hits would be inhibitors of factors with roles in longevity. This expectation was validated by the discovery of LS1, which we subsequently identified as an unusual inhibitor of topoisomerase 2 (Top2).

 

 

Figure 1: LS1 is structurally related to ellipticine. LS1 is a relatively weak Top2 poison and unlike ellipticine does not intercalate into DNA. Doxorubicin is a chemically unrelated Top2 poison that serves as a frontline chemotherapeutic, albeit with toxic side effects, including cardiotoxicity.

 

LS1 is an unusual Top2 poison that shortens replicative lifespan without affecting viability or proliferation.

Yeast Top2 is an essential enzyme that acts during transcription, replication, and DNA repair to relax supercoils and decatenate tangled DNA strands. Classic Top2 poisons, including frontline chemotherapeutic drugs doxorubicin (daunarubicin) and ellipticine (Fig. 1), represent a special class of inhibitor that stabilizes the intermediate(s) wherein one or both homodimeric subunits of the enzyme are covalently attached via tyrosine residues to the 5′-ends of a single or double strand break (DSB) (Fig 2). Top2 covalent complexes (Top2cc) are highly toxic to proliferating cells; hence their efficacy as anti-cancer drugs.

LS1, which resembles ellipticine (Fig. 1), exhibits several hallmarks of known Top2 poisons. But LS1 is not a simple Top2 poison, because it nontoxic to dividing cells at concentrations where it is biologically active as a lifespan shortener. Thus LS1 seems to act selectively on old cells. In contrast, doxorubicin and ellipticine are equally toxic to young and old yeast cells. It remains to be determined mechanistically how it is that LS1, a bona fide, albeit weak Top2 poison, displays no toxicity to exponentially dividing cells at concentrations where it serves as a potent lifespan shortening agent. The catalytic cycle of Top2 is complex, and the exact mechanisms by which various Top2 inhibitors and poisons act is nuanced and not always well understood. It is possible that the unusual biological properties of LS1 are not due to a novel kind of interaction with Top2, but rather is related to quantitative aspects of its particular binding kinetics. Certainly, LS1 is a poor Top2 poison, with little obvious therapeutic value (but see below), and would not have previously attracted anyone’s interested had it not been discovered in a phenotypic screen for RLS.

One possible explanation for the nontoxicity of LS1 is that its interaction with Top2 is transient. We hypothesized that transient LS1-Top2cc adducts might “stage” the enzyme in an intermediate that promotes access to a second, more potent Top2 poison that enters the complex and forms stable Top2cc adducts (Fig. 1). In fact, nontoxic concentrations of LS enhanced the toxicity of doxorubicin and etoposide in yeast. Furthermore, LS1 increased the potency of doxorubicin to a human cancer cell line, but showed no effect in primary cells. This model posits that LS1 promotes the transient formation of Top2cc adducts that are themselves short-lived to be toxic to young proliferating cells. Besides being kinetically interesting, this finding suggests a route to enhancing the potency of front line chemotherapeutics like doxorubicin, which at current therapeutic doses is fraught with toxic side effects such as cardiotoxicity.

 

Indirect antagonistic pleiotropy: LS1 becomes toxic as aging cells lose the capacity for efficient DNA repair.

Although wild-type cells tolerate 10-20-fold higher concentrations of LS1 needed to significantly reduce RLS, cells that are deficient in homologous recombination (HR) (e.g. rad52D cells) are hypersensitive to LS1. This observation, coupled with the knowledge that the efficacy of multiple DSB repair systems, including homologous recombination, decline as cells age, could explain the selective toxicity of LS1 to older cells. In yeast, the decline in DNA repair means that older cells will increasingly phenocopy HR-deficient rad52D cells, which are hypersensitive to LS1. We hypothesize that LS1 shortens RLS because aging cells lose the capacity to recognize and repair LS1-stabilized Top2cc adducts.

A prediction of our model for the mechanism of lifespan shortening by LS1 is that yeast RLS is normally limited by the natural occurrence of Top2cc complexes. It only takes a single unrepaired DSB to kill a yeast cell. The natural occurrence of Top2cc adducts is supported by the existence of conserved Tdp tyrosyl-DNA phosphodiesterases that function specifically to participate in the removal and repair of these adducts. If the spontaneous generation of gratuitous Top2cc adducts, which we propose become selectively toxic to aging cells. is proportional to cellular levels of Top2, then modest reductions in Top2, to levels that suffice for housekeeping duties, would reduce the occurrence of Top2cc adducts in aging cells and extend RLS. In fact, a ~3-fold reduction of TOP2 expression extended both mean and maximum RLS. This result suggests that a small molecule inhibitor of Top2, that reduces catalytic activity without generating toxic intermediates, would extend yeast RLS. Beside our demonstration that LS1 has clinical potential to enhance chemotherapeutics like doxorubicin, it remains to be determined whether the targeting of Top2 in mammals with inhibitors that reduce, but do not poison, Top2 activity can mitigate mammalian aging and age-associated diseases remains to be determined.

 

 

Figure 2: Top2 poisons stabilize covalent Top2cc adducts. Top2 poisons stabilize the catalytic intermediate containing covalent links between tyrosyl residues with the 5′ ends of DSBs and prevents relegation. Cells express tyrosyl-DNA phosphodiesterases (Tdp) that assist in the repair of naturally occurring Top2cc adducts. We propose that transient Top2cc complexes containing LS1 increase the potency of doxorubicin (Dox) by staging the enzyme in a susceptible complex.

 

 

 

Figure 3. The indirect antagonistic pleiotropy model for the role of Top2 in aging. Aging cells lose multiple systems required for efficient genome maintenance (blue line). When the capacity to recognize and repair DNA damage drops below a certain level, the accumulation of DNA damage becomes maladaptive. As DNA damage accumulates in older cells it ultimately contributing to age-associated diseases and death (solid red line). A moderate reduction in the expression of Top2 delays the accumulation of Top2-associated DNA damage, thereby extending lifespan (dotted red line). The normal generation and repair of DSBs may also contribute to aging via epigenetic changes (see Kim et al, 2016).

 

 

References:

Kim, J., Sturgill, D. Tran, A.D., Sinclair, D.A. and P. Oberdoerffer. (2016) Controlled double strand break in mice reveals post-damage transcriptome stability. Nucleic Acids Res. 44: e64

Jarolim, S., Millen, J., Heeren, G., Laun, P., Goldfarb, D.S. and M. Breitenbach. (2004). A novel assay for replicative lifespan in Saccharomyces cerevisiae. FEMS Yeast Res. 5, 169-177.