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Title: Natural variation in a single amino acid substitution underlies physiological responses to topoisomerase II poisons}
Article:
Abstract:
Many chemotherapeutic drugs are differentially effective from one patient to the next. Understanding the causes of this variability is a critical step towards the development of personalized treatments and improvements to existing medications. Here, we investigate sensitivity to a group of anti-neoplastic drugs that target topoisomerase II using the model organism Caenorhabditis elegans. We show that wild strains of C. elegans vary in their sensitivity to these drugs, and we use an unbiased genetic approach to demonstrate that this natural variation is explained by a methionine-to-glutamine substitution in topoisomerase II( TOP-2). The presence of a non-polar methionine at this residue increases hydrophobic interactions between TOP-2 and its poison etoposide, as compared to a polar glutamine. We hypothesize that this stabilizing interaction results in increased genomic instability in strains that contain a methionine residue. The residue affected by this substitution is conserved from yeast to humans and is one of the few differences between the two human topoisomerase II isoforms( methionine in hTOPIIα and glutamine in hTOPIIβ). We go on to show that this amino acid difference between the two human topoisomerase isoforms influences cytotoxicity of topoisomerase II poisons in human cell lines. These results explain why hTOPIIα and hTOPIIβ are differentially affected by various poisons and demonstrate the utility of C. elegans in understanding the genetics of drug responses.
Introduction:
Antineoplastic regimens used to treat cancer are often associated with poor prognoses and severe side effects. Ideally, antineoplastic regimens can be tailored to an individual patient based on various genetic markers known to be associated with drug response to maximize therapeutic effectiveness and minimize unwanted side effects. Advances in sequencing technologies over the course of the past decade promised the discovery of many genetic variants that contribute to human health. Though large-scale sequencing projects have led to the identification of many genetic variants associated with disease risk[1], relatively few variants have been identified that contribute to clinically relevant traits such as response to antineoplastic compounds. In fact, only 71 of over 500 FDA-approved antineoplastic compounds use genetic information to affect treatment efficacy( www. fda. gov). Unfortunately, the predictive power of these identified genetic variants can be inconsistent due to biases in the sampled population[2] and other key limitations of clinical genome-wide association( GWA) studies that attempt to link genetic variants with treatment outcomes. The major factor limiting the efficacy of these studies is sample size because it is difficult to identify large numbers of individuals exposed to the same antineoplastic regimens. This limitation is compounded when considering environmental[3, 4] and tumor heterogeneity[5]. As a result, most variants discovered to be associated with outcomes in clinical GWA studies offer low predictive power for patient responses to treatment[6]. These limitations and others emphasize the need for novel approaches to identify variants that predict patient outcomes to antineoplastic compounds. Studies of model organisms have greatly facilitated our understanding of basic cellular processes. In recent years, Saccharomyces cerevisiae and Drosophila melanogaster have been used to understand the physiological effects of small molecules and repurposed as screening platforms to identify new antineoplastic compounds[7–9]. The ability to generate extremely large numbers of recombinant yeast facilitates the identification of genomic regions that are predictive of drug response[10, 11]. Furthermore, the specific genes and variants within regions can be identified and functionally validated in yeast[12–14]. By contrast, D. melanogaster studies offer the ability to study the physiological responses to drugs in the context of multiple tissue types, but functional validation of specific genes and variants associated with drug responses has been more limited[9]. The roundworm Caenorhabditis elegans has the advantages of both S. cerevisiae and D. melanogaster because large cross populations can be generated to study the physiological responses to drugs in a metazoan. These attributes have made C. elegans an important model for connecting differential drug responses with genetic variants present in the species[15, 16]. Here, we take advantage of natural genetic variation present in C. elegans to identify the genetic basis underlying susceptibility to a panel of clinically relevant antineoplastic compounds that poison the activity of topoisomerase II enzymes. The inhibition of these enzymes by topoisomerase II poisons results in the accumulation of double-stranded breaks and genome instability[17–19]. Topoisomerase II enzymes are targeted by antineoplastic regimens because proliferative cell populations require their enzymatic activity to relieve topological stress ahead of the replication fork[20]. Using two unbiased genetic mapping approaches, we show that divergent physiological responses to the topoisomerase II poison etoposide are determined by natural genetic variation in a C. elegans topoisomerase II enzyme. Furthermore, we show using CRISPR/Cas9-mediated genome editing that variation in a specific amino acid( Q797M) underlies the observed phenotypic variation in response to etoposide. This residue is conserved in humans and is one of the few differences between the putative drug-binding pockets of the two topoisomerase II isoforms( M762 in hTOPIIα and Q778 in hTOPIIβ). Previous structural studies on hTOPIIβ implicated this glutamine residue in etoposide binding because of its proximity to the drug-binding pocket[21, 22]. However, a study on hTOPIIα suggested that the corresponding methionine residue has no functional role in drug binding[23]. We present a mechanistic model to explain how variation at this residue underlies differential responses to etoposide and other topoisomerase II poisons. Finally, we use genome-edited human cell lines to show that this residue in hTOPIIα contributes to differential toxicity of various topoisomerase II poisons. These results demonstrate the power of using C. elegans natural genetic variation to identify mechanisms of drug susceptibility in human cells that could inform human health decisions based on genetic information.
Discussion:
Few genetic markers have been identified that predict patient responses to chemotherapeutic regimens[32, 33]. The goal of this study was to introduce new methods for the rapid and cost-effective identification of genetic variants that explain differences in chemotherapeutic response. Our approach leveraged genetic and phenotypic variation present in the model organism C. elegans to identify a single amino acid variant( Q797M) in the topoisomerase II enzyme that underlies differences in etoposide response. Mechanistic insights into differential etoposide binding between the glutamine or methionine alleles gave us the power to predict the physiological responses to an expanded panel of topoisomerase II poisons. These results highlight how the combination of a highly sensitive phenotyping assay with classical and quantitative genetics approaches in C. elegans can rapidly identify the mechanistic underpinnings of phenotypic variability in response to a key class of antineoplastic compounds. Our approach stands in stark contrast to previous underpowered human cell line[34] and clinical studies[35] that failed to identify any statistically significant associations between etoposide-induced cytotoxicity and genetic variation in the human population. However, the residue we identified in C. elegans does not vary in the human population[36], suggesting that GWA studies would not have identified this variant as a marker for etoposide sensitivity. Nevertheless, this residue is one of the few differences between the putative drug-binding pockets of the two human topoisomerase II isoforms( M762 in hTOPIIα and Q778 in hTOPIIβ), which allowed us to investigate the molecular underpinnings of drug binding. We verified that this single amino acid change in the human topoisomerase II isoforms results in profound differences in topoisomerase II poison-induced cytotoxicity using 293T cells. Though previous hTOPOIIβ structural studies have implicated this glutamine-methionine difference as functionally important for etoposide binding[21, 22], studies involving hTOPOIIα have argued that this residue is not involved[23]. The results presented here unequivocally show that this residue contributes to differential topoisomerase II poison-induced cytotoxicity and have important implications for targeted drug design. Although topoisomerase II poisons can bind and inhibit both hTOPOIIα and hTOPOIIβ, hTOPOIIα is the cellular target of poisons in most cancers because it is expressed in proliferating cells[17]. However, recent evidence suggests that side effects associated with these treatments are caused by inhibition of hTOPOIIβ in differentiated cells[37]. For example, antineoplastic treatment regimens that contain the epipodophyllotoxins( e. g. etoposide or teniposide) are hypothesized to increase the risk of developing secondary malignancies caused by hTOPOIIβ-dependent 11q23 translocations[38–41]. Additionally, the most severe side effects associated with treatments that contain an alternative class of topoisomerase II poisons( anthracyclines, e. g. doxorubicin or daunorubicin) include dose-dependent cardiotoxicity and heart failure dependent on hTOPOIIβ[42–44]. Therefore, optimal topoisomerase II poisons will maximize interactions with hTOPOIIα to inhibit proliferating cells and minimize hTOPOIIβ interactions to reduce side effects. With this goal in mind, others have identified etoposide analogues with different isoform specificities but have not determined the mechanism of specificity[45]. Our study functionally validates a key residue determining isoform specificity and is critical to the improvement of this widely administered drug class. The importance of such functional characterization is underscored by our observation that C. elegans strains and human cells with the methionine TOP-2 allele are more sensitive to XK469, despite this drug being shown to be a β-specific poison[29]. Though XK469 has been shown to be a β-specific poison, no information regarding its drug binding pocket or the mechanism driving isoform specificity is currently known. Our results indicate that XK469 occupies a similar drug-binding pocket of TOP-2 as other topoisomerase II poisons and interacts with residue 797. To date, no human genetic variants have been linked to topoisomerase II poison-induced cytotoxicity. Of the 291 and 279 respective SNVs in hTOPOIIα and hTOPOIIβ that encode missense mutations( S14 and S15 Tables) found in the ExAC database[36], some are near the highly conserved DNA-binding domains or drug-binding pockets( S11 Fig), which could affect drug response. However, the extent to which these variants impact responses to topoisomerase II poisons is unknown, so functional validation is required. The approach of editing human cells and following allele frequencies via sequencing represents a scalable method to assess the functional role of these variants and avoids single-cell cloning. Importantly, differences in responses to topoisomerase II poisons might not be affected by variation in the topoisomerase II isoforms but instead mediated by variation in cellular import, metabolism, or export. Pharmacogenomic data available for many antineoplastic compounds[46, 47], in combination with human variation data[36], can be used to prioritize and test variants in highly conserved regions of proteins known to be involved in these alternative processes. This biased approach focused on candidate variants is necessitated by the lack of power in clinical GWA studies and is not guaranteed to successfully connect variants to differences in drug response. For this reason, unbiased mapping approaches in model organisms combined with functional validation in genome-edited human cells will greatly expand our current understanding of how human genetic variation affects drug responses.
The severe cytotoxic effects associated with anti-neoplastic treatment regimens make it difficult to assess the contributions of genetic variation on treatment responses in clinical settings . Therefore , we leveraged genetic diversity present in the metazoan model nematode Caenorhabditis elegans to identify genetic variants that contribute to differential susceptibility to a broadly administered class of anti-neoplastic compounds that poison the activity of topoisomerase II enzymes . We show that wild C . elegans isolates contain either glutamine or methionine at a highly conserved residue of the topoisomerase II ( TOP-2 ) protein and that this substitution is predictive of animal responses to the topoisomerase II poisons etoposide , teniposide , dactinomycin , and XK469 . Interestingly , the two human versions of this protein , hTOPIIα and hTOPIIβ , contain a methionine or glutamine at the corresponding residue , respectively . We show that this difference between the two human topoisomerase II isoforms contributes to the differential cytotoxicity induced by these drugs . Taken together , our results highlight the power of studying the effects of natural genetic variation on drug responses in a model organism and propose methods to develop new drugs that have increased affinity for the desired hTOPIIα isoform expressed in tumor cells .