Kim Zayhowski

Topoisomerase 1 (Top1) is an enzyme that is involved in relieving torsional stress in DNA. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair protein that removes Top1 DNA intermediates created by Top1 in order to allow single and double stranded DNA repair. A rare neurological disease called spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is characterized by the loss of function of this protein. Little is known about how Tdp1 is involved in the neurodegeneration of SCAN1, though the leading hypothesis is that SCAN1 is caused by an increased stabilization of Tdp1 intermediates. Studies examining SCAN1 have shown that Tdp1 may be an important chemotherapeutic target. Many anticancer drugs are meant to poison Top1. Tdp1 seems to play an important role in repairing the damage caused by these drugs. Therefore, Tdp1 inhibitors combined with Top1 poisons may increase the efficacy and sensitivity of certain anticancer drugs. This review focuses on the function of Tdp1, Tdp1’s role in SCAN1, and how research on Tdp1 and SCAN1 is being utilized to increase the effectiveness of anticancer drugs.

Introduction

Tens of thousands of single-strand breaks (SSBs) arise in DNA in each cell every day (1). In order to combat SSBs, cells have employed mechanisms to repair DNA, a process called DNA single-strand break repair (4). Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a rare, autosomal recessive neurological disease (15). The only known mutation in SCAN1 is associated with tyrosyl-DNA phosphodiesterase 1 (Tdp1), which is a protein involved in repairing of DNA strand breaks that are created by topoisomerase 1 (Top1) (5). When functioning properly, Tdp1 catalyzes the removal of covalent 3’-DNA adducts. In SCAN1, there is a p.His493Arg mutation in the Tdp1 which impairs Tdp1 activity and makes DNA more susceptible to forming Top1-DNA adducts, damaging both nuclear and mitochondrial DNA (6). To date, there is no cure for SCAN1, and treatments are all symptomatic.

Recently, studies involving SCAN1 and Tdp1 have defined Tdp1 as a chemotherapeutic target, and suggest that understanding Tdp1 and its involvement in SCAN1 may lead to an effective cancer treatment (2). Top1 poisons are a type of anticancer drug, and Tdp1 is able to fix some of the damage that these drugs cause. Therefore, Tdp1 inhibitors may increase the efficacy of certain anticancer drugs (14). This review will focus on Tdp1’s function in single-strand break repair, Tdp1’s contribution to SCAN1, and how researchers are using Tdp1 and SCAN1 research to find therapeutic targets for cancer.

Tdp1 and DNA repair mechanisms

Tdp1 is implicated in the mechanism for SCAN1. In order to understand the pathology for SCAN1, and how Tdp1 may be used to find anticancer targets, it is important to understand the normal role of Tdp1 in DNA repair.

Topoisomerase 1 and SSBs – Top1 is involved in relieving torsional stress in DNA in transcription, replication, and condensation (15). DNA strands create SSBs by a nucleophilic attack of a tyrosine residue on Top1’s active site on a phosphodiester bond in the DNA backbone. This allows the double helix to rotate around the intact strand. During the relaxation process, a reversible intermediate called Top1 cleavage complex is created (4). Here, Top1 is covalently attached through a tyrosyl residue to the 3’ terminus of the single-stranded break. Once Top1 relieves torsional stress, Top1 is able to reseal the tear, restoring DNA to its double helix.

DNA damage and oxidative stress can cause the Top1 DNA intermediate to become trapped or long lived because of the misalignment of the 5’-hydroxyl or from binding to oxidative lesions (4,6). DNA can break when DNA replication machinery or when RNA polymerase hits the irreversible intermediate and forms Top1-DNA adducts (6). When this happens, a DNA repair process must remove the intermediate (11, Figure 1).

Tdp1 interactions with Top1 DNA intermediate – Tdp1 is in the phospholipase D superfamily (15). Enzymes in this superfamily are involved in catalyzing the cleavage of phosphodiester bonds. Tdp1 is involved in repairing Top1-DNA complexes because it has a high affinity for the 3’ tyrosine-DNA phosphodiester moieties that are characteristic of Top1 DNA intermediates (4, Figure 1). Tdp1 catalyzes the hydrolysis of 3’-phosphotyrosyl bonds, allowing it to function as a repair enzyme (12,8). Tdp1 disconnects the Top1 DNA intermediate by removing the Top1 peptide after the intermediate is proteolytically cleaved, and thus Tdp1 allows single and double stranded break repairs (6).

The molecular mechanism of SCAN1

As stated previously, SCAN1 is a neurodegenerative disorder (6). In SCAN1, there is a mutation in histidine 493 that converts it to arginine, which affects the active site on Tdp1 (16, Figure 2). This mutation reduces the catalytic activity of the protein about 25 fold and impairs the reaction that would normally release Tdp1 from DNA (10). The molecular basis for SCAN1 is still incompletely understood. This section focuses on the disease characteristics of SCAN1, what is known about Tdp1’s relationship to SCAN1, and how Tdp1’s dysfunction in repair may lead to the neurodegeneration in SCAN1.

SCAN1 disease characteristics – There have only been nine reported cases of SCAN1, all from one extended Saudi Arabian family (16,7). SCAN1 targets the nervous system (15). The disease is characterized by a gradual late-childhood onset, with cerebellar ataxia, peripheral neuropathy, axonal sensorimotor neuropathy, hypercholesterolemia, seizures, and hypoalbuminemia (15,16). Affected patients develop impaired pain, loss of touch, a distorted gait, and a constant tingling in their hands and legs (6). Patients are expected to be wheelchair bound by early adulthood (8). Patients do not show impaired cognitive function and have normal longevity and fertility (6). To date, there are no cures for SCAN1, and there are only symptomatic treatments such as physical therapy and walking aids to promote mobility.

Proposed SCAN1 mechanisms – The molecular basis of SCAN1 is still unknown, though it is agreed upon that Tdp1 plays a role in the neurodegeneration. There are two main hypotheses for SCAN1’s mechanism. The leading hypothesis is that a loss of functional Tdp1 is sufficient to causes the neurodegeneration in SCAN1 (17). A biochemical analysis of Tdp1 with a p.His493Arg mutation supported this idea (17). In this analysis, Tdp1 became trapped on the DNA as a reaction intermediate, even though it showed a significant reduction in catalytic activity (17,8, Figure 2). The only enzyme that was able to resolve the intermediate was wild type Tdp1, suggesting that SCAN1 might arise from stabilization of the intermediate.

Biochemical, genetic, and mass spectrometry analyses of yeast cells have shown the formation of a Tdp1 DNA intermediate could be catalyzed by a highly conserved nucleophilic histidine on Tdp1 (2). Spatial and temporal coordination of histidine activity was shown to be crucial for Tdp1 to catalyze resolution of unwanted substrates. This research suggests that mutant Tdp1 intermediates may be impaired by the flexibility of Tdp1 active site residues.

Alternatively, a dysfunctional Tdp1 may not be sufficient to cause the SCAN1 phenotype. When creating mouse models of the disease, various groups of researchers have all found that mouse models of the disease do not exhibit ataxia (3). In fact, Tdp1 deficient mice showed no phenotypic differences from wild type mice (8). These findings suggest that SCAN1 does not result from just a dysfunctional Tdp1, or that mice may have redundant pathways for repairing stalled Top1 (10).

Tdp1 may not be evolutionarily conserved; Tdp1 may have a function in humans that it does not in mice (6). Tdp1 exhibits nuclear expression in mouse cells, but demonstrates cytoplasmic expression in human cells (17). Cells with the most cytoplasmic expression are certain types of motor neurons, purkinje cells, and dentate nucleus neurons. These cells are likely affected in SCAN1, since SCAN1 is neurodegenerative. A Drosophila homolog of Tdp1, glaikit, is only detected in the cytoplasm, and is essential for neuronal development (8). Without glaikit, Drosophila show defective embryonic neurogenesis and embryonic lethality (17,8). In humans, SCAN1 patients show normal neurodevelopment. If human Tdp1 is analogous to glaikit, it is possible that a maintenance function leads to the neurodegeneration shown in SCAN1 (17).

Neurodegeneration in SCAN1 – SCAN1 is the first known example of a genetic disorder in humans that is the result of a failure to repair DNA-protein covalent complexes (10,8). Because it is so novel, it is unclear why the SCAN1 has a late onset and why the nervous system is sensitive to Tdp1 loss, but there are a few speculations.

The small amount of residual activity of Tdp1 may be sufficient to provide the majority of the repair necessary and may be the reason why there is a late onset in SCAN1 in individuals (10). Otherwise, there could be a functional overlap of other, unknown repair mechanisms with Tdp1.

Neurons may have a larger need for Tdp1-mediated repair than other cells, or could be hypersensitive to Top1-DNA adducts. Therefore, SCAN1 pathology may result because of elevated oxidative stress in post-mitotic neurons, which could cause many SSBs, or because unrepaired SSBs could be processed into DSBs during DNA replication with an absence of Tdp1 (4). Because Tdp1 is involved in the repair of SSBs and DSBs, and these breaks are the result of oxidative damage, the nervous system may be particularly susceptible to damage with a mutation in Tdp1, since the nervous system consumes a large amount of oxygen and encounters much oxidative stress (5). Unrepaired breaks may trigger cell death. Since the nervous system is limited in regeneration and is not readily replaceable, cell loss may be the cause of neurodegeneration in SCAN1.

Additionally, DNA repair is costly. Therefore, if the expense of energy used to repair the DNA were greater than the energy that the cells contain through ATP or NADH, cell death would occur (6). Depletion of these energy reserves in the mitochondria triggers the release of cytochrome c and apoptosis-inducing factor, and in SCAN1 patients, the release of these factors would be at a lower threshold. The neurons with the least energy reserves would be sensitive and, unlike proliferating cells, hard to replace (6).

Tdp1 as a therapeutic target for anticancer drugs

Certain anticancer drugs, like camptothecins (CPT) and indenoisoquinolines, are meant to poison Top1 to prevent DNA from unwinding (13). They work by causing Top1 to stall on DNA (8). Because of the function of Tdp1, cell lines from SCAN1 patients are hypersensitive to these anticancer drugs; Tdp1 is important in fixing Top1 DNA complexes. However, despite the role of Tdp1 in DNA repair, SCAN1 patients do not have increased incidences of cancer. Unrepaired SSBs can result in increases in DSBs in DNA replication, so it is surprising that SCAN1 is not associated with genetic instability or cancer (1). Similarly, cancer patients taking Top1 inhibitors do not seem to have an increased incidence of neurodegeneration (8). This section focuses on Top1’s role in cancer, and on how Tdp1 could be used to increase the efficacy of certain anticancer drugs.

Cancer and Top1 – Top1 has been shown to have a role in several cancers (3). Specifically, colorectal cancer, the third most common cancer in the world, is often treated with CPT based Top1 poisons such as irinotecan (14). These drugs cause cell death by trapping the intermediate on DNA. For decades, patients treated with irinotecan have had an overall improvement in survival, though some patients show no response or develop resistance. Researchers have shown that Top1 levels inversely correlate with irinotecan sensitivity in colorectal cancer, and the effectiveness of Top1 poisons on cancer may be primarily due to levels of Top1 and the repair rate of Top1 DNA damage (14). High levels of Top1 or efficient Top1 DNA damage repair rates may make cells less sensitive to CPTs. Because Tdp1 works to fix damage caused by stalled Top1, Tdp1 could make cells less sensitive to CPTs.

Inhibiting Tdp1 as a chemotherapeutic strategy – Employing chemotherapeutic inhibitors with Tdp1 as a target could enhance the effect of Top1 poisons (13). Because mice with Tdp1 deficiencies do not show neurological disease, and SCAN1 has a late onset, it is speculated that Tdp1 could be inhibited for short periods without having severely negative penalties on the brain (6).

Cancer cells often induce Tdp1 overexpression when met with Top1 poisons, and therefore, would be especially susceptible to damage from Tdp1 poisons (9,3). This could help overcome cellular resistance to these chemotherapeutic strategies. Interthal et. al. performed a study where Tdp1 mutant cell lines derived from SCAN1 patients demonstrated CPT hypersensitivity, implicating Tdp1’s ability to act synergistically with CPTs (10). The Tdp1 deficient treated cells arrested growth in S phase. This study was the first to suggest that a mutation in Tdp1 alone could enhance cell sensitivity to CPT drugs. Huang et. al. also showed that Tdp1 in humans is capable of repairing the DNA damage induced by Top1 poisons, and therefore increasing drug resistance (9). Poisoning Tdp1 could produce synergistic toxicity when combined with Top1 poisons (3). Consistently, in vitro, Tdp1 knockout mouse cells demonstrate increased sensitivity to Top1 poisons (3). However, in vivo, the knockout mice show no increased sensitivity to pharmacologically relevant levels of CPTs.

Meisenberg et. al. conducted a study in which they examined how varying levels of Top1 and Tdp1 in colorectal cancer lines affects irinotecan sensitivity (14). They found that the majority of the samples from cancer patients expressed higher than average levels of Top1 than found in people without cancer. The researchers were not able to conclusively determine differences in Tdp1 levels between participants with cancer versus without cancer. Using Tdp1 modulation studies, their findings demonstrated that Tdp1 could influence the cancer’s response to irinotecan and irradiation. Overexpression of Tdp1 protects the cell from CPT-mediated cell death, while depletion sensitizes cells to CPT’s effects if Top1 is present. Developing small molecules to poison Tdp1, therefore stabilizing Tdp1 DNA intermediates, represents a promising chemotherapeutic strategy (2).

Future Directions

This review has detailed the mechanism for Tdp1 in DNA repair, and how this research interplays with the SCAN1 research and the improvement of anticancer drugs. Further research should focus on the role of Tdp1 in biological processes, the mechanism that underlies SCAN1, and using Tdp1 as a therapeutic target for cancer.

Much is to be determined on the relationship between Tdp1 and the neurodegeneration in SCAN1. More research should be done to examine how Tdp1 affects neuronal tissue and the contribution of SSBs and DSBs to neurodegeneration (4). Research should be conducted on the roles of Tdp1 in biological processes other than DNA repair (1,3). Data in both mice and yeast have suggested that Tdp1 interacts with many DNA repair processes, and understanding these pathways in the human brain might help to understand the increased sensitivity in humans to Tdp1 mutations (17). If researchers could create the SCAN1 phenotype in mouse models, they could potentially narrow focus onto the histidine mutation, and how that may be responsible for SCAN1, not just the loss of function Tdp1.

Research on the involvement of Tdp1 and chemotherapeutic targets is relatively new. Utilizing Tdp1 may increase the efficacy of Top1 poisons, as Tdp1 could counteract the effects of CPT drugs (17). Numerous questions remain unsolved. It would be useful to examine why mutant Tdp1 mouse models do not show increased sensitivity to CPT in vivo, but do in vitro (3). Further studies should examine the levels of Tdp1 in cancer patients. Tdp1 inhibition needs to be studied more in the context of chemotherapy (14). Otherwise, increasing Tdp1 function may be an effective therapeutic target for SCAN1 patients to reduce atrophy (14). Ultimately, further research is vital in order to use Tdp1 in finding treatments for both SCAN1 and certain types of cancer.

References

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