Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide

  1. Robert P Fuchs
  2. Asako Isogawa
  3. Joao A Paulo
  4. Kazumitsu Onizuka
  5. Tatsuro Takahashi
  6. Ravindra Amunugama
  7. Julien P Duxin
  8. Shingo Fujii

  • Curated by eLife

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    Evaluation summary:

    Glioblastomas, like many tumors, consist of a cohort of actively dividing cells and a substantially larger fraction of non-proliferating cells. The standard of care involves the administration of a chemotherapy drug (temozolomide (TMZ)) whose antitumor activity is thought to be dependent on a toxic intermediate produced during DNA replication. In this report, the authors show how this compound is also processed by the interaction of two DNA repair pathways which produce the same intermediate without the requirement for DNA replication. The paper will be of interest to those scientists concerned with the implications of DNA damage and repair for cancer chemotherapy, particularly for tumors as deadly as glioblastoma.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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Abstract

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O 6 -methylguanine (O 6 mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O 6 mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O 6 mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O 6 mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O 6 mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.

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  1. Version published to 10.7554/elife.69544 on eLife
  2. eLife

    Author Response:

    Joint Public Review:

    The Mismatch Repair (MMR) pathway removes mismatched bases from newly synthesized DNA strands. Strand discrimination is driven by single strand breaks in the daughter strands. MMR can also recognize some adducts formed by methylating chemotherapeutics, such as temozolomide (TMZ), the standard treatment for glioblastoma. TMZ, and the mimic N-methyl-N-nitrosourea (MNU), methylate guanine at N7 (7mG) and adenine at N3 (3mA). These account for 80-90% of total adducts and are repaired by the Base Excision Repair (BER) pathway. However, they also form 8-9% O6-methylguanine (O6-mG), which is cytotoxic and mutagenic and not repaired by BER. O6-mG can pair with T during replication giving rise to O6-mG:T lesions. This mismatch is recognized by MMR but provokes a "futile cycle" of repair in which the T, since it is in the daughter strand, is removed, after which repair synthesis restores the O6-mG:T. It has been proposed that, in the subsequent S phase, replication across gaps generated during the futile cycles results in toxic double strand breaks (DSBs). The key feature of this model is the requirement for two cycles of replication, the first to generate the provocative O6-mG: T mismatch, the second to produce the breaks. Versions of this scenario have been the primary concern of the field for many years.

    The submission from Fuchs and colleagues presents an additional and non-conventional model for MNU/TMZ toxicity. Their experimental approach departs from the requirement for replication and emphasizes the initial O6-mG:C lesion rather than O6-mG:T. They follow repair synthesis in plasmids treated with either MNU or methyl methane sulfonate (MMS) which produces high levels of 7mG and 3mA, but low levels of O6-mG:C. The plasmids were incubated in Xenopus egg extracts that support repair but not replication. They found that MMR proteins bound the MNU treated plasmid but not the MMS treated plasmid and that there was greater repair synthesis in the plasmid treated with MNU than with MMS. They also observed that the BER pathway was important for repair synthesis of the MNU treated plasmid. Experiments with a plasmid carrying a single defined O6-mG:C with or without MMS treatment supported this conclusion. Based on these and other observations they argue that BER of the 7mG and 3mA adducts introduced nicks that were exploited by MMR to drive gap formation and repair synthesis at sites of O6-mG:C. DSBs were formed in the plasmids undergoing both BER (against the N methyl adducts) and MMR against O6-mG:C. Their results support a model in which BER nicking at sites of N methyl adducts provides an enhanced opportunity for MMR of the O6-mG:C lesions. Extended exonuclease digestion by MMR reaches sites undergoing BER on the other strand thus generating DSBs.

    Although there is an extensive literature on replication-dependent production and processing of the MNU/TMZ O6-mG:T lesion, this report is novel in the attention to replication-independent repair of the primary mismatch product. Chemotherapy has typically been premised on targeting replicating cells. However, the majority of cells in a glioblastoma tumor are not proliferating, and insight into attacking non dividing cells might be very useful in treating this almost always fatal tumor. The author's data support their model, although some of the implications of their conclusions could be more fully developed. Additional data on two aspects would strengthen the paper.

    The first reflects the considerable interest in manipulations of DNA repair pathways that would enhance the toxicity towards tumors of DNA reactive chemotherapy drugs. The authors propose that the introduction of nicks during the early steps of BER are responsible for the enhanced efficacy of MMR in generating the DSBs. However, the later steps of BER act to reverse the nicks. The extract system would appear to lend itself to the identification of the later steps in the BER pathway which, if inhibited, would increase DSB formation by MMR mediated gap formation on one strand past nicks on the other.

    The second would extend the approach beyond the extracts. The authors have effectively exploited this system to identify key proteins responding to model substrates and address certain mechanistic questions with those substrates. However, the extracts cannot recapitulate all the features of repair/toxicity of MNU/TMZ adducts in the chromatin environment of the human genome. Although the authors allude to future cell-based assays, the paper would benefit by an initial test of the new model in a live cell system.

    The authors should also consider an apparent discrepancy with earlier work. Figure 1 describes the recovery of MMR proteins bound to the plasmid treated with MNU. This treatment would yield O6-mG:C in addition to the guanine and adenine N-alkylation products. Several years ago the Hsieh lab found that purified MutS alpha failed to bind O6-mG:C but recognized O6-mG:T (Mol Cell 22, 501, 2006). However, in this submission the authors report binding of MutS alpha to the plasmid with O6-mG:C. Current models suggest that mismatch binding by MutS alpha initiates the repair process (see Ortega, Cell Res. 31, 542, 2021). In the light of the report from the Hsieh lab the authors' results would seem to imply that something in the extract in addition to MutS alpha is required for that binding. The recognition of O6-mG:C is central to their model, and it would be useful for them to discuss how they reconcile their results with those of the Hsieh lab. In addition, there is a discrepancy with an earlier publication (Olivera Harris et al. 2015 DNA Repair about the effectiveness of the MGMT inhibitor Patrin-2 in Xenopus extracts that should be reconciled.

    Indeed, in the Hsieh paper, purified MutSa, is shown not to bind O6mG:C pairs. Our experiments involve extracts containing many proteins and there is probably synergy between MutSa and MutLa to achieve full MMR (as supported by the 2021 Ortega paper). Moreover, activation of MMR by a single O6mG:C lesion has been reported previously by the Modrich group as referenced in our paper (p7) (Duckett et al., 1999).

  3. eLife

    Evaluation summary:

    Glioblastomas, like many tumors, consist of a cohort of actively dividing cells and a substantially larger fraction of non-proliferating cells. The standard of care involves the administration of a chemotherapy drug (temozolomide (TMZ)) whose antitumor activity is thought to be dependent on a toxic intermediate produced during DNA replication. In this report, the authors show how this compound is also processed by the interaction of two DNA repair pathways which produce the same intermediate without the requirement for DNA replication. The paper will be of interest to those scientists concerned with the implications of DNA damage and repair for cancer chemotherapy, particularly for tumors as deadly as glioblastoma.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

  4. eLife

    Joint Public Review:

    The Mismatch Repair (MMR) pathway removes mismatched bases from newly synthesized DNA strands. Strand discrimination is driven by single strand breaks in the daughter strands. MMR can also recognize some adducts formed by methylating chemotherapeutics, such as temozolomide (TMZ), the standard treatment for glioblastoma. TMZ, and the mimic N-methyl-N-nitrosourea (MNU), methylate guanine at N7 (7mG) and adenine at N3 (3mA). These account for 80-90% of total adducts and are repaired by the Base Excision Repair (BER) pathway. However, they also form 8-9% O6-methylguanine (O6-mG), which is cytotoxic and mutagenic and not repaired by BER. O6-mG can pair with T during replication giving rise to O6-mG:T lesions. This mismatch is recognized by MMR but provokes a "futile cycle" of repair in which the T, since it is in the daughter strand, is removed, after which repair synthesis restores the O6-mG:T. It has been proposed that, in the subsequent S phase, replication across gaps generated during the futile cycles results in toxic double strand breaks (DSBs). The key feature of this model is the requirement for two cycles of replication, the first to generate the provocative O6-mG: T mismatch, the second to produce the breaks. Versions of this scenario have been the primary concern of the field for many years.

    The submission from Fuchs and colleagues presents an additional and non-conventional model for MNU/TMZ toxicity. Their experimental approach departs from the requirement for replication and emphasizes the initial O6-mG:C lesion rather than O6-mG:T. They follow repair synthesis in plasmids treated with either MNU or methyl methane sulfonate (MMS) which produces high levels of 7mG and 3mA, but low levels of O6-mG:C. The plasmids were incubated in Xenopus egg extracts that support repair but not replication. They found that MMR proteins bound the MNU treated plasmid but not the MMS treated plasmid and that there was greater repair synthesis in the plasmid treated with MNU than with MMS. They also observed that the BER pathway was important for repair synthesis of the MNU treated plasmid. Experiments with a plasmid carrying a single defined O6-mG:C with or without MMS treatment supported this conclusion. Based on these and other observations they argue that BER of the 7mG and 3mA adducts introduced nicks that were exploited by MMR to drive gap formation and repair synthesis at sites of O6-mG:C. DSBs were formed in the plasmids undergoing both BER (against the N methyl adducts) and MMR against O6-mG:C. Their results support a model in which BER nicking at sites of N methyl adducts provides an enhanced opportunity for MMR of the O6-mG:C lesions. Extended exonuclease digestion by MMR reaches sites undergoing BER on the other strand thus generating DSBs.

    Although there is an extensive literature on replication-dependent production and processing of the MNU/TMZ O6-mG:T lesion, this report is novel in the attention to replication-independent repair of the primary mismatch product. Chemotherapy has typically been premised on targeting replicating cells. However, the majority of cells in a glioblastoma tumor are not proliferating, and insight into attacking non dividing cells might be very useful in treating this almost always fatal tumor. The author's data support their model, although some of the implications of their conclusions could be more fully developed. Additional data on two aspects would strengthen the paper.

    The first reflects the considerable interest in manipulations of DNA repair pathways that would enhance the toxicity towards tumors of DNA reactive chemotherapy drugs. The authors propose that the introduction of nicks during the early steps of BER are responsible for the enhanced efficacy of MMR in generating the DSBs. However, the later steps of BER act to reverse the nicks. The extract system would appear to lend itself to the identification of the later steps in the BER pathway which, if inhibited, would increase DSB formation by MMR mediated gap formation on one strand past nicks on the other.

    The second would extend the approach beyond the extracts. The authors have effectively exploited this system to identify key proteins responding to model substrates and address certain mechanistic questions with those substrates. However, the extracts cannot recapitulate all the features of repair/toxicity of MNU/TMZ adducts in the chromatin environment of the human genome. Although the authors allude to future cell-based assays, the paper would benefit by an initial test of the new model in a live cell system.

    The authors should also consider an apparent discrepancy with earlier work. Figure 1 describes the recovery of MMR proteins bound to the plasmid treated with MNU. This treatment would yield O6-mG:C in addition to the guanine and adenine N-alkylation products. Several years ago the Hsieh lab found that purified MutS alpha failed to bind O6-mG:C but recognized O6-mG:T (Mol Cell 22, 501, 2006). However, in this submission the authors report binding of MutS alpha to the plasmid with O6-mG:C. Current models suggest that mismatch binding by MutS alpha initiates the repair process (see Ortega, Cell Res. 31, 542, 2021). In the light of the report from the Hsieh lab the authors' results would seem to imply that something in the extract in addition to MutS alpha is required for that binding. The recognition of O6-mG:C is central to their model, and it would be useful for them to discuss how they reconcile their results with those of the Hsieh lab. In addition, there is a discrepancy with an earlier publication (Olivera Harris et al. 2015 DNA Repair about the effectiveness of the MGMT inhibitor Patrin-2 in Xenopus extracts that should be reconciled.

  5. Version published to 10.1101/2020.11.21.391326v3 on bioRxiv
  6. Version published to 10.1101/2020.11.21.391326v2 on bioRxiv
  7. Version published to 10.1101/2020.11.21.391326v1 on bioRxiv