| 1. |
- Bonagas, Nadilly, et al.
(author)
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Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress
- 2022
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In: NATURE CANCER. - : Springer Science and Business Media LLC. - 2662-1347. ; 3:2, s. 156-
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Journal article (peer-reviewed)abstract
- The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors. Helleday and colleagues describe a nanomolar MTHFD2 inhibitor that causes replication stress and DNA damage accumulation in cancer cells via thymidine depletion, demonstrating a potential therapeutic strategy in AML tumors in vivo.
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| 2. |
- Bräutigam, Lars, et al.
(author)
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Hypoxic Signaling and the Cellular Redox Tumor Environment Determine Sensitivity to MTH1 Inhibition
- 2016
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In: Cancer Research. - 0008-5472 .- 1538-7445. ; 76:8, s. 2366-2375
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Journal article (peer-reviewed)abstract
- Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1 alpha response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance.
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| 3. |
- Pudelko, Linda, et al.
(author)
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An orthotopic glioblastoma animal model suitable for high-throughput screenings
- 2018
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In: Neuro-Oncology. - : Oxford University Press. - 1522-8517 .- 1523-5866. ; 20:11, s. 1475-1484
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Journal article (peer-reviewed)abstract
- Background. Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens. Methods. We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy. Results. We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease. Conclusion. We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens.
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| 4. |
- Pudelko, Linda
(author)
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Fishing for cures : the zebrafish as a powerful tool to identify novel therapies against glioblastoma by targeting MTH1 and beyond
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Glioblastoma (GBM) is the most aggressive form of brain cancer. Despite today’s combinatory therapy consisting of surgery, radio- and chemotherapy, the prognosis remains dismal. Fostered by extensive tumor heterogeneity, cancer cell plasticity and the presence of cancer stem cells, GBM evades almost any therapeutic strategy, leading to high mortality. Thus, the development of novel therapies is of urgent need. With the identification of the Hallmarks of Cancer several cancer specific characteristics have been described that could serve as promising anti-cancer targets, including the combination of an elevated proliferation rate, crucial changes in cancer metabolism and consequently, an altered redox environment. Cancer cells and GBM in particular depend on effective anti- oxidant defense systems and non-oncogenic addiction enzymes such as MTH1, an enzyme that detoxifies oxidized bases to prevent DNA damage and subsequent cell death. While potential anti-cancer targets are constantly being identified, the development of novel therapies against GBM is, amongst other reasons, hampered by the lack of orthotopic animal models that support large drug discovery screens. During the last decade, the zebrafish has been introduced as a clinically relevant model for human malignancies including cancer. Owing its biological and technical advantages, the zebrafish is the only vertebrate animal suitable for automated drug discovery screens to facilitate the identification and validation of novel cancer therapies. In this thesis, we primarily focused on complementing established biochemical and cellular assays with a broad application of the zebrafish model to: 1. Describe factors that render cancer cells sensitive to MTH1 inhibitors 2. Validate MTH1 as a arget in GBM and GBM stem cells 3. Develop a new orthotopic in vivo model for GBM In Paper I we have demonstrated that the cellular redox environment and activation of the hypoxia signaling axis determine sensitivity to MTH1 inhibition in vitro and in vivo, thus suggesting that MTH1 inhibition may present a promising approach to treat cancers characterized by deregulated hypoxia signaling and redox imbalance. In Paper II we have tested this hypothesis and showed that depletion or inhibition of MTH1 efficiently reduces viability of patient-derived GBM cultures independent of aggressiveness i in vitro and in vivo, thus providing supporting data that MTH1 represents a promising target for GBM therapy in particular. In Paper III we addressed the lack of an orthotopic animal model for GBM which is suitable for large drug discovery screens. We found that GBM cultures transplanted into the blastoderm of zebrafish embryos form a congregated tumor in the central nervous system, fully recapitulating the human disease. As no intracranial transplantation is required, we have developed an orthotopic animal model for GBM that could readily be implemented in fully automatable drug discovery screens in order to accelerate the identification and development of novel therapies against GBM.
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| 5. |
- Pudelko, Linda, et al.
(author)
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Glioblastoma and glioblastoma stem cells are dependent on functional MTH1
- 2017
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In: Oncotarget. - : Impact Journals LLC. - 1949-2553. ; 8:49, s. 84671-84684
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Journal article (peer-reviewed)abstract
- Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease.
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| 6. |
- Zhang, Si Min, et al.
(author)
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Development of a chemical probe against NUDT15
- 2020
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In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 16:10, s. 1120-1128
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Journal article (peer-reviewed)abstract
- The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
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