Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients. Cancer Res; 76(18); 5501-11. Ó2016 AACR.
Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.
The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.
With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADP-ribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism.
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