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Background: Previously we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently several new and potent inhibitors of MTH1 were demonstrated to be non toxic to cancer cells challenging the utility of MTH1 inhibition as a target for cancer treatment. Material and methods: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8 oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling proteomics cellular thermal shift assays kinase and CEREP panel were used for target engagement mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. Results: Here we demonstrate that recently described MTH1 inhibitors which fail to kill cancer cells also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579 (Karonudib) an analogue of TH588 which is a potent selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti cancer properties in vivo. Conclusion: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore we describe TH1579 as a best in class MTH1 inhibitor which we expect to be useful in order to further validate the MTH1 inhibitor concept. Key words: MTH1 reactive oxygen species cancer small molecule inhibitors DNA damage

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. ©2016 AACR.

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXϕ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls(AEGL) was rescued by the insertion of a classical YXXϕ motif in the cytoplasmic tail. Significantly, a Drosophila Wls(AEGL) mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXϕ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.

The TGF-beta superfamily of secreted signalling molecules plays a pivotal role in the regulation of early embryogenesis, organogenesis and adult tissue homeostasis. Here we report the identification of Xenopus N-acetylgalactosaminyltransferase-like 1 (xGalntl-1) as a novel important regulator of TGF-beta signalling. N-acetylgalactosaminyltransferases mediate the first step of mucin-type glycosylation, adding N-acetylgalactose to serine or threonine side chains. xGalntl-1 is expressed in the anterior mesoderm and neural crest territory at neurula stage, and in the anterior neural crest, notochord and the mediolateral spinal cord at tailbud stage. Inhibition of endogenous xGalntl-1 protein synthesis, using specific morpholino oligomers, interfered with the formation of anterior neural crest, anterior notochord and the spinal cord. Xenopus and mammalian Galntl-1 inhibited Activin as well as BMP signalling in the early Xenopus embryo and in human HEK 293T cells. Gain- and loss-of-function experiments showed that xGalntl-1 interferes with the activity of the common TGF-beta type II receptor ActR-IIB in vivo. In addition, our biochemical data demonstrated that xGalntl-1 specifically interferes with the binding of ActR-IIB to Activin- and BMP-specific type I receptors. This inhibitory activity of xGalntl-1 was dependent on mucin-type glycosylation, as it was sensitive to the chemical inhibitor benzyl-GalNAc. These studies reveal an important role of a N-acetylgalactosaminyltransferase in the regulation of TGF-beta signalling. This novel regulatory mechanism is evolutionarily conserved and, thus, might provide a new paradigm for the regulation of TGF-beta signalling in vertebrates.