Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5'-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS.

Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor a (TNF-a). HepG2 cells were treated with diclofenac followed by TNF-a challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-a. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-a-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-a-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-a-mediated nuclear factor kappaB (NF-jB) translocation oscillation in association with reduced NF-jB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-ainduced phosphorylation of the inhibitor of NF-jB alpha (IjBa). Finally, inhibition of IjB kinase b (IKKb) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNFa-induced cytotoxicity. Conclusion: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-a-induced survival signaling routes and sensitizes cells to apoptosis. (HEPATOLOGY 2011;53:2027-2041 A dverse drug reactions are an important cause of morbidity and mortality in humans and druginduced liver injuries (DILIs) are the leading cause of acute liver failure. 1 In addition, DILI accounts for most of the drug attritions 2 and more than 10% of the occurring liver failures happen due to idiosyncratic DILIs. 1 We propose that the crosstalk between drug reactive metabolite-mediated stress responses and cytokine-mediated pro-and antiapoptotic signaling is an important component in the pathophysiology of DILI.Abbreviations: AIF, apoptosis inducing factor; AnxV, annexin V; APAF1, apoptotic protease activating factor 1; c-FLIP, cellular FLICE-like inhibitory protein; DILI, drug-induced liver injury; GFP, green fluorescent protein; IjBa, inhibitor of NF-jB a; IKKb, IjB kinase b; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen activated protein kinase; NF-jB, nuclear factor-jB; NSAID, nonsteroidal antiinflammatory drug; PUMA, p53 up-regulated modulator of apoptosis; ROS, reactive oxygen species; RAIDD, RIP-associated protein with a death domain, siRNA, short interfering RNA; TNFR-1, TNF receptor-1; TNF-a, tumor necrosis factor a.From the