Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O6-Methylguanine Methyltransferase, MGMT.

DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct lesions that can be both toxic and mutagenic. Foremost among the mutagenic lesions is O6-methylguanine (m6G), which base pairs with thymine during replication to cause GC → AT mutations. The gpt delta C57BL/6J mouse strain of Nohmi et al. (Mol. Mutagen 1996, 28, 465-70) reliably produces mutational spectra of many DNA damaging agents. In this work, mouse embryo fibroblasts (MEFs) were made from gpt delta C57BL/6J mice and evaluated as a screening tool to determine the qualitative and quantitative features of mutagenesis by N-methyl-N-nitrosourea (MNU), a direct-acting DNA alkylator that serves as a model for environmental N-nitrosamines, such as N-nitrosodimethylamine and therapeutic agents such as Temozolomide. The DNA repair protein MGMT (O6-methylguanine DNA methyltransferase) protects against environmental mutagenesis by DNA methylating agents and, by removing m6G, limits the therapeutic potential of Temozolomide in cancer therapy. The gpt delta MEFs were treated with MNU to establish dose-dependent toxicity. In parallel, MNU mutagenicity was determined in the presence and absence of the MGMT inhibitor AA-CW236 (4-(2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole). With and without the inhibitor, the principal mutagenic event of MNU was GC → AT, but more mutations were observed when the inhibitor was present. Evidence that the mutagenic lesion was m6G was based on mass spectral data collected using O6-methyl-d3-guanine as an internal standard; m6G levels were higher in AA-CW236 treated MEFs by an amount proportional to the higher mutation frequency seen in the same cells. This work establishes gpt delta MEFs as a versatile tool for probing mutagenesis by environmental and therapeutic agents and as a cell culture model in which chemical genetics can be used to determine the impact of DNA repair on biological responses to DNA damaging agents.

Structural characterization of new deoxycytidine kinase inhibitors rationalizes the affinity-determining moieties of the molecules.

Deoxycytidine kinase (dCK) is a key enzyme in the nucleoside salvage pathway that is also required for the activation of several anticancer and antiviral nucleoside analog prodrugs. Additionally, dCK has been implicated in immune disorders and has been found to be overexpressed in several cancers. To allow the probing and modulation of dCK activity, a new class of small-molecule inhibitors of the enzyme were developed. Here, the structural characterization of four of these inhibitors in complex with human dCK is presented. The structures reveal that the compounds occupy the nucleoside-binding site and bind to the open form of dCK. Surprisingly, a slight variation in the nature of the substituent at the 5-position of the thiazole ring governs whether the active site of the enzyme is occupied by one or two inhibitor molecules. Moreover, this substituent plays a critical role in determining the affinity, improving it from >700 to 1.5 nM in the best binder. These structures lay the groundwork for future modifications that would result in even tighter binding and the correct placement of moieties that confer favorable pharmacodynamics and pharmacokinetic properties.

Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

IMPORTANCE: H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

Molecular origins of mutational spectra produced by the environmental carcinogen N-nitrosodimethylamine and SN1 chemotherapeutic agents.

DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O 6-methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of ∼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC→AT mutations in 5'-Pu-G-3' contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice.

N-Nitrosodimethylamine (NDMA) is a DNA-methylating agent that has been discovered to contaminate water, food, and drugs. The alkyladenine DNA glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we study Aag-knockout (Aag-/-) and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine [3MeA]) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switches from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrate a suite of molecular, cellular, and physiological analyses. We find that unrepaired 3MeA is somewhat toxic, but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tip the balance between blocks and breaks and thus dictate the disease consequences of DNA damage.

Structure-guided development of deoxycytidine kinase inhibitors with nanomolar affinity and improved metabolic stability.

Recently, we have shown that small molecule dCK inhibitors in combination with pharmacological perturbations of de novo dNTP biosynthetic pathways could eliminate acute lymphoblastic leukemia cells in animal models. However, our previous lead compound had a short half-life in vivo. Therefore, we set out to develop dCK inhibitors with favorable pharmacokinetic properties. We delineated the sites of the inhibitor for modification, guided by crystal structures of dCK in complex with the lead compound and with derivatives. Crystal structure of the complex between dCK and the racemic mixture of our new lead compound indicated that the R-isomer is responsible for kinase inhibition. This was corroborated by kinetic analysis of the purified enantiomers, which showed that the R-isomer has >60-fold higher affinity than the S-isomer for dCK. This new lead compound has significantly improved metabolic stability, making it a prime candidate for dCK-inhibitor based therapies against hematological malignancies and, potentially, other cancers.

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication.

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

Development of new deoxycytidine kinase inhibitors and noninvasive in vivo evaluation using positron emission tomography.

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1-12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.

Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress.

Nucleotide deficiency causes replication stress (RS) and DNA damage in dividing cells. How nucleotide metabolism is regulated in vivo to prevent these deleterious effects remains unknown. In this study, we investigate a functional link between nucleotide deficiency, RS, and the nucleoside salvage pathway (NSP) enzymes deoxycytidine kinase (dCK) and thymidine kinase (TK1). We show that inactivation of dCK in mice depletes deoxycytidine triphosphate (dCTP) pools and induces RS, early S-phase arrest, and DNA damage in erythroid, B lymphoid, and T lymphoid lineages. TK1(-/-) erythroid and B lymphoid lineages also experience nucleotide deficiency but, unlike their dCK(-/-) counterparts, they still sustain DNA replication. Intriguingly, dCTP pool depletion, RS, and hematopoietic defects induced by dCK inactivation are almost completely reversed in a newly generated dCK/TK1 double-knockout (DKO) mouse model. Using NSP-deficient DKO hematopoietic cells, we identify a previously unrecognized biological activity of endogenous thymidine as a strong inducer of RS in vivo through TK1-mediated dCTP pool depletion. We propose a model that explains how TK1 and dCK "tune" dCTP pools to both trigger and resolve RS in vivo. This new model may be exploited therapeutically to induce synthetic sickness/lethality in hematological malignancies, and possibly in other cancers.