Origins of nonsense mutations in human tumor suppressor genes.

Understanding the origins of mutations in tumor suppressor genes and oncogenes associated with cancers in different tissues is critical to the development of potential prevention strategies. Analysis of >10,000 nonsense mutations in 63 tumor suppressor genes based on the ratio of the number of nonsense mutations per codon type is reported for each gene. The ratio for C•G→T•A nonsense mutations at Arg CGA codons to the number of CGA codons in all cancers is 23 (3088 total nonsense mutations for 134 CGA codons in the 63 suppressor genes). The ratio for this codon, which is attributed to hydrolytic deamination of 5-methylcytosine at CpG sites based on the sequence context, is 6-fold higher than the next highest ratio that involves a C•G→T•A transition at Trp TGG codons. C•G→A•T transversions at Glu, Ser, Tyr, Gly and Cys codons account for 25 % of the total nonsense mutations but the mutation per codon ratio for these codons is 1.0. Analysis of the bases 5' of the mutated CGA codons in the 63 tumor suppressor genes in all cancers shows a preference of 5'-G > C ∼ T ∼ A, which is not indicative of a role for enzymatic deamination by deaminases. Overall C•G→T•A mutations account for 61 % of all of the nonsense mutations in the collection of tumor suppressor genes. It is demonstrated that the ratio of C•G→T•A deamination-associated nonsense mutations at CGA codons (hydrolytic deamination) to the number of frame shift insertion/deletion mutations (i.e., replication based) for 5 major tumor suppressors genes are very similar in 3 different tissues that undergo a wide range of stem cell divisions. Therefore, the frequency of deamination mutations parallels the number of stem cell replications. This may reflect the generation of more solvent accessible single-stranded DNA regions during polymerization that are kinetically more prone to deamination.

Origin of mutations in genes associated with human glioblastoma multiform cancer: random polymerase errors versus deamination.

The etiology of glioblastoma multiforme (GBM), the most serious form of brain cancer, remains obscure, although it has been proposed that cancer risk is a function of random polymerase errors that occur during stem cell division and the resulting mutations in oncogenes and tumor suppressor genes. Analysis of the 8 genes (PTEN, TP53, EGFR, PIK3R1, PIK3CA, NF1, RB1, IDH1) that are mutated in at least 5% of GBM tumors indicates a non-random mutation pattern that reflects a significant role for hydrolytic deamination at CpG sites. The formation of activating mutations in some genes, e.g., IDH1, where a very limited set of mutations are oncogenic, statistically cannot involve random mutagenesis due to polymerase errors that occur during each stem cell replication. Comparison of the in vitro misincorporation tendencies of three replicative polymerases and the "random" mutation pattern in a subset of genes indicates non-polymerase based pathways are involved. Analysis of the mutation patterns shows that chemical deamination that occurs at a slow rate at each CpG is favored over random polymerase errors by a factor of more than 10 million. Therefore, if a truncating nonsense mutation in a tumor suppressor, or an activating missense mutation in an oncogene, can occur due to a C > T base substitution at a CpG sequence, it is highly favored over other mutation pathways.

Thermodynamic Stability of DNA Duplexes Comprising the Simplest T → dU Substitutions.

Members of the uracil-DNA glycosylase (UDG) enzyme family recognize and bind uracil, sequestering it within the binding site pocket and catalyzing the cleavage of the base from the deoxyribose, leaving an abasic site. The recognition and binding are passive and rely on innate dynamic motions of DNA wherein base pairs undergo thermally induced breakage and conformational fluctuations. Once the uracil breaks from its base pair, it can be recognized and bound by the enzyme, which then alters its conformation for sequestration and catalysis. Our results suggest that the thymine to uracil substitution, which differs only by a single methyl group, causes a destabilization of the duplex thermodynamics, which would lead to an increase in the population of the extrahelical state and increase the probability of uracil being recognized and excised from DNA by UDG. This destabilization is dependent on the identity of the nearest-neighbor base-pair stacks; a G·C nearest neighbor leads to thermal and enthalpic destabilization that is weaker that that seen with two A·T neighbors. In addition, uracil substitution yields a nearest-neighbor increase in the counterion uptake of the duplexes but decreases the level of immobilization of structural water for all substituted duplexes regardless of the neighbor identity or number of substitutions.

The Adenomatous Polyposis Coli (APC) mutation spectra in different anatomical regions of the large intestine in colorectal cancer.

The enhanced incidence of colorectal cancer (CRC) in the U.S.A. has been linked to promutagens, such as heterocyclic aromatic amines, in the western diet that are produced by high temperature cooking of meat. However, a prior analysis of driver nonsense mutations in the Adenomatous Polyposis Coli (APC) gene, which is mutated in 75% of human CRC, indicated that the C·G → A·T transversions produced by this class of mutagens were not enriched but actually lower than what would be statistically anticipated. Moreover, the APC mutation patterns in the U.S.A. vs. China were indistinguishable despite differences in diet. In the present study, we have dissected the APC mutation pattern in tumors that arise in the different anatomical regions of the large intestine. The results show that the nonsense mutation pattern in APC differ in the different regions and that there is a statistically significant increase in C·G → A·T transversions in the rectum vs. the other regions, albeit, the percent of C·G → A·T mutations still remains lower than predicted based on random mutagenesis.

Origin of Somatic Mutations in ß-Catenin versus Adenomatous Polyposis Coli in Colon Cancer: Random Mutagenesis in Animal Models versus Nonrandom Mutagenesis in Humans.

Wnt signaling is compromised early in the development of human colorectal cancer (CRC) due to truncating nonsense mutations in adenomatous polyposis coli (APC). CRC induced by chemical carcinogens, such as heterocyclic aromatic amines and azoxymethane, in mice also involves dysregulation of Wnt signaling but via activating missense mutations in the ß-catenin oncogene despite the fact that genetically modified mice harboring an inactive APC allele efficiently develop CRC. In contrast, activating mutations in ß-catenin are rarely observed in human CRC. Dysregulation of the Wnt signaling pathway by the two distinct mechanisms reveals insights into the etiology of human CRC. On the basis of calculations related to DNA adduct levels produced in mouse CRC models using mutagens, and the number of stem cells in the mouse colon, we show that two nonsense mutations required for biallelic disruption of APC are statistically unlikely to produce CRC in experiments using small numbers of mice. We calculate that an activating mutation in one allele near the critical GSK3ß phosphorylation site on ß-catenin is >105-times more likely to produce CRC by random mutagenesis due to chemicals than inactivating two alleles in APC, yet it does not occur in humans. Therefore, the mutagenesis mechanism in human CRC cannot be random. We explain that nonsense APC mutations predominate in human CRC because of deamination at 5-methylcytosine at CGA and CAG codons, coupled with the number of human colonic stem cells and lifespan. Our analyses, including a comparison of mutation type and age at CRC diagnosis in U.S. and Chinese patients, also indicate that APC mutations in CRC are not due to environmental mutagens that randomly damage DNA.

Somatic mutations in cancer: Stochastic versus predictable.

The origins of human cancers remain unclear except for a limited number of potent environmental mutagens, such as tobacco and UV light, and in rare cases, familial germ line mutations that affect tumor suppressor genes or oncogenes. A significant component of cancer etiology has been deemed stochastic and correlated with the number of stem cells in a tissue, the number of times the stem cells divide and a low incidence of random DNA polymerase errors that occur during each cell division. While somatic mutations occur during each round of DNA replication, mutations in cancer driver genes are not stochastic. Out of a total of 2843 codons, 1031 can be changed to stop codons by a single base substitution in the tumor suppressor APC gene, which is mutated in 76% of colorectal cancers (CRC). However, the nonsense mutations, which comprise 65% of all the APC driver mutations in CRC, are not random: 43% occur at Arg CGA codons, although they represent <3% of the codons. In TP53, CGA codons comprise <3% of the total 393 codons but they account for 72% and 39% of the mutations in CRC and ovarian cancer OVC, respectively. This mutation pattern is consistent with the kinetically slow, but not stochastic, hydrolytic deamination of 5-methylcytosine residues at specific methylated CpG sites to afford T·G mismatches that lead to C→T transitions and stop codons at CGA. Analysis of nonsense mutations in CRC, OVC and a number of other cancers indicates the need to expand the predictable risk factors for cancer to include, in addition to random polymerase errors, the methylation status of gene body CGA codons in tumor suppressor genes.

Colon carcinogenesis in wild type and immune compromised mice after treatment with azoxymethane, and azoxymethane with dextran sodium sulfate.

The association between inflammation and the risk of colorectal cancer (CRC) is well documented in animal models and in humans, but the mechanistic role of inflammation in CRC is less well understood. To address this question, the induction of colon tumors was evaluated in (i) wild type (WT) and athymic BALB/c mice treated with the colon carcinogen azoxymethane (AOM) as a single agent, and (ii) in an inflammation model of colon cancer employing AOM and dextran sodium sulfate (DSS) in WT, athymic, TCRß(-/-) , TCRδ(-/-) and TCRß(-/-) TCRδ(-/-) C57Bl/6 mice. The athymic BALB/c mice treated with only AOM developed 90% fewer tumors than the WT mice. The difference in response was not due to metabolic activation of AOM or repair of DNA adducts. In the inflammation model using a standard sequential exposure to AOM followed by DSS treatment, the tumor incidence in WT mice was 58% with 7 adenomas and 6 adenocarcinomas. In contrast, the TCRß(-/-) , TCRδ(-/-) and TCRß(-/-) TCRδ(-/-) C57Bl/6 mice showed adenoma incidences of 10, 33, and 11%, respectively, and none of the immune compromised mice developed adenocarcinomas. When the DSS exposure was increased and the AOM lowered, no difference was observed between WT and TCRß(-/-) mice due to an increase in the incidence in the TCR null mice without concomitant increase in the WT mice. No tumors were observed in mice treated with AOM or DSS alone. © 2015 Wiley Periodicals, Inc.

Quantification of Colonic Stem Cell Mutations.

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.(1) This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 µm thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 µm apart were prepared to assess a total of >10(4) crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts÷by the number of wild type (blue) crypts in a treatment group.

Design and activity of AP endonuclease-1 inhibitors.

Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1) is a critical component of base excision repair that excises abasic lesions created enzymatically by the action of DNA glycosylases on modified bases and non-enzymatically by hydrolytic depurination/depyrimidination of nucleobases. Many anticancer drugs generate DNA adducts that are processed by base excision repair, and tumor resistance is frequently associated with enhanced APE-1 expression. Accordingly, APE-1 is a potential therapeutic target to treat cancer. Using computational approaches and the high resolution structure of APE-1, we developed a 5-point pharmacophore model for APE-1 small molecule inhibitors. One of the nM APE-1 inhibitors (AJAY-4) that was identified based on this model exhibited an overall median growth inhibition (GI50) of 4.19 µM in the NCI-60 cell line panel. The mechanism of action is shown to be related to the buildup of abasic sites that cause PARP activation and PARP cleavage, and the activation of caspase-3 and caspase-7, which is consistent with cell death by apoptosis. In a drug combination growth inhibition screen conducted in 10 randomly selected NCI-60 cell lines and with 20 clinically used non-genotoxic anticancer drugs, a synergy was flagged in the SK-MEL-5 melanoma cell line exposed to combinations of vemurafenib, which targets melanoma cells with V600E mutated BRAF, and AJAY-4, our most potent APE-1 inhibitor. The synergy between AJAY-4 and vemurafenib was not observed in cell lines expressing wild-type B-Raf protein. This synergistic combination may provide a solution to the resistance that develops in tumors treated with B-Raf-targeting drugs.

T-cells enhance stem cell mutagenesis in the mouse colon.

A role of inflammation in the etiology of cancer is attributed to the production of reactive oxygen/nitrogen species that can damage DNA. To test this hypothesis, we determined the mutation frequency (MF) in colonic stem cells in C57Bl/6 mice exposed to azoxymethane (AOM), dextran sulfate sodium (DSS) and a combination of AOM and DSS (AOM+DSS). AOM+DSS efficiently and rapidly produces colon tumors in B6 mice. AOM produces promutagenic O(6)-methylguanine lesions in DNA but does not induce colon tumors in C57Bl/6 mice as a single agent. DSS produces inflammation in the colon but does not produce tumors except upon multiple cycles of treatment in some DNA repair deficient mouse models. In addition, using TCRß null mice we tested whether α/ß T cells have any effect on the colonic stem cell MF in mice treated with AOM, DSS and AOM+DSS. The TCRß(-/-) mice are devoid of the critical receptor required for normal cytolytic and regulatory α/ß T-cell functions. The MF in the untreated and DSS treated WT and TCRß(-/-) mice was the same (<10(-5)) indicating that DSS and subsequent inflammation does not generate stem cell mutations in mice that are WT for DNA repair. AOM yielded mutant crypts in WT and TCRß(-/-) mice with MF's of ∼4×10(-4) and 2×10(-4), respectively, which represents a statistically significant decrease in the MF in the immune compromised mice. The combined treatment of AOM+DSS afforded fully mutated crypts in both strains with a statistically significant lower MF in the TCRß(-/-) mice. In addition, the MF in both strains of mice after the combination of AOM+DSS is lower than observed with AOM alone indicating that DSS inflammation destroyed pre-existing AOM mutated crypts. Using the MF in WT mice, the efficiency for the conversion of promutagenic O(6)-methylguanine lesions into a stem cell mutations was calculated to be ∼0.4%.

Looking for Waldo: a potential thermodynamic signature to DNA damage.

DNA in its simplest form is an ensemble of nucleic acids, water, and ions, and the conformation of DNA is dependent on the relative proportions of all three components. When DNA is covalently damaged by endogenous or exogenous reactive species, including those produced by some anticancer drugs, the ensemble undergoes localized changes that affect nucleic acid structure, thermodynamic stability, and the qualitative and quantative arrangement of associated cations and water molecules. Fortunately, the biological effects of low levels of DNA damage are successfully mitigated by a large number of proteins that efficiently recognize and repair DNA damage in the midst of a vast excess of canonical DNA. In this Account, we explore the impact of DNA modifications on the high resolution and dynamic structure of DNA, DNA stability, and the uptake of ions and water and explore how these changes may be sensed by proteins whose function is to initially locate DNA lesions. We discuss modifications on the nucleobases that are located in the major and minor grooves of DNA and include lesions that are observed in vivo, including oxidized bases, as well as some synthetic nucleobases that allow us to probe how the location and nature of different substituents affect the thermodynamics and structure of the DNA ensemble. It is demonstrated that disruption of a cation binding site in the major groove by modification of the N7-position on the purines, which is the major site for DNA alkylation, is enthalpically destabilizing. Accordingly, tethering a cationic charge in the major groove is enthalpically stabilizing. The combined structural and thermodynamic studies provide a detailed picture of how different DNA lesions affect the dynamics of DNA and how modified bases interact with their environment. Our work supports the hypothesis that there is a "thermodynamic signature" to DNA lesions that can be exploited in the initial search that requires differentiation between canonical DNA and DNA with a lesion. The differentiation between a lesion and a cognate lesion that is a substrate for a particular enzyme involves another layer of thermodynamic and kinetic factors.

Comparison of the biological effects of MMS and Me-lex, a minor groove methylating agent: clarifying the role of N3-methyladenine.

N3-methyladenine (3-mA), generated by the reaction of methylating agents with DNA, is considered a highly toxic but weakly mutagenic lesion. However, due to its intrinsic instability, some of the biological effects of the adduct can result from the formation of the corresponding depurination product [an apurinic (AP)-site]. Previously, we exploited Me-lex, i.e. {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2 carboxamido}propane, a minor groove equilibrium binder with selectivity for A/T rich sequences that efficiently reacts with DNA to afford 3-mA as the dominant product, to probe the biology of this lesion. Using human p53 cDNA as a target in a yeast system, a weak increase in mutagenicity was observed in the absence of Mag1 (3-methyladenine-DNA glycosylase 1, mag1), the enzyme devoted to remove 3-mA from DNA. Moreover, a significant increase in mutagenicity occurred in the absence of the enzymes involved in the repair of AP-sites (AP endonucleases 1 and 2, apn1apn2). Since methyl methanesulfonate (MMS) has been extensively used to explore the biological effects of 3-mA, even though it produces 3-mA in low relative yield, we compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. A mutagenesis reporter plasmid was damaged in vitro by MMS and then transformed into wild-type and Translesion Synthesis (TLS) Polζ (REV3) and Polη (RAD30) deficient strains. Furthermore, a mag1rad30 double mutant strain was constructed and transformed with the DNA plasmid damaged in vitro by Me-lex. The results confirm the important role of Polζ in the mutagenic bypass of MMS and Me-lex induced lesions, with Polη contributing only towards the bypass of Me-lex induced lesions, mainly in an error-free way. Previous and present results point towards the involvement of AP-sites, derived from the depurination of 3-mA, in the observed toxicity and mutagenicity.

Site-specific stabilization of DNA by a tethered major groove amine, 7-aminomethyl-7-deaza-2'-deoxyguanosine.

A cationic 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G¹A²G³A4X5C6G7C8T9C¹°T¹¹C¹²)2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X5 imino resonance remained sharp at 55 °C. Additionally, two 5'-neighboring base pairs, A4:T9 and G³:C¹°, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A4:T9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A4:T9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A4:T9 base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹° base pair, where αK(op) for the G³:C¹° base pair in the modified duplex was 3.0 × 106 versus 4.1 × 106 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X5:C8 and C6:G7 base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

Synthesis and characterization of DNA minor groove binding alkylating agents.

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Identification and characterization of human apurinic/apyrimidinic endonuclease-1 inhibitors.

The repair of abasic sites that arise in DNA from hydrolytic depurination/depyrimidination of the nitrogenous bases from the sugar-phosphate backbone and the action of DNA glycosylases on deaminated, oxidized, and alkylated bases are critical to cell survival. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1; aka APE1/ref-1) is responsible for the initial removal of abasic lesions as part of the base excision repair pathway. Deletion of APE-1 activity is embryonic lethal in animals and is lethal in cells. Potential inhibitors of the repair function of APE-1 were identified based upon molecular modeling of the crystal structure of the APE-1 protein. We describe the characterization of several unique nanomolar inhibitors using two complementary biochemical screens. The most active molecules all contain a 2-methyl-4-amino-6,7-dioxolo-quinoline structure that is predicted from the modeling to anchor the compounds in the endonuclease site of the protein. The mechanism of action of the selected compounds was probed by fluorescence and competition studies, which indicate, in a specific case, direct interaction between the inhibitor and the active site of the protein. It is demonstrated that the inhibitors induce time-dependent increases in the accumulation of abasic sites in cells at levels that correlate with their potency to inhibit APE-1 endonuclease excision. The inhibitor molecules also potentiate by 5-fold the toxicity of a DNA methylating agent that creates abasic sites. The molecules represent a new class of APE-1 inhibitors that can be used to probe the biology of this critical enzyme and to sensitize resistant tumor cells to the cytotoxicity of clinically used DNA damaging anticancer drugs.

Small-molecule inhibitors of DNA damage-repair pathways: an approach to overcome tumor resistance to alkylating anticancer drugs.

A major challenge in the future development of cancer therapeutics is the identification of biological targets and pathways, and the subsequent design of molecules to combat the drug-resistant cells hiding in virtually all cancers. This therapeutic approach is justified based upon the limited advances in cancer cures over the past 30 years, despite the development of many novel chemotherapies and earlier detection, which often fail due to drug resistance. Among the various targets to overcome tumor resistance are the DNA repair systems that can reverse the cytotoxicity of many clinically used DNA-damaging agents. Some progress has already been made but much remains to be done. We explore some components of the DNA-repair process, which are involved in repair of alkylation damage of DNA, as targets for the development of novel and effective molecules designed to improve the efficacy of existing anticancer drugs.