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 β-catenin oncogene despite the fact that genetically modified mice harboring an inactive APC allele efficiently develop CRC. In contrast, activating mutations in β-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 GSK3β phosphorylation site on β-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.

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.

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.

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.

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.

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2′-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2′-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme’s active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2′-deoxycytidine·2′-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2′-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.

In many high-resolution structures of DNA there are ordered waters associated with the floor of the minor groove and extending outward in several layers. It is thought that this hydration structure, along with cations, reduces the Coulombic repulsion of the interstrand phosphates. In previous studies, the replacement of the 3-N atom of adenine with a C−H to afford 3-deazaadenine was shown to decrease the thermodynamic stability of DNA via a reduction in the enthalpic term. Using spectroscopic and calorimetric methods, we report herein a rigorous examination of the thermodynamics of DNA with 3-deazaadenine modifications, and report for the first time how the presence of a minor groove methyl group, i.e., 3-methyl-3-deazaadeine, affects DNA stability, hydration, and cation binding. The methylation of adenine at the N3-position to yield N3-methyladenine represents an important reaction in the toxicity of many anticancer compounds. This minor groove lesion is unstable and cannot be readily studied in terms of its effect on DNA stability or structure. Our studies show that 3-methyl-3-deazaadenine, an isostere of N3-methyladenine, significantly destabilizes DNA (ΔΔG > 4 kcal·mol−1) due to a significant drop in the enthalpy (ΔH) term, which is associated with a lower hydration of the duplex relative to the unfolded state.

The replacement of the 7-N atom on guanine (G) with a C−H to give 7-deazaguanine (c7G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c7G, the tethering of a cation in the major groove using 1 affords DNA that is as, or more, stable than the corresponding unmodified DNA. The stabilization is associated with the folding enthalpy and hydration.

An efficient route to the preparation of 5-substituted 2-amino-7-((2R,4R,5R)-tetrahydro-4-hydroxy-5-(hydroxymethyl)furan-2-yl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one compounds has been developed by the condensation of ω-substituted aldehydes with 2,6-diaminopyrimidin-4(3H)-one, followed by Boc protection to afford the corresponding N2,N2,N7-tris-Boc-O4-t-Bu-5-substituted 2-amino-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one, which is amenable to direct condensation with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-α-d-erythro-pentofuranose. This route affords an efficient synthesis to 2-amino-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one, 2-amino-5-alkyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one, and guanine nucleosides.

•We compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast.•Me-lex efficiently and selectively generates 3-mA (>90%) in contrast to MMS.•Polζ is involved in the mutagenic bypass of MMS and Me-lex induced lesions.•Polη contributes towards the error-free bypass of Me-lex induced lesions.•AP sites are often more toxic than the initiating DNA lesion (3-mA).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.

•A large component of cancer etiology has been deemed stochastic.•Analysis of APC and TP53 driver nonsense mutations in cancers appears predictable.•Methylation status of gene body CGA codons in tumor suppressors predicts mutations.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.

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 O6-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 TCRβ null mice we tested whether α/β T cells have any effect on the colonic stem cell MF in mice treated with AOM, DSS and AOM + DSS. The TCRβ−/− mice are devoid of the critical receptor required for normal cytolytic and regulatory α/β T-cell functions. The MF in the untreated and DSS treated WT and TCRβ−/− 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 TCRβ−/− 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 TCRβ−/− 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 O6-methylguanine lesions into a stem cell mutations was calculated to be ∼0.4%.

We recently demonstrated that Polζ and Rev1 contribute to alleviate the lethal effects of Me-lex, which selectively generates 3-methyladenine, by error prone lesion bypass. In order to determine the role of Polη in the biological fate of Me-lex induced lesions, the RAD30 (Polη) gene was deleted in the yIG397 parental strain and in its rev3 (Polζ) derivative, and the strains transformed with plasmid DNA damaged in vitro by Me-lex. While deletion of RAD30 increased the toxicity of Me-lex, the impact on mutagenicity varied depending on the concentration of Me-lex induced DNA damage and the overall TLS capacity of the cells. For the first time the Me-lex induced mutation spectrum in rad30 strain was determined and compared with the spectrum previously determined in WT strain. Overall, the two mutation spectra were not significantly different. The effect on mutation frequency and the features of the Me-lex induced mutation spectra were suggestive of error prone (significant decrease of mutation frequency and significant decrease of AT > TA at a mutation hotspot in rad30 vs RAD30) but also error free (significant increase of AT > GC in rad30 vs RAD30) Polη dependent bypass of lesions. In summary, our previous results with Polζ and Rev1 mutants, the present results with Polη, and the known physical and functional interactions among TLS proteins, lead us to propose that the bypass of Me-lex induced lesions is a multi-DNA polymerases process that is mostly effective when all three yeast TLS polymerases are present.