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Background: Breast cancer remains as one of the most lethal types of cancer in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat type of breast cancer. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and disease recurrence., Methods: We evaluated the mechanism of prexasertib-induced regulation of homologous recombination (HR) proteins using 20S proteasome inhibitors and RT-PCR. HR efficiency and DNA damages were evaluated using Dr-GFP and comet assays. DNA morphology and DNA repair focus studies were analyzed using immunofluorescence. UALCAN portal was used to evaluate the expression of RAD51 and survival probability based on tumor stage, subtype, and race in breast cancer patients., Results: Our results show that prexasertib treatment promotes both post-translational and transcriptional mediated regulation of BRCA1 and RAD51 proteins. Additionally, prexasertib-treated TNBC cells revealed over 55% reduction in HR efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) and thus should synergize with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of prexasertib and PARPi olaparib increased DNA strand breaks, γH2AX foci, and nuclear disintegration relative to single-agent treatment. Further, the prexasertib and olaparib combination was synergistic in multiple TNBC cell lines, as indicated by combination index (CI) values. Analysis of TCGA data revealed elevated RAD51 expression in breast tumors compared to normal breast tissues, especially in TNBC subtype. Interestingly, there was a discrepancy in RAD51 expression in racial groups, with African-American and Asian breast cancer patients showing elevated RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African-American and Asian TNBC patients show decreased survival., Conclusions: Based on these data, RAD51 could be a biomarker for aggressive TNBC and for racial disparity in breast cancer. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, the in vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved outcomes and reduced racial disparity in TNBC.

F10 is a novel polymeric fluoropyrimidine drug candidate with strong anticancer activity in multiple preclinical models. F10 has strong potential for impacting cancer treatment because it displays high cytotoxicity toward proliferating malignant cells with minimal systemic toxicities thus providing an improved therapeutic window relative to traditional fluoropyrimidine drugs, such as 5-fluorouracil. F10 has a unique mechanism that involves dual targeting of thymidylate synthase and Top1. In this review, the authors provide an overview of the studies that revealed the novel aspects of F10's cytotoxic mechanism and summarize results obtained in preclinical models of acute myeloid leukemia, acute lymphocytic leukemia, glioblastoma and prostate cancer that demonstrate the strong potential of F10 to improve treatment outcomes., Competing Interests: Financial & competing interests disclosure The authors advise Salzburg Therapeutics, which is involved in commercialization of F10 for cancer treatment and are inventors on issued patents and pending patent applications filed by Wake Innovations. The authors are grateful for support from the Wake Forest Baptist Comprehensive Cancer Center P30 CA012197. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

The polymeric fluoropyrimidine F10 displays excellent anti-leukemia activity in pre-clinical models of acute myelogenous leukemia (AML) through dual targeting of thymidylate synthase and DNA topoisomerase 1. Here we report that F10 activates the extrinsic apoptotic pathway in AML cells by enhancing localization of Fas and Fas ligand (FasL) at the plasma membrane and while reducing overall lipid raft levels promotes Fas/FasL co-localization in remaining lipid rafts. The HMG-CoA synthase inhibitor simvastatin was synergistic with F10 and induced cell death via similar apoptotic processes. Our results are consistent with diverse processes activating a common apoptotic pathway characterized by reduced overall levels of lipid rafts and Fas/FasL co-localization in the plasma membrane, including in remaining lipid rafts which may play a role in both cell-survival and cell death signaling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

F10 is a novel anti-tumor agent with minimal systemic toxicity in vivo and which displays strong cytotoxicity towards glioblastoma (GBM) cells in vitro. Here we investigate the cytotoxicity of F10 towards GBM cells and evaluate the anti-tumor activity of locally-administered F10 towards an orthotopic xenograft model of GBM. The effects of F10 on thymidylate synthase (TS) inhibition and Topoisomerase 1 (Top1) cleavage complex formation were evaluated using TS activity assays and in vivo complex of enzyme bioassays. Cytotoxicity of F10 towards normal brain was evaluated using cortices from embryonic (day 18) mice. F10 displays minimal penetrance of the blood-brain barrier and was delivered by intra-cerebral (i.c.) administration and prospective anti-tumor response towards luciferase-expressing G48a human GBM tumors in nude mice was evaluated using IVIS imaging. Histological examination of tumor and normal brain tissue was used to assess the selectivity of anti-tumor activity. F10 is cytotoxic towards G48a, SNB-19, and U-251 MG GBM cells through dual targeting of TS and Top1. F10 is not toxic to murine primary neuronal cultures. F10 is well-tolerated upon i.c. administration and induces significant regression of G48a tumors that is dose-dependent. Histological analysis from F10-treated mice revealed tumors were essentially completely eradicated in F10-treated mice while vehicle-treated mice displayed substantial infiltration into normal tissue. F10 displays strong efficacy for GBM treatment with minimal toxicity upon i.c. administration establishing F10 as a promising drug-candidate for treating GBM in human patients.

DNA and porphyrin based therapeutics are important for anti-cancer treatment. The present studies demonstrate single-stranded DNA (ssDNA) assembles with meso-tetra-4-pyridyl porphine (MTP) forming porphyrin:DNA nano-complexes (PDN) that are stable in aqueous solution under physiologically relevant conditions and undergo dissociation with DNA release in hydrophobic environments, including cell membranes. PDN formation is DNA-dependent with the ratio of porphyrin:DNA being approximately two DNA nucleobases per porphyrin. PDN produce reactive oxygen species (ROS) in a light-dependent manner under conditions that favor nano-complex dissociation in the presence of hydrophobic solvents. PDN induce light-dependent cytotoxicity in vitro and anti-tumor activity towards bladder cancer xenografts in vivo. Light-dependent, PDN-mediated cell death results from ROS-mediated localized membrane damage due to lipid peroxidation with mass spectrometry indicating the generation of the lipid peroxidation products 9- and 13-hydroxy octadecanoic acid. Our results demonstrate that PDN have properties useful for therapeutic applications, including cancer treatment., From the Clinical Editor: In this study, porphyrin-DNA nanocomplexes were investigated as anti-cancer therapeutics inducing ROS production in a light-dependent manner. Efficacy is demonstrated in vitro as well as a in a bladder cancer xenograft model., (© 2014.)

The simultaneous binding of netropsin in the minor groove and Zn(2+) in the major groove of a DNA hairpin that includes 10 consecutive FdU nucleotides at the 3'-terminus (3'FdU) was demonstrated based upon NMR spectroscopy, circular dichroism (CD), and computational modeling studies. The resulting Zn(2+)/netropsin: 3'FdU complex had very high thermal stability with aspects of the complex intact at 85 °C, conditions that result in complete dissociation of Mg(2+) complexes. CD and (19)F NMR spectroscopy were consistent with Zn(2+) binding in the major groove of the DNA duplex and utilizing F5 and O4 of consecutive FdU nucleotides as ligands with FdU nucleotides hemi-deprotonated in the complex. Netropsin is bound in the minor groove of the DNA duplex based upon 2D NOESY data demonstrating contacts between AH2 (1)H and netropsin (1)H resonances. The Zn(2+)/netropsin: 3'FdU complex displayed increased cytotoxicity towards PC3 prostate cancer (PCa) cells relative to the constituent components or separate complexes (e.g. Zn(2+):3'FdU) indicating that this new structural motif may be therapeutically useful for PCa treatment. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:32.

FdUMP[10] is a multimer of FdUMP, a suicide inhibitor of thymidylate synthase (TS), and was designed to bypass resistance to 5-fluorouracil (5FU). The aim of the study was to compare the effect of FdUMP[10] with 5FU and 5-fluoro-2-deoxyuridine (FUdR) in their efficacy to inhibit their target TS in resistant cells. Therefore cell lines FM3A/0, FM3A/TK- (deficient in thymidine kinase) and FM3A/TS- (deficient in thymidylate synthase) were used to determine TK dependency and specificity for TS inhibition. FdUMP[10] inhibited cell growth with greater potency than 5FU and FdUMP. Direct folate-based inhibitors Raltitrexed, GW1843U89 and Pemetrexed were also evaluated using these cell lines. In TK-deficient cells these folate-based inhibitors had greater potency than the fluoropyrimidines (FPs). Surprisingly, Pemetrexed even inhibited cell growth in TS-deficient cells. Incubation with nucleotidase and phosphatase inhibitors resulted in a reduction of cytotoxicity of FdUMP[10], indicating that the drug can be degraded outside the cells. In the TS in situ inhibition assay (TSIA) 24 h exposure of FM3A cells to 0.5 microM FdUMP and 0.05 microM FdUMP[10] decreased TSIA to 7 and 1% of control. Inhibition of nucleotidase and phosphatase activities reduced the effect of FdUMP[10], while the inhibitory effect was lower in cells lacking TK. FdUMP[10] can enter the cells intact, but also to some extent after dephosphorylation. In conclusion, FdUMP[10] can bypass resistance to FUdR by direct inhibition of TS.

FdUMP[10], a 10mer of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), the thymidylate synthase inhibitory metabolite of 5-fluorouracil (FU), is most closely correlated with the DNA topoisomerase I (Top1) inhibitor camptothecin in the National Cancer Institute COMPARE analysis, but not with FU. FdUMP[10] exhibits more potent antiproliferative activity than FdUMP or 5-fluoro-2'-deoxyuridine (FdU) and is markedly more active than FU. Camptothecin-resistant P388/CPT45 cells lacking Top1 are cross-resistant to FdUMP[10] as well as to FdUMP, FdU, and the thymidylate synthase inhibitor raltitrexed (Tomudex). FdUMP[10] induces DNA single-strand breaks and cellular Top1-DNA complexes. Such complexes are also observed in response to FdUMP, FdU, raltitrexed, and FU. The FdUMP[10]-induced Top1-DNA complexes are not inhibited by the caspase inhibitor z-VAD-fmk and form independently of apoptotic DNA fragmentation, indicating that they do not correspond to apoptotic Top1-DNA complexes. In biochemical assay, Top1 is directly trapped at uracil and FdU misincorporation sites. We propose that FdUMP[10] damages DNA by trapping Top1 at uracil and FdU misincorporation sites resulting from thymidylate synthase inhibition and thymine depletion.

Thymidylate synthase (TS) is a well-validated target for cancer chemotherapy. TS was established as the principal target of the widely used anticancer drug 5-fluorouracil (5FU). The 5FU metabolite FdUMP forms a covalent complex with TS that is stabilized by 5-formyl tetrahydrofolate (leucovorin; LV). Numerous chemical strategies have been employed to develop novel TS inhibitors that are superior to 5FU/LV. 5FU is non-ideal as a TS-inhibitory drug because it is only inefficiently converted to FdUMP, while the remainder of the administered dose is converted to toxic metabolites. My laboratory has explored the utility of FdUMP[N] compounds (oligodeoxynucleotides comprised of FdUMP nucleotides) as FdUMP pro-drugs. FdUMP[N] compounds result in potent TS-inhibition, and display many advantages relative to 5FU/LV. A number of other chemical strategies have also been employed to develop pro-drugs, or metabolic precursors of FdUMP, and several of these strategies will be reviewed. In addition to chemical strategies to develop FdUMP pro-drugs, a number of chemical strategies have been devised to develop molecules that resemble the reduced folate co-factor required for TS catalysis. The synthesis of antifolates that have TS-inhibitory activity, such as Raltitrexed, has resulted in compounds that are effective and specific TS-inhibitors and, in some cases, have clinical potential. Chemical strategies that target TS mRNA for destruction are also being explored as potential chemotherapeutics. These diverse chemical approaches to control TS activity in tumor cells for the treatment of cancer will be reviewed.

Current chemotherapy protocols that include fluoropyrimidines, such as 5-fluorouracil (5-FU), are limited by the development of chemoresistance during the course of treatment. Our laboratory has developed a novel class of fluoropyrimidines, FdUMP[N], that are oligodeoxynucleotides (ODNs) composed of some number, N, of 5-fluoro-2'-deoxyuridine-5'-O-monphosphate (FdUMP) nucleotides. Novel synthetic procedures are described that permit conjugation of folic acid to the 5'-OH of FdUMP[10] via a phosphodiester linkage using automated synthesis. The synthetic methods developed are generally applicable for ODN conjugation with folic acid. The folic acid conjugate FA-FdUMP[10] showed improved cytotoxicity toward human colorectal tumor cells (H630), and 5-FU-resistant colorectal tumor cells (H630-10). Enhanced cytotoxicity was observed for FA-FdUMP[10] relative to nonconjugated FdUMP[10] for cells grown under folate-restricted conditions, consistent with cellular uptake being, in part, receptor-mediated. Folate receptor alpha (FRalpha) mRNA was shown by RT-PCR to be overexpressed 26.3-fold in 5-FU-resistant H630-10 cells relative to H630 cells. Thus, FA-FdUMP[N] may prove useful for the treatment of 5-FU-resistant malignancies.

Gemcitabine (Gem) is a deoxycytidine analog that is effective against pancreatic cancer and other malignancies following conversion to the 5'-O-mono-, di- and tri-phosphate forms. We evaluated the cytotoxicity of GemMP[10], a novel multimeric form of 2'-deoxy-2',2"-difluorocytidine-5'-O-monophosphate (gemcitabine monophosphate) against three thyroid carcinoma cell lines established from anaplastic (8505C), papillary (B-CPAP) and poorly-differentiated papillary (BHT-101) cancer. GemMP[10] decreased tumor cell growth at concentrations ranging from 1 to 50 nM. These concentrations were 5- to 10-fold lower than those required for inhibition of tumor cell growth by monomeric Gem. GemMP[10] cytotoxicity occurred via induction of apoptosis. Flow cytometric analysis of GemMP[10] treated cells revealed growth arrest in S-phase. Fas-antigen expression was increased in thyroid cancer cells treated with GemMP[10], whereas Fas-L and Bcl-2 expression were not significantly affected. These results demonstrated that GemMP[10] is a potent cytotoxic agent that serves to induce apoptosis in association with increased Fas expression in cultured thyroid cancer cell lines.

The cytotoxicity of the 10mer ODN FdUMP[10] towards human colorectal tumor cells was evaluated using a clonogenic assay. FdUMP[10] was more than 100-fold more active than 5-FU at inhibiting colony formation of H630 cells. FdUMP[10] was also evaluated for cytotoxicity in the NCI 60 cell line screen, and showed markedly improved activity relative to 5-FU against numerous tumor cell lines. The in-vivo tolerance of FdUMP[10] is more than three-fold greater per mole fluorinated pyrimidine, than 5-FU.

NMR spectroscopy has been shown to be useful in determining the structures of nucleic acid fragments in solution. Over the last several years NMR spectroscopy, in conjunction with restrained molecular dynamics, has been employed to understand the 3D structures of a number of anticancer drugs and to rationalize their DNA binding behavior. In this review we address the methodologies used most frequently to determine nucleic acid structures in solution. In subsequent sections, we examine how these methods have been applied to rationalize the activities of a number of anticancer agents that target duplex DNA such as cisplatin, bleomycin and calicheamicin. Non-duplex DNA and RNA also represent interesting nucleic acid targets for anticancer drug design and applications of solution NMR spectroscopy to understanding the structures of these types of molecules (e.g. Okazaki fragments, DNA tetraplexes) are also reviewed. In the final sections, advances in NMR methodologies (e.g. linear prediction, superconducting probes) that are likely to impact the research conducted in this area are reviewed. The success of NMR spectroscopy in understanding the structural basis for clinically useful anticancer drugs bodes well for future applications of this methodology not only in rationalization of existing biological activity, but in the design of novel agents that will be useful in treating neoplastic disease.