Sheryl A. Flanagan

Depletion of Deoxyribonucleotide Pools Is an Endogenous Source of DNA Damage in Cells Undergoing Oncogene-Induced Senescence

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.

Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers

Radiation sensitization by 2′,2′-difluoro-2′-deoxycytidine (dFdCyd) has correlated with dATP depletion [dFdCDP-mediated inhibition of ribonucleotide reductase (RR)] and S-phase accumulation. We hypothesized that radiosensitization by dFdCyd is due to nucleotide misincorporations in the presence of deoxynucleotide triphosphate pool imbalances, which, if not repaired, augments cell death following irradiation. The ability of dFdCyd to produce misincorporations was measured as pSP189 plasmid mutations in hMLH1-deficient [mismatch repair (MMR) deficient] and hMLH1-expressing (MMR proficient) HCT116 cells. Only MMR-deficient cells showed a significant increase in nucleotide misincorporations (2- to 3-fold increase; P ≤ 0.01) after radiosensitizing concentrations of dFdCyd ± 5 Gy radiation, which persisted for at least 96 h. dFdCyd (10 nmol/L) did not radiosensitize MMR-proficient HCT116 or A549 cells, but following small interfering RNA–mediated suppression of hMLH1, this concentration produced excellent radiosensitization (radiation enhancement ratios = 1.6 ± 0.1 and 1.5 ± 0.1, respectively; P < 0.05) and a 2.5-fold increase in mutation frequency in A549 cells. Cytosine arabinoside (1-β-d-arabinofuranosylcytosine), which can be incorporated into DNA but does not inhibit RR, failed to radiosensitize MMR-deficient cells or increase mutation frequency in the MMR-deficient and MMR-proficient cells. However, the RR inhibitor hydroxyurea radiosensitized MMR-deficient cells and increased nucleotide misincorporations (≥5-fold increase; P < 0.05), thus further implicating the inhibition of RR as the mechanism underlying radiosensitization by dFdCyd. These data showed that the presence and persistence of mismatched nucleotides is integral to radiosensitization by dFdCyd and suggest a role for hMLH1 deficiency in eliciting the radiosensitizing effect. [Mol Cancer Ther 2007;6(6):1858–68]

Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity.

The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC50 in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV.

MLH1 Deficiency Enhances Radiosensitization with 5-Fluorodeoxyuridine by Increasing DNA Mismatches

The antitumor drug 5-fluoro-2′-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. Although radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches that, if not repaired before irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells that lack the expression of the required MMR protein MLH1 (inactive MLH1), and in MMR-proficient (wild-type MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC50 = 3.5 μM) versus HCT116 1-2 cells (IC50 = 0.75 μM), when irradiation followed FdUrd (IC50) the MLH1-inactivated cells exhibited greater radiosensitization compared with MMR-wild-type cells [radiation enhancement ratio (RER) = 1.8 ± 0.28 versus 1.1 ± 0.1, respectively] and an increase (≥8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wild-type cells, FdUrd (IC50) did not produce radiosensitization nor did it increase the mutation frequency, but after short hairpin RNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 ± 0.10 and 1.5 ± 0.06, respectively) and an increase in nucleotide misincorporations (8-fold and 6-fold, respectively). Incubation with higher concentrations of FdUrd (IC90) after suppression of MLH1 produced a further increase in ionizing radiation sensitivity in both SW620 and HCT116 1-2 cells (RER = 1.8 ± 0.03 and 1.7 ± 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.

Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule

Significance The search for anticancer drugs continues to be greatly pursued. The nucleoside analog gemcitabine, which targets ribonucleotide reductase (RR) as a diphosphate and DNA polymerases as a triphosphate, is the standard first-line treatment in patients with pancreatic cancer. However, its cytotoxicity to normal dividing tissues leads to unwanted side effects. Here, we have discovered a nonnucleoside RR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), that has efficacy similar to gemcitabine and the potential to be modified to provide safer and more effective cancer therapies. Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.

Drug Metabolism and Homologous Recombination Repair in Radiosensitization with Gemcitabine

Gemcitabine (difluorodeoxycytidine; dFdCyd) is a potent radiosensitizer, noted for its ability to enhance cytotoxicity with radiation at noncytotoxic concentrations in vitro and subchemotherapeutic doses in patients. Radiosensitization in human tumor cells requires dFdCyd-mediated accumulation of cells in S phase with inhibition of ribonucleotide reductase, resulting in ≥80% deoxyadenosine triphosphate (dATP) depletion and errors of replication in DNA. Less is known of the role of specific DNA replication and repair pathways in the radiosensitization mechanism. Here the role of homologous recombination (HR) in relationship to the metabolic and cell cycle effects of dFdCyd was investigated using a matched pair of CHO cell lines that are either proficient (AA8 cells) or deficient (irs1SF cells) in HR based on expression of the HR protein XRCC3. The results demonstrated that the characteristics of radiosensitization in the rodent AA8 cells differed significantly from those in human tumor cells. In the AA8 cells, radiosensitization was achieved only under short (≤4 h) cytotoxic incubations, and S-phase accumulation did not appear to be required for radiosensitization. In contrast, human tumor cell lines were radiosensitized using noncytotoxic concentrations of dFdCyd and required early S-phase accumulation. Studies of the metabolic effects of dFdCyd demonstrated low dFdCyd concentrations did not deplete dATP by ≥80% in AA8 and irs1SF cells. However, at higher concentrations of dFdCyd, failure to radiosensitize the HR-deficient irs1SF cells could not be explained by a lack of dATP depletion or lack of S-phase accumulation. Thus, these parameters did not correspond to dFdCyd radiosensitization in the CHO cells. To evaluate directly the role of HR in radiosensitization, XRCC3 expression was suppressed in the AA8 cells with a lentiviral-delivered shRNA. Partial XRCC3 suppression significantly decreased radiosensitization [radiation enhancement ratio (RER) = 1.6 ± 0.15], compared to nontransduced (RER = 2.7 ± 0.27; P = 0.012), and a substantial decrease compared to nonspecific shRNA-transduced (RER = 2.5 ± 0.42; P = 0.056) AA8 cells. Although the results support a role for HR in radiosensitization with dFdCyd in CHO cells, the differences in the underlying metabolic and cell cycle characteristics suggest that dFdCyd radiosensitization in the nontumor-derived CHO cells is mechanistically distinct from that in human tumor cells.

Late DNA Damage Mediated by Homologous Recombination Repair Results in Radiosensitization with Gemcitabine

Gemcitabine (dFdCyd) shows broad antitumor activity in solid tumors in chemotherapeutic regimens or when combined with ionizing radiation (radiosensitization). While it is known that mismatches in DNA are necessary for dFdCyd radiosensitization, the critical event resulting in radiosensitization has not been identified. Here we hypothesized that late DNA damage (≥24 h after drug washout/irradiation) is a causal event in radiosensitization by dFdCyd, and that homologous recombination repair (HRR) is required for this late DNA damage. Using γ-H2AX as a measurement of DNA damage in MCF-7 breast cancer cells, we demonstrate that 10 or 80 nM dFdCyd alone produced significantly more late DNA damage compared to that observed within 4 h after treatment. The combination of dFdCyd treatment followed by irradiation did not produce a consistent increase in DNA damage in the first 4 h after treatment, however, there was a synergistic increase 24–48 h later relative to treatment with dFdCyd or radiation alone. RNAi suppression of the essential HRR protein, XRCC3, significantly decreased both radiosensitization and late DNA damage. Furthermore, inhibition of HRR with the Rad51 inhibitor B02 prevented radiosensitization when added after, but not during, treatment with dFdCyd and radiation. To our knowledge, this is the first published study to show that radiosensitization with dFdCyd results from a synergistic increase in DNA damage at 24–48 h after drug and radiation treatment, and that this damage and radiosensitization require HRR. These results suggest that tumors that overexpress HRR will be more vulnerable to chemoradiotherapy, and treatments that increase HRR and/or mismatches in DNA will enhance dFdCyd radiosensitization.

Abstract 2999: Cytotoxicity with hydroxamic acid HDACis in combination with antimetabolites is highly sequence dependent in solid tumor cells

Used alone or in combination with other chemotherapeutics, the antimetabolites 2’, 2’-difluoro-2’-deoxycytidine (gemcitabine, dFdCyd), and 5-fluorouracil (5-FU)/fluorodeoxyuridine (FdUrd) comprise the mainstay of treatment for gastrointestinal malignancies, such as pancreatic and colorectal cancer. Gemcitabine with cisplatin is also first-line therapy for non-small cell lung cancer (NSCLC). While these treatments can prolong survival, many patients still relapse and there is need for improvement to current therapies. Cytotoxicity elicited by dFdCyd and 5-FU/FdUrd is influenced by drug metabolism/activation, DNA repair proteins, cell cycle progression, and apoptosis. Histone deacetylase inhibitors (HDACi) target histone deacetylases, which regulate gene expression through deacetylation of lysine residues on histones and some non-histone proteins. HDACis have been shown to increase expression of p21 and increase apoptosis which could enhance cytotoxicity with antimetabolites. The success of HDACis, including vorinostat and panobinostat, in the treatment of hematologic malignancies has encouraged their use in solid tumors. However, results from clinical trials in patients treated with HDACis alone or with other drugs, including antimetabolites have been mixed and the mechanism(s) underlying successful therapy with HDACis is largely unknown. In the present studies, we used solid tumor cell lines that represent tumors commonly treated with antimetabolites; A549 (NSCLC) and PANC1 (pancreatic cancer) cells, and HT29 (colon cancer) cells to investigate the effect of an HDACi with either dFdCyd or FdUrd, respectively, and we measured clonogenic survival. The data demonstrate that cytotoxicity with an HDACi and an antimetabolite is highly sequence dependent; vorinostat added concurrently with dFdCyd or FdUrd or prior to either antimetabolite resulted in antagonistic cytotoxicity, whereas the addition of the antimetabolite followed by HDACi was additive/synergistic with dFdCyd. Cytotoxicity with panobinostat and antimetabolites was also sequence dependent. Cell cycle distribution studies revealed the accumulation of cells in G1 and G2/M following HDACi addition (≥90% of cells), thus rendering S-phase specific dFdCyd less cytotoxic upon its subsequent addition. Similarly, concurrent addition of HDACi and dFdCyd produced less S-phase accumulation than observed with dFdCyd alone. These data demonstrate that administration of the HDACis and antimetabolites must be sequenced appropriately so that cell cycle effects complement rather than antagonize drug mechanisms. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Aaron Kramer, Jennifer Feigin, Donna S. Shewach. Cytotoxicity with hydroxamic acid HDACis in combination with antimetabolites is highly sequence dependent in solid tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2999.

Abstract 847: Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which augment sensitivity to subsequent ionizing radiation (IR) but are not required to elicit cytotoxicity. We have proposed that RR suppression would be as effective as dFdCyd for radiosensitization. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. We used a RNAi approach to suppress either R2 or p53R2 (≥ 90% suppression of either R2 or p53R2, with little effect (≤20%) on expression of its homolog) in two p53 wild type cell lines, MCF7 breast carcinoma and A549 lung carcinoma. This approach produced equivalent radiosensitization, dATP depletion, cytotoxicity and increase in DNA mismatches compared to inactivation of RR by dFdCyd (IC50). These results reinforce our prior finding that radiosensitization with dFdCyd is the result of inhibition of RR and not incorporation into DNA or cytotoxicity. We then proposed that R2 but not p53R2 suppression would radiosensitize mutant p53 tumor cells. Interestingly, R2 shRNA suppression did not radiosensitize mutant p53 MCF7/ADR cells (radiation enhancement ratio (RER) = 1.07 ± 0.06). Despite the mutant p53 status of MCF7/ADR cells, p53R2 was elevated (≥ 50%) following suppression of R2 compared to untreated cells (no shRNA). We hypothesized that this increase in p53R2 permits RR to continue producing dNTPs for DNA replication and repair in the absence of R2, thus preventing radiosensitization. Indeed, simultaneous suppression of R2 and p53R2 produced excellent radiosensitization (RER = 1.68 ± 0.04 (shRNAs) vs. 1.6 ± 0.01 (dFdCyd (IC50)), and similar cytotoxicity (surviving fraction (SF) = 76.5 ± 8.3% (shRNA) vs. 71.3 ± 6.7% (dFdCyd)). Impressively, suppression of p53R2 alone produced excellent radiosensitization (RER= 1.76 ± 0.30) with similar cytotoxicity (SF = 84 ± 20%) compared to the double knockdown or dFdCyd. Considering that most solid tumors express mutant p53, suppression of p53R2 instead of R2 may be a more effective method for radiosensitization. These studies suggest that deoxynucleotide biosynthesis is regulated differently in p53 wild type compared to mutant p53 tumor cells, and may be used to maintain DNA replication and repair and prevent anticancer efficacy after damage induced by IR or other DNA damaging agents. The mechanism by which mutant p53 tumor cells upregulate p53R2 warrants further investigation. Note: This abstract was not presented at the meeting. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Sudha Mannava, Mikhail A. Nikiforov, Donna S. Shewach. Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2014-847

Abstract 445: shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation.

Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which, if not repaired, augment sensitivity to subsequent ionizing radiation (IR). DNA mismatches were not required to elicit cytotoxicity but were necessary for radiosensitization to occur. We therefore hypothesized that RNAi mediated suppression of RR activity would also radiosensitize cells. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. To determine whether the suppression of either R2 or p53R2 produced a similar extent of radiosensitization compared to dFdCyd, two different shRNAs were used to suppress each subunit in wild-type p53 MCF7 breast carcinoma and A549 non-small cell lung carcinoma cells, producing a profound (≥ 90%) suppression of the target protein, R2 or p53R2, with little effect (≤20%) on expression of its homolog. shRNAs for both R2 and p53R2 produced effects similar to those observed with dFdCyd on dATP depletion (% control value) (MCF7: 66 ± 6% (shRNA) vs. 85 ± 10 % (dFdCyd); A549: 75 ± 20% (shRNA) vs. 80 ± 5% (dFdCyd), and cytotoxicity (MCF7: 35-60% (shRNA) vs. 40-50% (FdCyd); A549: 40-65% (shRNA) vs. 40-60% (dFdCyd)). When shRNA suppression was followed by IR, radiosensitivity was similar with the shRNAs (R2 and p53R2) vs. dFdCyd (MCF7: radiation enhancement ratio (RER)) = 1.45 ± 0.08 (shRNA) vs. 1.7 ± 0.14 (dFdCyd); A549: RER= 1.51 ± 0.09 (shRNA) vs. 1.55 ± 0.14(dFdCyd)), and a similar increase in DNA mismatches compared to untreated wild type cells (MCF7: ≥ 7-fold (dFdCyd) vs. 5-7 fold (shRNA); A549: ≥ 3 fold (dFdCyd) vs. 3-5 fold (shRNA)) was observed. Although p53R2 is thought to be induced primarily in response to DNA damage, its effects on dATP depletion and DNA mismatches prior to IR suggest that it may play a previously unidentified role in normal maintenance of DNA replication. These results reinforce our prior finding that the mechanism of radiosensitization with dFdCyd is the decrease in dATP and not its incorporation into DNA or its cytotoxicity. Excellent radiosensitization with suppression of either R2 or p53R2, compared to dFdCyd, suggests that this targeted approach merits in vivo evaluation. Elevated expression of R2 is known to enhance the invasiveness of cancer cells, and increase resistance to dFdCyd, thus its suppression would further enhance anticancer activity in addition to its effect on radiosensitization. Citation Format: Sheryl A. Flanagan, Kristin Cooper, Sudha Mannava, Mikhail Nikiforov, Donna S. Shewach. shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2013-445