Gemcitabine is an antimetabolite with broad solid tumor activity in patients. In addition, it is a potent radiation sensitizer. During the previous grant period, we investigated the metabolic and repair pathways important for cytotoxicity and radiosensitization with gemcitabine. Mechanistic studies demonstrated that inhibition of ribonucleotide reductase, mediated by the diphosphate of gemcitabine, was responsible primarily for inhibition of DNA synthesis, whereas incorporation of the analog into DNA contributed more to cytotoxicity. In addition, ribonucleotide reductase-mediated decrease in deoxynucleotides correlated strongly with radiosensitization. We tested the hypothesis that the imbalance in deoxynucleotides led to misincorporation of nucleotides into DNA which, if not repaired prior to irradiation, resulted in radiosensitization. Results demonstrated that gemcitabine produced mismatches in DNA, which occurred only at radiosensitizing concentrations and persisted only after irradiation. Radiosensitization with gemcitabine was enhanced by mismatch repair deficiency, which increased mismatches in DNA, and suppression of the p53-inducible ribonucleotide reductase subunit p53R2, which prolonged the deoxynucleotide pool imbalances. In contrast, mismatch repair deficiency decreased cytotoxicity with gemcitabine. We now propose that these mechanisms can be generalized to other antimetabolites that produce alterations in deoxynucleotide pools and function as radiosensitizers, such as hydroxyurea (ribonucleotide reductase inhibitor), and fluorodeoxyuridine, methotrexate and pemetrexed (thymidylate synthase inhibitors) to produce the first unifying hypothesis for antimetabolite radiosensitization. We will also evaluate cytotoxicity and radiosensitization of shRNA-mediated suppression of the enzymes targeted by these antimetabolites, and compare the in vivo antitumor efficacy of shRNA enzyme suppression with antimetabolites alone or with ionizing radiation. New data indicate that late- occurring DNA double strand breaks which are unable to be repaired by homologous recombination are responsible for radiosensitization with gemcitabine. The DNA damage and repair pathways, which may include ATM, ATR and homologous recombination, that produce radiosensitization will be evaluated. Preliminary results indicate that suppression of either thymidylate synthase or the R2 subunit of ribonucleotide reductase with shRNA is at least as effective as antimetabolites in producing radiosensitization. Mechanistic studies will aid in developing a general hypothesis for cytotoxicity and radiosensitization with the antimetabolites and shRNAs. In vivo animal studies will explore dosing antimetabolites or shRNA for radiosensitization based on the amount of drug or shRNA that will decrease dNTPs and produce misincorporated deoxynucleotides in tumor cell DNA. We further hypothesize that suppression of R2 in vivo will produce superior radiosensitization with lower normal tissue toxicity. These mechanism-based studies have high potential for translation to clinic to improve radiosensitization protocols for patients.

Public Health Relevance

These studies propose to test the hypothesis that, for anticancer drugs which target enzymes required to supply compounds for replication of DNA (gemcitabine, fluorodeoxyuridine, hydroxyurea, methotrexate and pemetrexed), the drug-mediated imbalance in these compounds produces mistakes in DNA which are most harmful to the cancer cell when combined with radiotherapy. Furthermore, a novel approach to cancer therapy is proposed in which we will decrease the proteins that supply the required compounds for DNA replication, which we predict will result in anticancer activity alone or in combination with radiotherapy. Understanding the mechanisms responsible for the activity of these common anticancer drugs or the novel protein suppression approach will help us to optimize their use in patients, with the ultimate goal of improving tumor control while minimizing unwanted side effects in normal tissues.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA083081-12
Application #
8278686
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Ahmed, Mansoor M
Project Start
1999-07-08
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
12
Fiscal Year
2012
Total Cost
$258,395
Indirect Cost
$78,624
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Ladd, Brendon; Ackroyd, Jeffrey J; Hicks, J Kevin et al. (2013) Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity. DNA Repair (Amst) 12:1114-21
Wawrzyniak, Joseph A; Bianchi-Smiraglia, Anna; Bshara, Wiam et al. (2013) A purine nucleotide biosynthesis enzyme guanosine monophosphate reductase is a suppressor of melanoma invasion. Cell Rep 5:493-507
Ladd, B; O'Konek, J J; Ostruszka, L J et al. (2011) Unrepairable DNA double-strand breaks initiate cytotoxicity with HSV-TK/ganciclovir. Cancer Gene Ther 18:751-9
O'Konek, Jessica J; Ladd, Brendon; Flanagan, Sheryl A et al. (2010) Alteration of the carbohydrate for deoxyguanosine analogs markedly changes DNA replication fidelity, cell cycle progression and cytotoxicity. Mutat Res 684:1-10
Flanagan, Sheryl A; Krokosky, Christina M; Mannava, Sudha et al. (2008) MLH1 deficiency enhances radiosensitization with 5-fluorodeoxyuridine by increasing DNA mismatches. Mol Pharmacol 74:863-71
Flanagan, Sheryl A; Robinson, Blaine W; Krokosky, Christina M et al. (2007) Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers. Mol Cancer Ther 6:1858-68
Robinson, Blaine W; Ostruszka, Leo; Im, Michael M et al. (2004) Promising combination therapies with gemcitabine. Semin Oncol 31:2-12
Ostruszka, Leo J; Shewach, Donna S (2003) The role of DNA synthesis inhibition in the cytotoxicity of 2',2'-difluoro-2'-deoxycytidine. Cancer Chemother Pharmacol 52:325-32
Robinson, Blaine W; Im, Michael M; Ljungman, Mats et al. (2003) Enhanced radiosensitization with gemcitabine in mismatch repair-deficient HCT116 cells. Cancer Res 63:6935-41
Robinson, B W; Shewach, D S (2001) Radiosensitization by gemcitabine in p53 wild-type and mutant MCF-7 breast carcinoma cell lines. Clin Cancer Res 7:2581-9

Showing the most recent 10 out of 11 publications