5-aza-2',2'-Difluoro Deoxycytidine (NUC013): A Novel Nucleoside DNA Methyl Transferase Inhibitor and Ribonucleotide Reductase Inhibitor for the Treatment of Cancer

Abstract

Tumor suppressor genes can be silenced genetically as well as epigenetically. One approach to reversing epigenetic suppression of tumor suppressor genes is to inhibit DNA methyl transferase. 5-aza-2',2'-diflurorodeoxycytidine (NUC013) is a novel DNA methyl transferase and ribonucleotide reductase inhibitor that is a more potent inhibitor of growth than decitabine in the NCI 60 cancer cell line panel. NUC013 is more active than decitabine against p53-null/mutant cancer cell lines (p = 0.027) but is even more so against p53 wild-type (WT) cell lines (p = 0.0025). The maximum tolerated dose in mice of NUC013 is greater than 120 mg/kg administered intravenously for three consecutive days a week for three weeks. With this regimen and a dose of 20 mg/kg in a human leukemia HL-60 (p53-null) NCr-nu/nu mouse xenograft model (n = 10/group), NUC013 demonstrated a survival benefit (saline median survival (MS) = 26.5 days, NUC013 MS = 32 days and hazard ratio (HR) = 0.26 (p = 0.032)). In a colon cancer LoVo (TP53 WT) xenograft, mice treated with decitabine at 5 mg/kg had worse survival than saline controls (decitabine MS = 31 days, saline MS > 60 days and HR = 26.89 (p < 0.0001)). At a dose of 20 mg/kg NUC013, mean tumor volume in the LoVo xenografts was lower than controls by 50.9% and at 40 mg/kg by 53.7% (both p < 0.0001).

Keywords: 5-azacytidine; DNA methyl transferase; NUC013; decitabine; gemcitabine; nucleoside; p53; ribonucleotide reductase.

Figure 1

Comparison of the structures of decitabine, gemcitabine and NUC013.

Figure 2

(A) Western blot comparing vehicle (veh) to decitabine (DAC) and NUC013 6 days after exposure to 0.1 µM and 0.2 µM of nucleoside and probing with DNA methyl transferase (DNMT1) antibody in human leukemia cells lines THP-1 and Kasumi-1; (B) Comparison of induction of apoptosis in THP-1 or Kasumi-1 cells by vehicle control, decitabine (0.2 µM) or NUC013 (concentrations as indicated in figure). Cells were co-stained with Annexin-V and PI on day 2 and analyzed by flow cytometry. Only Annexin-V data are presented because there were no changes in propidium iodide (PI) staining over the time period; (C) Photomicrographs of THP-1 cells treated with vehicle, decitabine or NUC013 on day 6 following drug exposure and stained with Giemsa (×630).

Figure 3

Survival proportions in HL-60 and LoVo xenografts treated with decitabine or NUC013 (n = 10/group). “Survival” refers to animals that were not removed from the study by death or per protocol euthanasia. (A) Survival proportion in HL-60 xenografts. Saline control compared to decitabine (5 mg/kg) and NUC013 (5.8 mg/kg). Saline median survival (MS) = 25 days, decitabine MS = 26 days, and hazard ratio (HR) = 0.92 (p = 0.64, Log-rank test); NUC013 MS = 28.5 days and HR = 0.59 (p = 0.19, Log-rank test); (B) Survival proportion in HL-60 xenografts. Saline compared to NUC013 (20 mg/kg). Saline MS = 26.5 days, NUC013 MS = 32 days and HR = 0.26 (p = 0.032, Log-rank test); (C) Survival proportion in LoVo xenografts. Saline control compared to decitabine (5 mg/kg) and NUC013 (5.8 mg/kg). Saline MS undefined, decitabine MS = 31 days and HR of 26.89 (p < 0.0001, Log-rank test); NUC013 MS = 60 days and HR = 1.64 (p = 0.48); (D) Survival proportion in LoVo xenografts. Saline control compared to 20 mg/kg and 40 mg/kg NUC013. MS undefined.

Figure 4

Tumor volume as a function of time since tumor implant. The first data point on each graph is on the day of study drug treatment initiation. Values are ± standard deviation. (A) HL-60 comparison of NUC013 5.8 mg/kg vs. saline control; (B) HL-60 comparison of NUC013 20 mg/kg vs. saline control; (C) LoVo comparison of NUC013 5.8 mg/kg vs. saline control; (D) LoVo comparison of NUC013 20 mg/kg and 40 mg/kg vs. saline control.

Figure 4

Tumor volume as a function of time since tumor implant. The first data point on each graph is on the day of study drug treatment initiation. Values are ± standard deviation. (A) HL-60 comparison of NUC013 5.8 mg/kg vs. saline control; (B) HL-60 comparison of NUC013 20 mg/kg vs. saline control; (C) LoVo comparison of NUC013 5.8 mg/kg vs. saline control; (D) LoVo comparison of NUC013 20 mg/kg and 40 mg/kg vs. saline control.