The Novel Fluoropyrimidine FdUMP[10] Is Highly Active Against Acute Myeloid Leukemia

Abstract 3302

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less then 10% of patients survive 5 years from diagnosis. There is a desperate need for the identification of new active agents with favorable toxicity profiles.

The novel polymeric fluoropyrimidine (FP) FdUMP[10] is an oligodeoxynucleotide pro-drug of the thymidylate synthase (TS)-inhibitory FP metabolite 5-fluoro-2'-deoxyuridine-5`-O-monophosphate (FdUMP). The observation that this compound was highly active against several leukemia lines in the NCI 60 cell line screen prompted us to evaluate its activity in several preclinical models of AML. In vitro, FdUMP[10] exhibited remarkable activity against 3 human acute leukemia cell lines, HL60, Jurkat and THP-1, with IC₅₀ values of 3.378 nM (95% CI 2.984 to 3.825), 5.438 nM (4.609 to 6.417) and 4.093 nM (3.413 to 4.907) respectively. We next tested its efficacy against a more genetically defined murine model of AML driven by expression of MLL-ENL. FdUMP[10] exhibited even greater activity against all murine lines tested. The IC₅₀ values of FdUMP[10] against two MLL-ENL driven murine AML cell lines were 214 pM (95%CI 178.9 to 255.9) and 292.3 pM (251.8 to 339.4). The IC₅₀ values observed for FdUMP[10] for all the murine lines tested were lower than both Ara-C (30-40 nM) and doxorubicin (2-4 nM).

We then determined the cytotoxic mechanism for FdUMP[10] in vitro. Upon treatment with FdUMP[10] both the human and murine cell lines undergo extensive apoptosis as indicated by Annexin V and propidium iodide staining. Treated cells developed γH2AX foci, rapid and complete TS inhibition and display trapped Topoisomerase I (Topo I) cleavage complexes. FdUMP[10]-mediated induction of apoptosis was p53 independent as murine AML cells that had p53 knocked down by RNAi demonstrated resistance to both Ara-C and doxorubicin, but not to FdUMP[10].

We next tested the efficacy of FdUMP[10] in vivo. The MLL-ENL driven murine AML model results in blasts that can be transplanted into sublethally irradiated, immunocompetent, syngeneic recipients. The recipients develop a fatal and therapy-resistant AML. Lines were generated that expressed a luciferase reporter. Animals were imaged 6–7 days after injection of the leukemias to ensure engraftment and then began treatment with either the combination of Ara-C plus doxorubicin, single-agent FdUMP[10], or observation. Studies were performed using 2 doses of FdUMP[10] at 150 or 300 mg/kg injected on days 1 and 3 and compared to animals treated with 100 mg/kg Ara-C and 3mg/kg doxorubicin injected on days 1 through 5. Both treatments resulted in a statistically significant survival advantage over observation.

A preliminary toxicology study compared FdUMP[10], 150 mg/kg daily, to 5-fluorouracil (5 FU), 150 mg/kg daily, or the combination of Ara-C at 100 mg/kg plus doxorubicin at 3 mg/kg daily. All groups were treated for 3, 4 or 5 days. On day 6 animals were sacrificed and organs harvested, sectioned, and stained. Slides were then reviewed by a veterinary pathologist. Tissues most affected were the small intestine, colon, and the bone marrow. The 5FU-treated animals had severe villous blunting and fusion with crypt necrosis in both large and small intestine. In contrast, FdUMP[10]-treated animals had only mild crypt epithelial apoptosis with mitoses. The 5 FU and Ara-C plus doxorubicin groups had a severe pan-cytopenia in the marrow compared to FdUMP[10] treated animals that showed only minimal to mild apoptosis. These data support the assertion that FdUMP[10] has lower toxicity then either Ara-C plus doxorubicin or identically dosed 5 FU.

In summary FdUMP[10] exhibited remarkable activity against AML cells in vitro and in vivo. Additionally, FdUMP[10] had decreased toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin.