Unique Dual Targeting of Thymidylate Synthase and Topoisomerase1 by FdUMP[10] Results in High Efficacy Against AML and Low Toxicity

Abstract 2584

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. In 2011 approximately 12,950 people will be diagnosed in the United States, and 9,050 will die from this disease. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less than 10% will 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). In vitro, FdUMP[10] exhibited remarkable activity against six human acute leukemia cell lines, HL60, Jurkat, K562, OCI-AML3, KG1a and THP-1 with and average IC₅₀ value of 7.47nM (Range 3.4–21.5nM). In three separate murine AML cell lines driven by expression of MLL-ENL, FdUMP[10] exhibited even greater activity with an average IC₅₀ value of 126.5 pM (Range 124.2–131.4pM). The IC₅₀ values observed for FdUMP[10] for all the cell lines tested were ∼1000 times lower than the corresponding values for 5-fluorouracil despite delivering only a tenfold increase in FP content. Likewise IC₅₀ values for FdUMP[10] were lower than those of Ara-C and doxorubicin. Additionally, FdUMP[10] inhibited colony formation of AML cell lines and primary patient samples at concentrations that did affect normal human hematopoietic cells.

Treated cells developed γH2AX foci, rapid and complete TS inhibition and displayed trapped Topoisomerase I (Topo I) cleavage complexes. This combination of DNA damage and TS inhibition lead to cells arresting in S phase and extensive apoptosis as indicated by Annexin V and propidium iodide staining. 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]. All cell lines and virtually every patient sample tested displayed expression of both TS and Topo I by western blot. Importantly, 5-fluorouracil was unable to demonstrate sustained TS inhibition, did not cause DNA damage and did not lead to S phase arrest indicating a novel mechanism of FdUMP[10].

In vivo FdUMP[10] treatment provided a statistically significant increase in survival for two separately derived MLL-ENL driven syngeneic AML models. Additionally, the survival benefit conferred was statistically indistinguishable from that conferred by the combination of Ara-C and doxorubicin. 5-FU dosed to deliver identical fluoropyrimidine content was toxic and did not confer a survival advantage.

A toxicology study compared FdUMP[10], the combination of Ara-C plus doxorubicin and 5-FU. All groups were treated as in the survival studies. After 72 hours following treatment 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. The effect of FdUMP[10] on normal hematopoietic stem cells was assessed by performing transplant experiments using bone marrow from mice untreated, treated with FdUMP[10], 5-FU or Ara-C plus doxorubicin. Marrow from FdUMP[10] treated animals engrafted well and was comparable to untreated marrow while 5-FU treated marrow showed only minimal engraftment, indicating substantial injury to the stem cell compartment.

In summary FdUMP[10] exhibited remarkable activity against AML cells and primary patient samples in vitro and in vivo. FdUMP[10] had only minimal effects on normal human and murine hematopoietic stem cells as well as decreased systemic toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin.