Unique dual targeting of thymidylate synthase and topoisomerase1 by FdUMP[10] results in high efficacy against AML and low toxicity

Abstract

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to marrow failure and death. There is a desperate need for new therapies. The novel fluoropyrimidine, FdUMP[10], was highly active against both human AML cell lines, (IC(50) values, 3.4nM-21.5nM) and murine lines (IC(50) values, 123.8pM-131.4pM). In all cases, the IC(50) of FdUMP[10] was lower than for cytarabine and ∼ 1000 times lower than 5-fluorouracil (5-FU). FdUMP[10] remained effective against cells expressing the Flt3 internal tandem duplication, BCR-ABL, MN1, and an shRNA against p53. It had activity against patient samples at concentrations that did not affect normal hematopoietic cells. FdUMP[10] inhibited thymidylate synthase (TS) and trapped topoisomerase I cleavage complexes (Top1CCs), leading to DNA damage and apoptosis. All cell lines and nearly all primary AML samples examined expressed both TS and Top1. In vivo, FdUMP[10] was active against a syngeneic AML model with a survival advantage equivalent to doxorubicin plus cytarabine. 5-FU treatment was toxic and did not improve survival. FdUMP[10] was better tolerated than 5-FU or cytarabine plus doxorubicin and did not affect normal HSCs, while 5-FU dramatically impaired their ability to engraft. In summary, FdUMP[10] was highly efficacious and better tolerated than standard therapies.

Figure 1

FdUMP[10] is active against cells expressing adverse prognostic factors. (A) Cytotoxicity assays of murine AML cells that knockdown p53. Cells were exposed to the indicated drugs for 72 hours and then assessed for viability. Viability is shown as percentage of control; error bars represent the SE. (B) Flow cytometry of annexin V assay. Cells expressing the p53 shRNA were exposed to the indicated drug for 48 hours and then labeled with annexin V and propidium iodide (PI). (C) Cytotoxicity assays of murine AML cells expressing either MN1 or BCR-ABL (p210). Cells were exposed to the indicated drugs for 72 hours and then assessed for viability. Viability is shown as percentage of control; error bars represent the SE.

Figure 2

FdUMP[10] is active against leukemia stem cells (LSCs) from cell lines and primary patient samples. (A) Colony formation assays. Primary patient samples or cell lines were incubated with the indicated drug for 24 hours and placed in methylcellulose media. Plates were read on or after day 7. All experiments were done in triplicate. Primary AML is the combined results of 3 independent primary patient samples. Colony numbers are normalized to controls, and error bars represent the SE. (B) Colony formation assays. As in panel A, the result shown is the combination of 4 separate normal HSC donors.

Figure 3

FdUMP[10] is a potent inhibitor of TS and traps Top1CCs. (A) TS inhibition assay. HL60 cells were exposed to 10nM FdUMP[10] or 100nM 5-FU for the indicated time, lysed, and assayed for TS activity. Activity is plotted as percentage of control. (B) ICE bioassay for Top1CCs. THP-1, Jurkat, and HL60 cells were incubated with 100nM FdUMP[10] for 48 hours. Cells were lysed and subjected to ICE bioassay (see “ICE bioassay/Top1 cleavage complex detection”), and DNA-containing fractions were blotted for Top1. (C) Murine AML TS competition assays. Murine AML cells were partially infected with TS and GFP-expressing retrovirus and treated with the indicated drug for 72 hours. All experiments were performed in triplicate. The GFP⁺ percentage in the viable cell population is indicated; representative histograms are shown. (D) HL60 TS competition assays. HL60 cells were partially infected with TS and GFP-expressing retrovirus and exposed to the indicated drug for 72 hours. Percentage of GFP⁺ cells was determined as in panel C and normalized to untreated controls. All experiments were done in triplicate. (E) Results of TS and Top1 Western blot analyses. K562 (K), HL60 (H), Kg1a (Kg), MLL-ENL murine AML (M3), or 3 primary AML patient samples (B1-3) were blotted with the indicated Ab. Error bars represent SE. *P < .05.

Figure 4

FdUMP[10] causes S-phase arrest regardless of p53 status. (A) EdU (5-ethynyl-2′-deoxyuridine) incorporation assays. MLL-ENL–driven murine AML cells were incubated with the indicated drug for 8 hours and subjected to an EdU incorporation assay. S-phase cells were gated as shown. Percentages shown are for cells in S phase. (B) EdU incorporation assay as in panel A. Cells were infected with either a p53-targeting shRNA or a control vector and exposed to the indicated drug for 8 hours.

Figure 5

FdUMP[10] causes DNA damage and induces apoptosis. (A) Immunofluorescence for γH2AX foci. K562 cells were treated with the indicated drug for 24 hours and then assayed for the presence of γH2AX foci. Secondary Ab was conjugated with Alexa Fluor 594, and images were captured with an Olympus IX70 inverted fluorescent microscope equipped with a Retiga 2000R digital color camera and using an LPlanFl 40×/0.40 objective. Images were analyzed with Image Pro Plus 5.1 software. (B) Flow cytometry of annexin V assay. THP-1 or HL60 cells were treated with the indicated drug for 48 hours and then labeled with annexin V and propidium iodide (PI) and analyzed by flow cytometry. Numbers in bold denote percentage of cells in each quadrant.

Figure 6

FdUMP[10] confers a survival benefit equivalent to the combination of Ara-C and Dox. (A) Schema of treatment trial. C57/Bl6 mice were sublethally irradiated to 4.5 Gy and injected with an MLL-ENL and Flt3 ITD syngeneic leukemia. Once engraftment was established by bioluminescence imaging, mice were treated with either saline (S), FdUMP[10] at 300 mg/kg (Fd), 5-FU at 121 mg/kg or Ara-C at 125 mg/kg plus Dox at 3.75 mg/kg (AD) on days 1, 3, 5, and 7. (B) Bioluminescent image of mice on day 6 after treatment. (C) Kaplan-Meier curves for animals treated with Fd, 5-FU, or AD as in panel B.

Figure 7

FdUMP[10] induces less toxicity than either 5-FU or Ara-C plus Dox. (A) H&E staining of organs from mice treated with either FdUMP[10], 5-FU, or Ara/Dox. Mice were treated in the same manner as in Figure 6 and killed 72 hours after the last dose, and organs were harvested, fixed, paraffin-embedded, sectioned, and stained. Slides were imaged with a Nikon Eclipse 50i light microscope, magnification as indicated. Photographs of tissues were taken with the NIS Elements D3.10 camera and software system. (B) BM transplantation. Donor mice were treated with 5-FU, FdUMP[10], or Ara-C/Dox as in panel A, and 72 hours after last doses animals were killed and b/l femur cells were harvested. Ly5.1⁺ C57/Bl6 recipient mice were irradiated to 8 Gy and injected with donor cells. After 3 weeks recipients were harvested, and degree of femur engraftment by donor cells was determined by staining with Ly5.2 Ab and analyzed by flow cytometry. *P < .0001 and #P = .0827.