Chelation of intracellular iron with the antifungal agent ciclopirox olamine induces cell death in leukemia and myeloma cells by Yanina Eberhard, Sean P. McDermott, Xiaoming Wang, Marcela Gronda, Amudha Venugopal, Tabitha E. Wood, Rose Hurren, Alessandro Datti, Robert A. Batey, Jeffrey Wrana, William E. Antholine, John E. Dick, and Aaron D. Schimmer Blood Volume 114(14):3064-3073 October 1, 2009 ©2009 by American Society of Hematology

Screens for off-patent drugs with unrecognized anticancer activity identify the antifungal CPX. (A) Compounds (n = 45) identified as hits in a primary screen for inhibitors of the survivin promoter were tested for their effects on survivin transactivation a... Screens for off-patent drugs with unrecognized anticancer activity identify the antifungal CPX. (A) Compounds (n = 45) identified as hits in a primary screen for inhibitors of the survivin promoter were tested for their effects on survivin transactivation and cytotoxicity in HeLa cells stably overexpressing the survivin promoter driving luciferase. HeLa cells were treated with the hit compounds (5 μM) for 24 hours. After incubation, luciferase expression was measured as described in “Luciferase assay,” and cell viability was measured by the MTS assay. Data represent the percentage of viable cells (y-axis) and the percentage luciferase decrease (x-axis) compared with buffer control. (B) Chemical structure of CPX. (C) HeLa cells stably overexpressing the survivin promoter driving luciferase were treated with increasing concentrations of CPX. Twenty-four hours after incubation, luciferase activity was measured. Data represent the mean percentage of luciferase expression ± SD after CPX treatment compared with buffer control from one of 3 representative experiments. (D) HeLa cells were treated with CPX (5 μM). At increasing times after incubation, cells were harvested and total proteins were isolated. Expression of survivin and GAPDH was measured by immunoblotting. (E) TEX and M9-ENL1 cells were treated with increasing concentrations of CPX. Seventy-two hours after incubation, cell viability was measured by the Alamar Blue assay. Data represent the mean percentage of viable cells ± SD from 1 of 3 representative experiments. Yanina Eberhard et al. Blood 2009;114:3064-3073 ©2009 by American Society of Hematology

CPX induces cell death in malignant cell lines. CPX induces cell death in malignant cell lines. Leukemia (A), myeloma (B), solid tumor (C), and nonmalignant lung fibroblast (D) cell lines were treated with increasing concentrations of CPX. Seventy-two hours after incubation, cell viability was measured by MTS assay. Data represent the mean percentage of viable cells ± SD from 1 of at least 3 representative experiments. (E) Primary AML cell samples (n = 12) were treated with increasing concentrations of CPX. Seventy-two hours after incubation, cell viability was measured by the Alamar Blue assay. Data represent the mean percentage of viable cells ± SD where each sample was tested in triplicate. (F) Normal mononuclear cells derived from bone marrow (n = 3) or peripheral blood stem cells (n = 2) were plated in a methylcellulose colony-forming assay with increasing concentrations of CPX. Myeloid (granulocyte macrophage-colony forming units) and erythroid (erythroid-burst forming units) colonies were counted 14 days after plating and normalized to cultures treated with buffer alone. Data represent the mean ± SD of 5 independent experiments performed in duplicate. Yanina Eberhard et al. Blood 2009;114:3064-3073 ©2009 by American Society of Hematology

CPX delays tumor growth in mouse models of leukemia. CPX delays tumor growth in mouse models of leukemia. Sublethally irradiated NOD/SCID mice were injected intraperitoneally with (A) MDAY-D2 murine leukemia cells (n = 30; 15 per group), or subcutaneously with (B) K562 cells (n = 30; 15 per group) or (C) OCI-AML2 human leukemia cells (n = 24; 12 per group). After implantation, mice were treated with CPX (25 mg/kg) or vehicle control by oral gavage daily. After 8 (MDAY-D2), 30 (K562), and 16 (OCI-AML2) days, mice were killed and tumors were excised, measured, and weighed. The mean weight and volume ± SD are shown. Means were compared by the Student t test. ***P < .001, **P < .01 (Student t test). One of at least 2 representative experiments is shown. (D) Primary cells from one patient with AML and normal cytogenetics were injected intrafemorally into the right femur of female sublethally irradiated nude/NOD/SCID mice. Four weeks after injection, mice were treated by oral gavage once daily with vehicle (n = 3) or CPX (20 mg/kg; n = 3) for 4 weeks. After treatment, human leukemia cell engraftment in the injected right femur was measured by flow cytometry for human CD45. Data represent the mean ± SD of engrafted human cells from all mice. *P < .05 (Student t test). Yanina Eberhard et al. Blood 2009;114:3064-3073 ©2009 by American Society of Hematology

CPX-induced cell death is iron dependent. CPX-induced cell death is iron dependent. (A) Leukemia (NB4, MDAY-D2, and OCI-M2) and solid tumor (OVCAR-3 and PPC-1) cells were loaded with the intracellular iron-chelating fluorescent dye calcein-AM. Cells were then treated with CPX (10 μM) or DFX (100 μM) for 2 minutes. Intracellular iron bound by calcein was measured by flow cytometry. Percentage increase ± SD in the geometric mean intracellular calcein fluorescence is represented *P < .05 (Student t test). One of at least 2 representative experiments performed in triplicate is shown. (B) HT-29(i) and HepG2 (ii) cells were treated with increasing concentrations of CPX with or without iron supplementation with iron in a complex with a transferrin-replacement compound (8 μM). Seventy-two hours after incubation, cell viability was measured by MTS. Data represent the mean ± SD percentage of viable cells from 1 of 3 representative experiments. (C) Jurkat and NB4 leukemia cells were treated with increasing concentrations of CPX or DFX. Seventy-two hours after incubation, cell viability was measured by the MTS assay. Data represent the mean ± SD percentage viable cells from 1 of at least 3 representative experiments performed in triplicate. (D) NB4 cells were loaded with the intracellular iron-chelating fluorescent dye calcein-AM (i). Cells were then treated with 10 μM of CPX or the structurally CPX-related analogs DPOH, ADP, and MDP for 2 minutes. Intracellular iron bound by the compounds was measured by flow cytometry as described in panel A. (ii) NB4 cells were treated with increasing concentrations of CPX or the analogs in panel Di. Seventy-two hours after incubation, cell viability was measured by the MTS assay. Data represent the mean ± SD percentage viable cells from 1 of at least 2 representative experiments performed in triplicate. Yanina Eberhard et al. Blood 2009;114:3064-3073 ©2009 by American Society of Hematology

CPX inhibits ribonucleotide reductase. CPX inhibits ribonucleotide reductase. (A) MDAY-D2 and OCI-M2 leukemia cells were treated with CPX (5 and 10 μM) or buffer control for 24 hours. After incubation, cells were harvested, rapidly frozen, and the tyrosyl radical activity (↑) was measured by EPR at a microwave frequency of 9.12 GHz and 77 Kelvin. The averaged of 9 scans is presented. One of 2 representative experiments is shown. (Insets) Mean percentage viability of cells treated with increasing concentrations of CPX. (B) HeLa cervical cancer cells were transfected with cDNA corresponding to RRM2 or vector control and incubated with CPX at increasing concentrations. Seventy-two hours after incubation, cell viability was assessed by the MTS assay. Data represent the mean ± SD percentage viable cells from 1 of 3 representative experiments performed in triplicate; *P < .05 (Student t test). (C) U937 leukemia and KMS18 myeloma cells were treated with increasing concentrations of CPX or hydroxyurea (HU). Seventy-two hours after incubation, cell growth and viability were measured by the MTS assay. Data represent the mean percentage ± SD relative to control cells of 1 of 3 representative experiments performed in triplicate. Yanina Eberhard et al. Blood 2009;114:3064-3073 ©2009 by American Society of Hematology

CPX synergizes with cytarabine to induce cell death in leukemia cells. CPX synergizes with cytarabine to induce cell death in leukemia cells. The effects of different concentrations of CPX in combination with cytarabine on the viability of OCI-AML2 cells were measured by MTS assay after 72 hours of incubation. Data were analyzed with Calcusyn software. Combination index (CI) versus fractional effect (FE) plot showing the effect of the combination of CPX and cytarabine. CI < 1 indicates synergism. One of 2 representative isobologram experiments performed in triplicate is shown. Yanina Eberhard et al. Blood 2009;114:3064-3073 ©2009 by American Society of Hematology