Eltrombopag inhibits the proliferation of leukemia cells via reduction of intracellular iron and induction of differentiation

Abstract

Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G(1) phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EP's antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia.

Figure 1

EP inhibits cell cycling and leads to a block in the G₁ phase. (A) HL60 and URE cells were treated with increasing concentrations of EP. Cell viability was measured by cell counts with trypan blue exclusion (top panel), and MTS assays (bottom panel) were performed every 24 hours for 72 hours. Data represent the means ± SD of viable cells performed in triplicate and MTS proliferative index. *P < .05; **P < .01. (B) HL60 cells (i) and URE cells (ii) were incubated in 10μM Cell Tracker Orange for 30 minutes, washed, and analyzed by FACS (red line = hour 0). Cells were treated without EP (blue line) or with 5 μg/mL of EP (orange line) and FACS analysis was performed every 24 hours for 3 days. Lower mean fluorescence indicates increased cell division. The fold change of HL60 cells (iii) and URE cells (iv) of FACS mean fluorescence intensity (MFI) ± SD (n = 3) of Cell Tracker Orange–labeled HL60 cells treated with 5 μg/mL of EP relative to untreated cells is shown. *P < .05; **P < .01; ***P < .001. EP slows cell division in HL60 and URE cells because higher MFI represents slower cell division. (C) Cell-cycle analysis of HL60 cells (i) and URE cells (ii) with or without 5 or 10 μg/mL of EP for 48 hours. EP induces a cell-cycle block in G₁ phase with a subsequent decrease in the S phase. (D) The fold change variation of gene expression by microarray in HL60 cells treated with 3 μg/mL of EP for 36 hours relative to untreated cells. EP down-regulated genes necessary for the transition from the G₁ to the S phase.

Figure 2

EP induces differentiation of leukemia cell lines. (A) Representative morphology of HL60 cells treated for 72 hours without EP, with EP, or with TPO shown at 20× (left panel) and 63× (right panel). Cells treated with EP portray reorganization of the nuclear contents compared with untreated cells (red arrows) and cells treated with TPO. (B) The fold change of FACS MFI ± SD (n = 3) of CD11B expression (i) and CD14 (ii) in HL60 cells treated with the above conditions relative to untreated cells for 72 hours. *P < .05; **P < .01; ***P < .001. FACS analysis of CD11B expression (iii) in untreated HL60 cells (blue line) versus cells treated with 5 μg/mL of EP (orange line) for 72 hours. FACS analysis of CD14 expression (iv) in untreated HL60 cells (green line) versus cells treated with 5 μg/mL of EP (orange line) for 72 hours. CD11B and CD14 are overexpressed with EP treatment. (C) Representative morphology of U937 cells and URE cells treated for 72 hours under the above conditions. U937 cells treated with EP demonstrate increased size with increased vacuoles in the cytoplasm. (D) The fold change of FACS MFI ± SD (n = 3) of CD11B expression in U937 cells and URE cells treated with or without EP for 72 hours. *P < .05; **P < .01.

Figure 3

EP depletes intracellular iron in leukemia cell lines. (A) HL60 cells were labeled with a 0.25μM concentration of the intracellular iron-chelating dye calcein-AM for 5 minutes. Cells were washed then treated with 0 μg/mL of EP, 5 μg/mL of EP, 10 μg/mL of EP, 25 ng/mL of TPO (− control), 100μM SIH (+ control), or 100μM DFO (+ control) for 1 hour (left panel) or 4 hours (right panel) at 37°C and cells were analyzed by FACS. Data represent the change in the MFI ± SD (n = 3) compared with untreated HL60 cells *P < .05; **P < .01; ***P < .001. (B) Five leukemia/lymphoma cell lines were labeled with calcein-AM and then treated with 5 μg/mL of EP for 4 hours. Cells were analyzed by FACS. Data represent the change in the MFI± SD (n = 3) compared with untreated cells. (C) The fold change variation of gene expression in HL60 cells treated with 3 μg/mL of EP or 100 ng/mL of TPO for 12 hours (left panel) or 36 hours (right panel) relative to untreated cells. EP up-regulated the transferrin receptor (TFRC) and down-regulated the ferritin light chain (FTL) and heavy chain (FTH1). (D) FACS analysis of CD71 expression in untreated HL60 cells (blue line) versus cells treated with 5 μg/mL of EP (orange line) for 24 hours (left panel). FACS analysis of CD71 expression in HL60 cells treated with 5 μg/mL of EP (orange line) versus cells preloaded with 500 μg/mL of ferric ammonium citrate (FAC) for 24 hours and then treated with 5 μg/mL of EP (blue line) for 24 hours (middle panel). FACS MFI ± SD (n = 3) of CD71 expression in HL60 cells and URE cells untreated, treated with 5 μg/mL of EP, or preloaded with 500 μg/mL of ferric ammonium citrate followed by treatment with 5 μg/mL of EP for 24 hours (right panel). P value (by t test) represents the difference in MFI between treated and untreated cells *P < .05; **P < .01; ***P < .001. CD71 is overexpressed in response to EP treatment and expression is decreased when cells are preloaded with iron.

Figure 4

EP-induced cell death is iron dependent. (A) HL60 cells were untreated or treated with 5 μg/mL of TPO, 25 ng/mL of EP, or 100u DFO for 72 hours preloaded or not with 500 μg/mL of ferric ammonium citrate (FAC) for 24 hours. Cell viability was measured by cell counts with trypan blue exclusion (top panel) and MTS assays (bottom panel) performed at 72 hours. Data represent the means ± SD of viable cells performed in triplicate. P values represent the difference between iron-loaded and noniron-loaded cells. *P < .05; **P < .01. (B) URE^−/− cells were untreated or treated with 5 or 10 μg/mL of EP for 72 hours preloaded or not with 500 μg/mL of ferric ammonium citrate for 24 hours. Cell viability was measured by cell counts with trypan blue exclusion (top panel) and MTS assays (bottom panel) performed at 72 hours. Data represent the means ± SD of viable cells performed in triplicate. P values represent the difference between iron-loaded and noniron-loaded cells. *P < .05; **P < .01; ***P < .001. (C) HL60 Cells were incubated in 10μM Cell Tracker Orange for 30 minutes, washed, and analyzed by FACS. Cells were treated without EP, with 5 μg/mL of EP, or were preloaded with 500 μg/mL of ferric ammonium citrate and then treated with or without 5 μg/mL of EP. FACS analysis was performed at 72 hours. The percent change in FACS MFI ± SD (n = 3) of Cell Tracker Orange labeled HL60 cells treated with 5 μg/mL of EP with or without iron preload relative to untreated cells, corrected for hours 0 MFI. The P value represents the difference between the MFI of the iron-preloaded cells treated with EP and the cells treated with EP without iron preload. *P < .05; **P < .01. EP slows cell division in HL60 cells and cell division is rescued by preloading cells with iron. (D) Representative morphology of HL60 cells treated for 72 hours under the above conditions shown at 20× (left panel) and 63× (right panel). HL60 cells preloaded with iron and subsequently treated with EP display less segmented nuclei than EP-treated cells without iron (cells with increased nuclear segmentation are indicated by arrows). (E) FACS analysis of CD11b expression (i) in HL60 cells treated with 5 μg/mL of EP (orange line) versus cells preloaded with 500 μg/mL of ferric ammonium citrate (FAC) for 24 hours and then treated with 5 μg/mL of EP (blue line) for 72 hours. FACS analysis of CD14 expression (ii) in HL60 cells treated with 5 μg/mL of EP (orange line) versus cells preloaded with 500 μg/mL of FAC for 24 hours and then treated with 5 μg/mL of EP (blue line) for 72 hours. CD11b and CD14 are overexpressed with EP treatment and rescued by preloading cells with iron. The fold change of FACS MFI ± SD (n = 3) of CD11b expression (iii) and CD14 (iv) in HL60 cells preloaded with 500 μg/mL of FAC and then treated with 5 μg/mL of EP compared with cells treated with 5 μg/mL of EP without iron load is shown. P values represent the difference between iron-loaded and noniron-loaded cells. *P < .05; **P < .01.

Figure 5

EP prolongs survival in mouse models of leukemia. (A) Ten million HL60 cells were transplanted into the tail veins of NSG mice (n = 10) 4 hours after sublethal irradiation. Mice were divided into 2 groups, one receiving untreated drinking water, and the other receiving 1 mg/mL of EP in the drinking water starting at day +3. BM aspirates were performed on day +21 and FACS analysis was performed assessing donor cell chimerism (CD15⁺Ly5.1⁻; i). Kaplan Meier survival curve of HL60-transplanted mice (ii) treated with 1 mg/mL or 3.0 mg/mL EP in the drinking water versus mice with untreated drinking water. *P < .05; **P < .01. (B) One million URE cells were transplanted into the tail veins of NSG mice 4 hours after sublethal irradiation. Mice were divided into 3 groups, with one group receiving untreated drinking water (n = 7), one group receiving 0.4 mg/mL of EP in the drinking water (n = 5), and the other receiving 1 mg/mL of EP in the drinking water (n = 4) starting at day +3. Kaplan-Meier survival curve of URE transplanted mice treated with 0.4 or 1.0 mg/mL of EP in the drinking water versus mice with untreated drinking water is shown. **P < .01. (C) HL60 cells were treated with or without 20nM cytarabine (i), with or without 24 ng/mL of doxorubicin (ii) ± 5 μg/mL of EP for 72 hours. Cell viability was measured by cell counts with trypan blue exclusion. Data represent the means ± SD of viable cells performed in triplicate. **P < .01; ***P < .001.