Effect of the nonpeptide thrombopoietin receptor agonist Eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome

Abstract

Thrombocytopenia is a frequent symptom and clinical challenge in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Eltrombopag is a small molecule thrombopoietin receptor agonist that might be a new option to treat thrombocytopenia in these diseases, provided that it does not stimulate malignant hematopoiesis. In this work, we studied the effects of Eltrombopag on proliferation, apoptosis, differentiation, colony formation, and malignant self-renewal of bone marrow mononuclear cells of patients with AML and MDS. Malignant bone marrow mononuclear cells did not show increased proliferation, or increased clonogenic capacity at concentrations of Eltrombopag ranging from 0.1 to 30 microg/mL. On the contrary, we observed a moderate, statistically nonsignificant (P = .18), decrease of numbers of malignant cells (mean, 56%; SD, 28%). Eltrombopag neither led to increased 5-bromo-2-deoxyuridine incorporation, decreased apoptosis, an increase of malignant self-renewal, nor enhanced in vivo engraftment in xenotransplantations. Furthermore, we found that Eltrombopag was capable of increasing megakaryocytic differentiation and formation of normal megakaryocytic colonies in patients with AML and MDS. These results provide a preclinical rationale for further testing of Eltrombopag for treatment of thrombocytopenia in AML and MDS.

Figure 1

Eltrombopag does not enhance growth of malignant BM cells from patients with MDS/AML. (A) Cumulative cell numbers after 12-day culture in semisolid methylcellulose medium (n = 5 normal controls; n = 10 MDS/AML). BM-MNC were seeded in methylcellulose containing hrIL-3, hrSCF, hrIL-6, insulin, and transferrin. Cultures were supplemented with either 0, 0.1, 1, 3, 10, or 30 μg/mL Eltrombopag, or 100 ng/mL hrTPO. (B) BrdU incorporation in MDS/AML BM-MNC in the presence of either 0, 3, or 10 μg/mL Eltrombopag. BM-MNC of MDS/AML patients were kept in liquid cultures containing BSA, insulin, and transferrin, and cultures were supplemented with hrIL-3, hrIL-6, hrSCF, and hrFLT3L. Cells were incubated with the different concentrations of Eltrombopag for 1, 2, 6, or 12 days. BrdU incorporation was measured by FACS analysis. Averages and SDs (error bars) are indicated (n = 6). (C) Apoptotic activity upon exposure to increasing concentrations of Eltrombopag. BM-MNC of MDS/AML patients were kept in liquid cultures containing BSA, insulin, and transferrin, supplemented with hrIL-3, hrIL-6, hrSCF, and hrFLT3L. Cells were incubated with either 0, 0.1, 1, 3, 10, or 30 μg/mL Eltrombopag for 24 and 72 hours and analyzed for apoptotic cells (Annexin V⁺) by FACS. Averages and error bars (SD) are shown (n = 6).

Figure 2

Eltrombopag does not increase relative percentages of leukemic blasts. (A) Relative numbers of CD33-expressing myeloid cells in cultures of MDS/AML patient-derived BM-MNC in the presence of Eltrombopag. BM-MNC of MDS/AML patients were seeded in liquid cultures containing BSA, insulin, and transferrin, supplemented with hrIL-3, hrIL-6, hrSCF, and hrFLT3L. Cells were incubated with either 0 or 3 μg/mL Eltrombopag or 100 ng/mL hrTPO for 12 days. CD33⁺ cells were assessed by FACS analysis. Results from 7 individual patients are shown. (B) Relative abundance of leukemic blasts and CD33⁺ cells in cultures of MDS/AML patient-derived BM-MNC in presence of Eltrombopag. BM-MNC of MDS/AML patients were grown in cytokine-supplemented medium and incubated with either 0 or 3 μg/mL Eltrombopag for 5 days. Relative blast counts were assessed from cytospin preparations, and CD33⁺ cells were assessed by FACS analysis. Results from 4 individual patients are shown. (C) Relative abundance of leukemic blasts after plating of MDS BM-MNC in methylcellulose. BM-MNC were cultured in cytokine-supplemented methylcellulose for 12 days and then replated, in the presence or absence of Eltrombopag or 100 ng/mL hrTPO. Cytospins were prepared and stained with Wright-Giemsa after the primary plating as well as the replating. Pictures of a representative MDS patient are shown. Blasts are indicated by arrows.

Figure 3

Eltrombopag does not increase long-term self-renewal of normal or MDS/AML patient-derived BM-MNC. Serial replating assays of BM-MNC were performed in methylcellulose containing hrIL-3, hrSCF, hrIL-6, insulin, and transferrin. Cultures were supplemented with 0, 3, or 10 μg/mL Eltrombopag or 100 ng/mL hrTPO. After 12 days of culture, cells were isolated from the methylcellulose medium and replated onto the next plate. Serial replatings were carried out for 4 rounds of replating or until colony formation exhausted. (A) Serial relating of BM-MNC from healthy donors (n = 5). (B) Serial replating of BM-MNC from patients with MDS/AML (n = 13). (C) Xenotransplantation assays of BM-MNC from MDS/AML patients (n = 3). BM-MNC were transplanted into NOG mice after sublethal irradiation. Successfully engrafted mice were either treated with Eltrombopag (0.3 mg/mL) in the drinking water or left untreated. After 12 weeks of Eltrombopag treatment, all mice were killed and analyzed for human CD45-expressing cells by FACS. (Left) Representative dot plot of human CD45 analysis. (Right) Chimerism of human CD45⁺ cells in xenografted mice (n = 20 total) with or without Eltrombopag treatment. Averages and SDs of mouse cohorts of each of the 3 transplanted samples are shown. None of the cohorts showed higher engraftment upon treatment with Eltrombopag.

Figure 4

Eltrombopag stimulates megakaryopoietic progenitor cells in normal BM-MNC. (A) Total number of Mk colonies in the presence of increasing concentrations of Eltrombopag (n = 3). BM-MNC were seeded in semisolid collagen-based medium containing hrIL-3 and hrIL-6. Cultures were supplemented with either 0, 0.1, 1, 3, or 10 μg/mL Eltrombopag or 100 ng/mL hrTPO. Mk colonies were enumerated after 12 days of culture. (B) Differential colony count of Mk progenitor-derived colonies. Relative number of colonies from most immature bipotent Mk progenitors (□), immature Mk progenitors and precursors (), and mature Mk progenitor cells (■) are shown (see inset for colony morphology). Error bars indicate SD (n = 3). (C) Formation of non-Mk, myeloid colonies is not altered in the presence of Eltrombopag. BM-MNC were seeded in cytokine-supplemented methylcellulose medium. After 12 days of culture, CFU from granulocytic and monocytic progenitor cells (CFU-G, CFU-GM, CFU-M), erythrocytic progenitor cells (BFU-E, CFU-E), and granulocyte/erythrocyte/Mk/monocyte progenitor cells (CFU-EMM) were enumerated. Averages are shown. Error bars indicate SD (n = 3).

Figure 5

Eltrombopag stimulates megakaryopoiesis in CD34⁺ BM-MNC cells from MDS/AML patients. (A) Number of Mk CD41a-expressing cells upon Eltrombopag treatment of CD34⁺ BM-MNC from MDS/AML patients (n = 5). Immunomagnetically enriched CD34⁺ BM-MNC were seeded in liquid cultures containing BSA, insulin, transferrin, hrIL-3, hrIL-6, hrSCF, hrFLT3L, and human low-density lipoproteins. Cells were incubated with either 0 or 3 μg/mL Eltrombopag, or 100 ng/mL hrTPO alone, or a combination of 3 μg/mL Eltrombopag and 20 ng/mL hrTPO for 5 days. CD41a⁺ cells were assessed by FACS analysis. The fold of change of CD41a⁺ cells in comparison with 100 ng/mL hrTPO-treated cultures of 5 individual patients is shown. Four of the 5 samples were treated with the combination of Eltrombopag plus TPO (one patient data not analyzed [n/a]). (B) Representative FACS histogram plot of cells upon incubation with either 0 or 3 μg/mL Eltrombopag, 100 ng/mL TPO alone, or 3 μg/mL Eltrombopag and 20 ng/mL hrTPO. (C) Eltrombopag increases the number of Mk colonies in cultures of CD34⁺ BM-MNC from MDS/AML patients. CD34⁺ BM-MNC were seeded in semisolid collagen-based medium containing hrIL-3, hrIL-6, hrSCF, hrFLT3L, and human low-density lipoproteins. Cultures were supplemented with either 0 or 3 μg/mL Eltrombopag, 100 ng/mL hrTPO alone, or 3 μg/mL Eltrombopag in combination with 20 ng/mL hrTPO. Mk colonies were enumerated after 12 days of culture. The fold of change compared with 100 ng/mL hrTPO-treated CD34⁺ cells is shown (n = 5). (D) Mk colony morphology of Mk colonies derived from healthy persons' and MDS/AML patients' BM-MNC. (E) Differential colony counts of Mk progenitor-derived colonies after 12 days of culture in the presence of either 0 or 3 μg/mL Eltrombopag, 100 ng/mL hrTPO alone, or 3 μg/mL Eltrombopag in combination with 20 ng/mL hrTPO. Colonies from bipotent Mk progenitors, immature Mk progenitors, and mature Mk progenitors were scored. The fold change compared with cells cultured in presence of 100 ng/mL hrTPO is shown. Results from 5 individual patients are shown.

Figure 6

Eltrombopag spares Stat3 phosphorylation. Flow cytometric phosphoprotein analysis of MDS/AML cells treated with either 0 or 3 μg/mL Eltrombopag or 100 ng/mL hrTPO. (A) Representative histogram plot. Cells were prestarved in serum-free RPMI medium for 4 hours, treated with Eltrombopag or hrTPO for 60 minutes, fixed, permeabilized, stained with phospho-specific antibodies against p-Stat3 and p-Stat5, and analyzed by FACS. (B) Mean values and SD of p-Stat5 and p-Stat3 analysis of 4 MDS/AML samples upon treatment with either Eltrombopag or hrTPO. ***Statistical significance (P < .001).