Reduced proliferation of non-megakaryocytic acute myelogenous leukemia and other leukemia and lymphoma cell lines in response to eltrombopag
Introduction
Thrombocytopenia in leukemia patients can result from incompetent marrow due to the disease or damage of the marrow due to chemotherapy. The development of targeted thrombopoietic agents has implications for improving therapy for patients suffering low platelet counts due to their disease. Approximately 40–65% of patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) are reported to be thrombocytopenic [1], [2]. Eltrombopag (SB-497115, PROMACTA®; GlaxoSmithKline, USA) represents the first oral, small molecule, non-peptide thrombopoietin receptor (TpoR) agonists. Eltrombopag has been approved for treatment of adults with chronic idiopathic thrombocytopenic purpura (ITP) in the USA, and is in clinical trials for the treatment of thrombocytopenia associated with chronic liver disease, Hepatitis C antiviral therapy, MDS and chemotherapy induced thrombocytopenia (www.clinicaltrials.gov). Eltrombopag increased platelet counts in normal subjects, as well as in patients with chronic ITP and chronic Hepatitis C patients [3], [4], [5], [6]. Eltrombopag stimulates differentiation of normal CD34+ marrow cells into megakaryocytes, but its requirement for TpoR expression limits its specificity to the megakaryocyte lineage [7], [8]. However, there remains the question of whether eltrombopag might have the potential to induce proliferation of any TpoR expressing cell. Various reports found approximately 50% of AML patient blood or marrow expresses MPL, the gene for TpoR [9], [10]. Additional reports investigating the proliferative response of AML patient samples to thrombopoietin (Tpo) or megakaryocyte growth and development factor (MGDF) demonstrated a range of proliferation responses (0–40%) in AML samples, although these did not correlate with the patient's French American British (FAB) classification [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. Several megakaryocytic cell lines were used to identify and characterize eltrombopag [8], but its effects on other leukemia cell lines had not been examined. This manuscript describes the expression of MPL on a number of AML and other leukemia and lymphoma cell lines and reports how eltrombopag affects the proliferation, apoptosis and differentiation of these cell lines.
Section snippets
Cytokines and compound
rhTpo, recombinant human stem cell factor (rhSCF), and recombinant murine interleukin-3 (rmIL-3) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Recombinant human erythropoietin (rhEpo) and G-CSF were obtained from Amgen, Inc. (Thousand Oaks, CA, USA). Cytokines were diluted in Iscove's Modified Dulbecco's Medium (IMDM) and used at final concentrations of 1, 3, 10, 30 or 100 ng/mL. SB-497115-GR, the monoethanolamine salt form, was resuspended in water and diluted in IMDM with 1%
Proliferation results
The human leukemia and lymphoma cell lines tested, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, OCI-AML2, OCI-AML3, ML-2, THP-1, F-36P, U937 and PLB-985, demonstrated no increase in proliferation following 72 h treatment with eltrombopag. In fact, there was a decrease in proliferation in these cell lines, generally at eltrombopag concentration greater than 1 μg/mL (Fig. 1A–C). These data were fit to curves and IC50 values for the decrease in proliferation ranging from 0.5 to 21 μg/mL (Table 1).
Discussion
Patients with AML or MDS frequently have neutropenia or thrombopcytopenia [1], [2]. The first step in determining the utility of the non-peptide, small molecule TpoR agonist, eltrombopag, in these patients is to determine its effects on AML cell lines. Eltrombopag did not increase the proliferation of the 15 leukemia and lymphoma cell lines that were not of megakaryocytic origin. Interestingly, there was a decrease in proliferation (thymidine incorporation) at higher concentrations of
Conflict of interest statement
CLEM, JK, MA and AC are employees of GlaxoSmithKline, CLEM, MA and AC own GSK stock, CLEM is named on GSK patents. RM and PP have no conflicts of interest.
Author contributions
CLEM designed research, analyzed data, supervised the project and wrote the manuscript. JK and PP carried out research and analyzed data. MA critically reviewed the manuscript and analyzed data. RM designed experiments and analyzed data. AC designed experiments, carried out research and analyzed data.
Acknowledgments
Disclosures: This work was fully funded by GlaxoSmithKline.
References (28)
- et al.
Establishment of baseline toxicity expectations with standard frontline chemotherapy in acute myelogenous leukemia
Blood
(2007) - et al.
Phase I clinical study of eltrombopag, an oral, nonpeptide thrombopoietin receptor agonist
Blood
(2007) - et al.
Effect of eltrombopag on platelet counts and bleeding during treatment of chronic idiopathic thrombocytopenic purpura: a randomized, double-blind, placebo-controlled trial
The Lancet
(2009) - et al.
The discovery of eltrombopag, an orally bioavailable TpoR agonist
- et al.
Analysis of thrombopoietin receptor (c-mpl) mRNA expression in de novo acute myeloid leukemia
Leukemia Research
(2000) - et al.
Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin
Blood
(1995) - et al.
Proliferative reaction of myelogenous leukemia cells with cytokines G-CSF, GM-CSF, M-CSF, SCF and TPO
Leukemia Research
(1998) - et al.
A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion
Cell
(2007) Growth factors in acute myeloid leukemia
Best Pract Res: Clin Haematol
(2001)- et al.
Phase 1/2 study of AMG531 in thrombocytopenic patients (pts) with low-risk myelodysplastic syndrome (MDS): update including extended treatment
Blood
(2007)