In vitro effect of eltrombopag alone and in combination with azacitidine on megakaryopoiesis in patients with myelodysplastic syndrome
Francesco D’Alò, Ilaria Zangrilli, Elisa Cupelli, Luana Fianchi, Marianna Criscuolo, Giulia Falconi, Emiliano Fabiani, Livio Pagano, Stefan Hohaus & Valerio De Stefano
To cite this article: Francesco D’Alò, Ilaria Zangrilli, Elisa Cupelli, Luana Fianchi, Marianna Criscuolo, Giulia Falconi, Emiliano Fabiani, Livio Pagano, Stefan Hohaus & Valerio De Stefano (2020): In vitro effect of eltrombopag alone and in combination with azacitidine on megakaryopoiesis in patients with myelodysplastic syndrome, Platelets, DOI: 10.1080/09537104.2020.1742312
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In vitro effect of eltrombopag alone and in combination with azacitidine on megakaryopoiesis in patients with myelodysplastic syndrome
ImageImageImageImageImageImageImageImageImageFrancesco D’Alò 1,2, Ilaria Zangrilli1, Elisa Cupelli 1, Luana Fianchi 1, Marianna Criscuolo 1, Giulia Falconi 3, Emiliano Fabiani 3, Livio Pagano 1,2, Stefan Hohaus 1,2, & Valerio De Stefano 1,2
1Dipartimento Di Diagnostica per Immagini, Radioterapia Oncologica Ed Ematologia, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma, Italy, 2Sezione Di Ematologia, Dipartimento Di Scienze Radiologiche Ed Ematologiche, Università Cattolica Del Sacro Cuore, Roma, Italy, and 3Dipartimento Di Biomedicina E Prevenzione, Università Di Roma Tor Vergata, Roma, Italy
Abstract
Thrombocytopenia is a severe complication for patients with myelodysplastic syndrome (MDS). Eltrombopag increases platelet count in MDS patients but its combination with azacitidine elicited controversial results. We aimed to quantify the colony forming units of megakaryocytes (CFU-Mk) obtained from CD34+ bone marrow cells isolated from patients with MDS and from healthy donors that were cultured in vitro in the presence or absence of azacitidine and with or without the sequential addition of eltrombopag to the culture medium. CD34+ bone marrow cells from 6 MDS patients and 3 controls were expanded in vitro and cultured for 3 days with or without azacitidine. Subsequently, a CFU-Mk assay was performed in presence or absence of eltrombopag. The addition of eltrombopag in the CFU-Mk assay after mock treatment of CD34+ cells increased the number of CFU-Mk in both controls and patients. On the contrary, using azacitidine pretreated CD34+ cells, eltrombopag minimally increased CFU-Mk in controls and produced heterogeneous response in MDS patients with no change in two patients and CFU-Mk increase in four patients. In vitro CFU-Mk assay suggest that some MDS patients are likely to benefit from the sequential addition of eltrombopag after azacitidine treatment, in the context of a personalized medicine.
Introduction
Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal hematopoietic neoplasms characterized by per- ipheral cytopenias, dysplasia in one or more myeloid lineages, recurrent genetic aberrations and an increased risk of developing acute myeloid leukemia (AML). Thrombocytopenia is a serious and life-threatening complication in MDS patients, as it occurs in 40-65% of cases, is severe in 20-25% of cases, and is an inde- pendent poor prognostic factor included in the R-IPSS score [1,2]. Treatment with the hypomethylating agent azacitidine can induce transient thrombocytopenia; however, doubling of the platelet count before starting the 2nd cycle of therapy is a good prognostic factor associated with better survival[3].
Eltrombopag is a nonpeptide agonist of the thrombopoietin (TPO) receptor, has been approved for the treatment of autoimmune thrombocytopenia, and was shown to be effective in increasing the platelet count and reducing bleeding events in MDS patients [4–7]. A phase I study evaluated the feasibility and safety of a combined treatment with eltrombopag and azacitidine in vivo in thrombocyto- penic MDS patients[8]. However, a recent randomized double- blinded phase 3 studyinvestigating the platelet supportive effects of concomitant eltrombopag administration with azacitidine was prematurely stopped because efficacy outcomes crossed the prede- fined futility threshold as indicated by the lower response rate and Correspondence: Francesco D’Alò, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Università Cattolica Del Sacro Cuore, LargoA. Gemelli 8, Roma 00168, Italy. E-mail: [email protected] trend toward increased progression to AML in the eltrombopag arm versus the placebo arm[9].
The aim of our study was to assess the effect of a sequential in vitro treatment with azacitidine and eltrombopag on megakar- yopoiesis. CD34+ bone marrow cells were isolated from patients with MDS and from healthy donors, expanded and cultivated in the presence of azacitidine, and evaluated for the formation of megakaryocytic colonies in the presence of eltrombopag. The hypomethylating effect of azacitidine can reactivate silenced genes involved in megakaryocytopoiesis and induce megakaryo- cytic differentiation of leukemic myeloid cell lines [10,11]. We have shown that another hypomethylating agent, decitabine, can induce genes involved in megakaryocytic differentiation in vitro in CD34+ cells isolated from patients with MDS[12]. Here, we show that the number of colony forming units of megakaryocytes (CFU-Mk) is markedly reduced in 4 of 6 patients with MDS when compared to controls with a heterogeneous response to azaciti- dine, eltrombopag or their sequential treatment. Interestingly, in one patient, azacitidine-induced inhibition of CFU-Mk and mega- karyocytic colony formation could be restored by treatment with eltrombopag. The heterogeneity of MDS limits the possibility of developing treatment strategies that work for all patients and highlights the need to single out small subgroups of patients who might benefit from a personalized treatment.
Patients and methods
The study population included 15 patients with untreated MDS (female 9, male 6; median age 75 years, range 38–87). Cases were
classified according to the 2016 WHO classification [13] as follows: MDS with single lineage dysplasia (MDS-SLD), 6 cases; MDS with multilineage dysplasia (MLD), 4 cases; MDS with excess blast type 1 (MDS-EB1), 2 cases; MDS with excess blast type 2 (MDS-EB2), 1 case; and therapy-related AML post- MDS (therapy-related myeloid neoplasm, t-MN), 2 cases. All patients but one were thrombocytopenic with a median platelet count of 55 x 109/L, ranging between 34 and 246 x 109/L.
The control population consisted of 6 healthy bone marrow donors, including 4 females and 2 males (median age 31, range 22–47).
Informed consent for the collection of personal and sensitivedata was obtained by all patients.This study follows the principles of the Declaration of Helsinki and was approved by the local Institutional Board.Azacitidine (Vidaza, Celegne) was kindly provided by the local pharmacy, dissolved to 5 mg/ml in 50% glacial acetic acid and stored at −20°C. Eltrombopag (Revolade, Novartis) was kindly provided by Novartis, dissolved in pure water to 10 mg/ ml, aliquoted in multiple vials and stored at −20°C.The leukemic cell lines Raji and HL60 were cultured in RPMImedium supplemented with 10% FBS and treated with different concentrations of azacitidine (100 ng/ml, 250 ng/ml, 500 ng/ml and 1000 ng/ml) for three days. Mock-treated cells were included in each experiment. Medium with fresh drug was replaced daily, and cells were harvested each day for DNA collection. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen). The EZ DNA Methylation-Gold™ Kit was used for bisulfite conversion accord-ing to the manufacturer’s instructions. To confirm the effective- ness of the hypomethylating treatment at different drug concentrations, methylation-specific PCR (MS-PCR) was per-formed for the DAP-K gene, which is known to be methylated in HL60 and Raji cells, as previously described[14].Mononuclear cells were isolated from diagnostic bone marrow samples by Ficoll gradient centrifugation, and CD34+ cells were positively selected by immunomagnetic separation using a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’sinstructions. CD34+ cells were expanded in vitro for 7–10 daysusing the StemSpan™ SFEM II (StemCellTM Technologies).
After a sufficient number of cells (at least 1 × 106 cells) was reached, CD34+ cells (3-5×105 cells/ml in 1 ml) were pretreated for 3 days with 250 ng/ml azacitidine or the corresponding amount of solvent (mock treatment). Medium and drug were replaced daily. To detect and quantify megakaryocytic colonies, we used the MegaCult™-C kit (Stem Cell Technologies), a collagen-based system that uses culture medium containing human recombinant IL3, IL6 and TPO. A megakaryocytic colony assay to detect CFU-MK was performed according to the manufacturer’s instructions, starting with 1.1 × 105 azacitidine-pretreated or mock-pretreated CD34+ cells resuspended in final culture mixture containing collagen and cytokines, in the presence or absence of 1 µg/ml eltrombopag. The concentration of eltrombopag chosen for this study was in the range of concentrations reported in previous studies [15–17]. CD34+ cells were incubated for 10 days in collagen-containing medium on dual-chamber slides at a concentration of 2.5 × 103 CD34+ cells per chamber. Each treatment was performed in duplicate. After dehydration and fixa- tion, the number of CFU-Mk was detected by staining cells with primary antibody targeting the Mk-specific antigen GPllb/llla (CD41) followed by treatment with a biotinylated secondary anti- body and an avidin-conjugated alkaline phosphatase. The number and size of CFU-Mk were scored using a light microscope.CFU-MK were defined as clusters of more than 3 CD41+ cells and subdivided into groups by size: small (3–20 cells per colony, arising from a mature Mk progenitor cell), medium (21–49 cells per colony, arising from an immature Mk progenitor cell), large (more than 50 cells per colony arising from the most immature Mk progenitor cell), and mixed CFU-Mk/non-Mk (containing both CD41+ and CD41- cells, arising from the most immature progenitors capable of differ- entiating into erythroid and Mk cells) (Figure 1).The results are expressed as the mean of two duplicates and the standard deviation (SD). Differences in the CFU-Mk number according to different treatments were expressed as fold change and percent variance and analyzed by nonparametric Mann- Whitney test. GraphPad Prism Version 8.1.0 was used to perform the statistical analysis and construct the graphs.
Results
Preliminary experiments on HL60 and Raji cell lines were carried out to define the optimal azacitidine concentration for hypo- methylating in vitro. Compared to mock treatment, azacitidine induced cell growth inhibition in both cell lines and hypomethy- lation at the DAP-K promoter at a concentration of 100 ng/ml (Supplementary Figures 1 and 2).CD34+ cells from all 6 healthy control subjects were efficiently expanded in culture to obtain at least 1 × 106 cells (starting cell concentration for azacitidine and mock treatment was 3–5 × 105 cells/ml in 1 ml medium). In contrast, CD34+ cells from only 7 out of 15 MDS patients were successfully expanded in vitro to reach therequired cell number. Characteristics of the patients with successful CD34+ cell expansion are summarized in Table I.Treatment of CD34+ bone marrow cells with 250 ng/ml aza- citidine induced growth inhibition in cell cultures of samples from both patients and controls compared to the mock treatment (Figure 2).The CFU-Mk assay was performed starting with the same number of mock-treated or azacitidine-treated CD34+ cells that were cultured in the presence or absence of 1 µg/ml eltrombopag. The number of CFU-Mk was assessed under the following conditions: CD34+ cells were first treated with azacitidine or mock treated and then cultured with or without the addition of eltrombopag. The results are expressed as the mean of a duplicate count for each treatment condition. For the analysis, we consid- ered the total number of pure CFU-Mk, including small, medium
and large CFU-Mk.
azacitidine
MDS CTRL
Pretreatment of CD34+ cells with azacitidine before the CFU- Mk assay was performed produced heterogeneous results in patients and control subjects. In the absence of eltrombopag, azacitidine pretreatment reduced the CFU-Mk number in two control samples (fold change 0.68 and 0.72), while it increased it in the third control sample (fold change 1.73). Azacitidine pretreatment reduced the number of CFU-Mk in two patients with t-MN (fold change 0.53 and 0.28, respectively) and produced a minimal increase in three MDS patients (fold change 1.03, 1.02 and 1.14, respectively) but a more consistent increase in one thrombocytopenic patient with MDS with single lineage dysplasia (fold change 2.16, percent variance 116.49%, p = .069).
The addition of eltrombopag to azacitidine-pretreated CD34
+ cells increased the number of CFU-MK in the control subjects (mean percent variance 7.35%, SD 4.27) and even more in
. Growth inhibition effect of azacitidine on CD34+ cells from MDS patients and control subjects expanded in vitro.
One patient with MDS-EB2 (patient ID MDS5) was excluded because no CFU-Mk developed. Three controls were used to set up the conditions of in vitro treatment, and the number of CFU- Mk was not assessed under all treatment conditions. The CFU-Mk assay was successfully carried out in 3 controls and in 6 patients for all treatment conditions. The absolute number of CFU-Mk from each patient at all treatment conditions is represented in Figure 3.
In the mock-treated groups, the mean CFU-Mk number appeared to be higher in control subjects (mean 789, range 513 to 1108) than the patients (mean 357, ranging from 49.5 to 1044) without reaching statistical significance (p = .2).The addition of eltrombopag to the CFU-Mk assay after mock treatment of CD34+ cells increased the number of CFU-Mk in both control subjects (mean percent variance 31.37%; SD 44.19) and patients (mean percent variance 17.95%; SD 31.56) without any significant difference (p = .9048).patients (mean percent variance 42.89%, SD 71.37). However, the effect of eltrombopag on azacitidine-pretreated CD34+ cells was highly heterogeneous in the patient group, as two patients did not show any increase in the number of CFU-Mk (fold change0.91 in both) while four patients showed an increase (fold change 1.07, 1.3, 1.6 and 2.78, respectively) (Figure 4). In particular, one patient with t-MN and del(5q31) who had a reduction in the CFU- Mk number after azacitidine pretreatment in the absence of eltrombopag (fold change 0.28, percent variance −72.46%,p = .069) showed a significant increase in the number CFU-MKafter the addition of eltrombopag (fold change 2.78, percent var- iance 178.43%, p = .009).
Discussion
Preclinical studies reported that eltrombopag is effective in increasing megakaryocytic differentiation and the formation of normal megakaryocytic colonies in patients with AML and MDS without stimulating proliferation of a malignant leukemic clone [17,18]. study is the first to evaluate the effect of eltrombopag and azacitidine on megakaryocytic colony formation in vitro in nor- mal control subjects and in patients with MDS and t-MN. A sequential schedule was chosen because it closely reflects the clinical setting of treatment with a disease-modifying agent, such as azacitidine, that is followed by support with a thrombopoiesis- stimulating agent. In addition, the collagen-based semisolid sys- tem of the megakaryocytic colony assay would render impossible the replacement of the medium with azacitidine, which has a short half-life. Of note, the MegaCultTM-C medium with Cytokines used for the CFU-Mk assay already includes rhTPO at a concentration of 50 ng/ml. Although this can in part reduce the magnitude of fold changes observed with eltrombopag, the inclusion of this molecule nevertheless reflects real clinical con- ditions because serum TPO levels in thrombocytopenic MDS/ AML patients are usually not reduced but are often even higher than normal controls[19]. Moreover, eltrombopag binds the trans- membrane domain of the TPO receptor and does not compete with TPO for receptor binding but rather enhances endogenous TPO function as opposed to replacing it[20]
In our study, pretreatment with azacitidine without the addition of eltrombopag increased CFU-MK numbers in 1 of 3 healthy controls and in 4 of 5 patients with MDS. Similarly, previous studies have shown the ability of a hypomethylating treatment to induce megakaryocytic differentiation of theleukemic myeloid cell lines 416B and ELF-153, as well as of hematopoietic pro-genitor cells from healthy control subjects and ITP and MDS patients [10–12,21].
The phase 3 randomized, double-blind, placebo-controlledSUPPORT study, which investigated the platelet-supportive effects of eltrombopag given concomitantly with azacitidine, sur- prisingly found that eltrombopag plus azacitidine worsened plate- let recovery (transfusion independence 16% and 31% in the eltrombopag and placebo groups, respectively), reduced response rates (20% and 35%, respectively) and was associated with a trend toward increased progression to AML (15% and 9%, respectively) [9]. These results contrast with the proven inhibitory effect of eltrombopag on leukemic myeloid blast proliferation and its abil- ity to stimulate megakaryocytic differentiation and formation of normal megakaryocytic colonies in patients with AML and MDS [17,18]. Single agent trials in MDS and AML patients have shown the ability of eltrombopag to increase platelet count and
reduce clinically relevant thrombocytopenic events without any increase in the rate of disease progression [5–7]. Interestingly, in
ing the CFU-Mk number, in at least some MDS patients and all the control subjects, and could represent an alternative approach to be explored in a clinical context.
A limitation of our study is the small number of patients included and the heterogeneous response obtained by the in vitro treatment. Indeed, this heterogeneity reflects disease biology, being MDS patients widely heterogeneous for clinic and molecular features, and drug response. Our findings suggest that some patients with MDS could achieve an increase in the number of CFU-Mk after treatment with only the hypomethylat- ing treatment without any benefit from the addition of eltrombo- pag, while other patients, in particular those whose CFU-Mk number is reduced after hypomethylating treatment, could increase the number of CFU-Mk with eltrombopag. Further stu- dies will be necessary with larger sample numbers and a more extensive molecular characterization in order to identify the sub- group of patients that might have a benefit from sequential addi- tion of eltrombopag to azacitidine.
Our study serves as a proof of concept to study megakaryopoi- esis in vitro. This assay could be included into clinical trials on the combination of azacitidine and eltrombopag to assess its predictive power for clinical response. This might offer a possibility to individualize treatment of MDS patients increas- ing the therapeutic benefit/risk ratio.
Authors’ Contribution
F.D. designed the project, analyzed data and wrote the paper; I.Z. and E. paper; V.D., G.F. and E.F. designed the project, revised and finally approved the paper; L.F., M.C. and L.P. designed the project, provided patients’ samples and data, revised and finally approved the paper; S. H. analyzed data, revised and finally approved the paper.
Acknowledgements
Financial support for the preclinical study was partially provided by Novartis Farma SpA. The Eltrombopag active substance was generously supplied by Novartis Pharma AG. We acknowledge Prof. Bacigalupo and colleagues at the Bone Marrow Transplantation Unit for providing the control samples.
Disclosure of Interest
The authors report no conflict of interest.
Funding
This work was supported by the Novartis.
Supplementary material
Supplemental data for this article can be accessed on the publisher’s website.
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