Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells from Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo

Abstract

Current understanding suggests that malignant stem and progenitor cells must be reduced or eliminated for prolonged remissions in myeloid neoplasms such as acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). Multicolor flow cytometry has been widely used to distinguish stem and myeloid progenitor cells from other populations in normal and malignant bone marrow. In this study, we present a method for assessing drug sensitivity in MDS and AML patient hematopoietic stem and myeloid progenitor cell populations ex vivo using the investigational Nedd8-activating enzyme inhibitor MLN4924 and standard-of-care agent cytarabine as examples. Utilizing a multicolor flow cytometry antibody panel for identification of hematopoietic stem cells, multipotent progenitors, common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythroid progenitors present in mononuclear cell fractions isolated from bone marrow aspirates, we compare stem and progenitor cell counts after treatment for 24 hours with drug versus diluent. We demonstrate that MLN4924 exerts a cytotoxic effect on MDS and AML stem and progenitor cell populations, whereas cytarabine has more limited effects. Further application of this method for evaluating drug effects on these populations ex vivo and in vivo may inform rational design and selection of therapies in the clinical setting. Stem Cells Translational Medicine 2017;6:840-850.

Keywords: Acute myelogenous leukemia; Hematopoietic stem cells; MLN4924; Myelodysplastic syndrome; Myeloid progenitor cells.

Figure 1

Quantification of hematopoietic stem and progenitor cell populations in cord blood units and relative survival of populations in MLN4924‐treated versus control samples. (A): Gating scheme for identification of hematopoietic stem and progenitor cell populations from initial CD34⁺ CD45^dim population. See text for details. (B): Antigen profiles for each population of interest. (C): Survival of CD34⁺ CD45^dim cord blood populations in MLN4924 or AraC compared with diluent control. Kruskal‐Wallis test, 6 sample groups, p value = NS (.71). Error bars represent ± SEM from four independent cord blood samples. Abbreviations: AraC, cytarabine; CMP, common myeloid progenitor; DMSO, dimethyl sulfoxide; GMP, granulocyte‐monocyte progenitor; HSC, hematopoietic stem cell; MEP, megakaryocyte‐erythroid progenitor; MPP, multipotent progenitor; NS, not significant.

Figure 2

Survival of AML hematopoietic stem and progenitor cells in MLN4924 or cytarabine versus diluent. Aliquots of bone marrow mononuclear cells from AML patients were treated for 24 hours with increasing concentrations of MLN4924, 100 nM AraC, or diluent (0.1% DMSO). Cells were harvested, stained using the fluorescent antibody panel for identification of live stem and progenitor cells (see Methods), and analyzed by flow cytometry. HSC and progenitor populations were quantified using the gating scheme outlined in Figure 1A. Summary of survival of AML stem and progenitor cells relative to populations in diluent treated samples from seven AML patient BMs. Patient data and cell count in each population are reported in Table 1 and supplemental online Table 1 (de novo AML, n = 5, patients 1 through 5; post‐MDS AML, n = 2, patients 6 and 7). Several populations could not be gated for quantification from patient 4 nor could the MPP from patient 3 (Table 1). CD34⁺ CD45^dim = all stem and myeloid progenitor populations; stem cells = HSC + MPP; myeloid progenitors = CMP + GMP + MEP. Abbreviations: AraC, cytarabine; AML, acute myelogenous leukemia; CMP, common myeloid progenitor; DMSO, dimethyl sulfoxide; GMP, granulocyte‐monocyte progenitor; HSC, hematopoietic stem cell; MDS, myelodysplastic syndrome; MEP, megakaryocyte‐erythroid progenitor; MPP, multipotent progenitor.

Figure 3

MLN4924 induces apoptosis in post‐MDS AML. (A–C): Bone marrow mononuclear cells from a patient with post‐MDS AML were treated with increasing concentrations of MLN4924 versus diluent (0.1% DMSO) for 24 hours. Parallel samples were assayed for subdiploid (apoptotic) cells by propidium iodide staining (A); Cullin1, c‐Myc, and Bcl‐2 family member expression by immunoblotting (B); and Noxa mRNA levels (expressed relative to diluent control) by quantitative RT‐PCR (C). Note the increase in c‐Myc and Noxa as previously seen in de novo AML [27]. Abbreviations: AML, acute myelogenous leukemia; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MDS, myelodysplastic syndrome.

Figure 4

Quantification and relative survival of MDS hematopoietic stem and progenitor cells treated with MLN4924 versus diluent. Bone marrow mononuclear cells were treated for 24 hours with MLN4924 or diluent (0.1% DMSO), harvested, stained using the fluorescent antibody panel for identification of live stem and progenitor cells (see Methods) and analyzed by flow cytometry. HSC and myeloid progenitor populations were quantified using the gating scheme outlined in Figure 1A. (A–E): Survival for each population in samples treated with MLN4924 relative to corresponding diluent‐treated population for MDS patients 1 through 5. Patient data and cell count in each population are reported in Table 2 and supplemental online Table 2. CD34⁺ CD45^dim = all stem and myeloid progenitor populations; stem cells = HSC + MPP; myeloid progenitors = CMP + GMP + MEP. Abbreviations: CMP, common myeloid progenitor; DMSO, dimethyl sulfoxide; GMP, granulocyte‐monocyte progenitor; HSC, hematopoietic stem cell; MEP, megakaryocyte‐erythroid progenitor; MPP, multipotent progenitor; ND, population not detected.