The New CD95 Ligand Inhibitor APG101 Leads To Decreased Apoptosis and Improved Erythroid Differentiation In Primary CD34+ Cells From Patients With Low Risk Myelodysplastic Syndrome (MDS)

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid malignancies characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. Particularly early stage MDS are at least in part characterized by an increased apoptosis of myeloid and erythroid progenitors that causes peripheral cytopenia. APG101 is a glycosylated fusion protein consisting of the extracellular domain of human CD95 (Fas receptor) and the Fc domain of human IgG1. APG101 effectively binds to the CD95 ligand (CD95L) expressed on effector cells as well as to functionally active ligand in solution, by that blocking the interaction between CD95 and its ligand. The aim of our study was to evaluate whether APG101 treatment of primary CD34+ reduces the apoptotic rate and improves the differentiation capacity of these cells.

Bone marrow cells were obtained during routine bone marrow aspiration after all patients gave their written informed consent. Isolated primary CD34+ cells from 11 MDS patients were cultured in complete supplemented IMDM medium for 6 days with increasing concentrations of APG101 (1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL). After incubation time, cells were multicolor- stained with the following dye combination: Annexin-FITC + CD235a-PE + CD34-PECy7 + CD71-APC + 7-AAD and analyzed immediately on a flow cytometer (FACSCanto, BD Bioscience, Heidelberg, Germany). Analysis of raw FACS data was done with the FACSDiva software. To analyze the differentiation capacity of CD34+ progenitors, methylcellulose assays were performed in parallel to the aforementioned experiments. However, due to limited cell numbers, colony assays were performed on 9 MDS patients only. Cells were cultured in triplicates with increasing concentrations of APG101 for 14 days. Colonies were counted and the mean number of colonies was determined.

Treatment of differentiating CD34+ cells with APG101 led to a decreased apoptosis in both CD34+ cells and CD71+ cells, respectively, indicated by decreased Annexin-FITC fluorescence. Interestingly, this effect was particularly seen at low APG101 concentrations (maximum of 10 µg/ml), while the effect was abrogated at higher APG101 concentrations. The anti-apoptotic effect was more pronounced in low risk MDS patients compared to high risk MDS patients. No effect was seen when the CD235a+ fraction of cells was analyzed. With regard to colony formation, an improvement of erythroid differentiation, indicated by an increase in CFU-E, was found in 3 out of 4 low risk patients (less than 5% blasts in the bone marrow). No effect was seen on erythroid differentiation in high risk patients (more than 5% blasts in the bone marrow).

APG101 shows promising in vitro activity in viable CD34+ cells with regard to inhibition of apoptosis and promotion of differentiation. The observation that the anti-apoptotic effect was more pronounced in low risk MDS patients as compared to high risk MDS patients supports the concept of increased apoptosis particularly in early stage MDS progenitors. Although the numbers in the differentiation experiments are small, we found a promising effect of APG101 on CFU-E formation at lower doses in patients with less than 5% bone marrow blasts. Moreover, the same dose-dependent effect was observed in the apoptosis assays. Since the activation of the CD95 pathway seems not only to be involved in apoptosis induction, but is also required for terminal erythroid differentiation in normal hematopoiesis, this dose-dependent effect might particularly reflect these ambivalent roles of CD95 and its ligand in both MDS and healthy hematopoiesis, repsectively.

Kunz:APOGENIX GmbH: Employment. Fricke:APOGENIX GmbH: Employment.