Combining Effects of the SMO Inhibitor and BCL-2 Inhibitor in MDS-Derived Induced Potent Stem Cells (iPSC)

Background:

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). MDS clones acquire an apoptotic resistance upon disease progression, which coincides with the appearance of an elevated number of myeloid blasts and increased likelihood of disease acceleration into AML. Induced pluripotent stem cells (iPSCs) are promising for studying stem cell signaling pathways. When reprogramming is performed with MDS cells, MDS-derived iPSCs offer a unique system for modeling MDS pathogenesis and developing high throughput drug screening. Glasdegib is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. We have previously shown the persistent effects of glasdegib on long term self-renewing MDS-initiating cells (J Cancer Sci Ther. 2017, 9:479), however, quick clinical efficacy of glasdegib is only limitted. In the present study, we investigated the combining effects of venetolax (ABT-199), the BCL2-selective inhibitor, and glasdegib on phenotypically immature cell population of MDS-derived iPSCs.

Methods:

We generated iPSCs from bone marrow mononuclear cells of three MDS patients (MDS-MLD, MDS-EB1 and MDS-EB2 by WHO classification). Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. In vitro re-differentiation of MDS-iPSCs was performed with differentiation media (30 ng/ml VEGF, 30 ng/ml BMP-4, 40 ng/ml SCF, 50 ng/ml Activin) for 4 days. At day 14, a single cell suspension expressing CD34+CD38- was achieved with hematopoietic cytokine (300 ng/ml Flt-3 ligand, 10 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml G-CSF, 25 ng/ml BMP-4).

Results:

We generated the phenotypically immature cell population (CD34+CD38-CD90+CD45+) and mature cell population (CD34-CD45+) from MDS-derived iPSCs. The single treatment of venetoclax significantly reduced the numbers of mature cell population (CD34-CD45+) from MDS-derived iPSCs. Although the single treatment of venetoclax or glasdegib　alone have minimum effect on immature cell population (CD34+CD38-CD90+CD45+), co-treatment with venetoclax and glasdegib significantly reduce the numbers of cell population (CD34+CD38- CD90+CD45+) (p<0.001). Further, NOD/SCID mice were injected with MDS-derived iPSCs, and then treated with venetoclax and glasdegib on day 21 for 7 days. Co-treatments with venetoclax and glasdegib significantly reduced the population of CD34+CD38- CD90+ cells compared with venetoclax or glasdegib as single agents. Also co-treatment with venetoclax and glasdegib significantly reduced the colony formations of mature erythroid, granulocyte-macrophade, and mixed of these hematopoietic cells derived from MDS-EB2-iPSCs for CFC assay To identify the mechanisms that limit the MDS-EB2-derived immature cell population by venetoclax and glasdegib, CD34+CD38-CD90+CD45+ cell fractions from MDS-iPSCs were cultured with venetoclax and glasdegib for 72 hrs. Co-treatment with venetoclax and glasdegib reduced the expression of BMI1, c-Myc and nanog. Finally, we investigate the engraftment potential of MDS-EB1 derived iPSCs treated with venetoclax. One million of CD34+ cells derived from MDS-EB1 iPSCs treated with venetoclax were intravenously transplanted into NOD/SCID mouse. Transient engraft was observed from control mice, however, co-treatment of venetoclax never shows the engraftment.

Conclusion:

Our preclinical results indicate that the combination with venetoclax and glasdegib have potential as an important option for controlling the immature MDS cell population. It is expected that the combination with venetoclax and glasdegib may become extremely useful therapeutic interventions to obtain the quick clinical efficacy for MDS patients.