Abstract
Background:
Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). Even in low risk MDS, clonal hematopoiesis already dominates at diagnosis, and clones found in secondary AML originate from the MDS stage of disease, highlighting the need to specifically target the MDS-initiating clone. PF-0449913 is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. PF-04449913 demonstrated preliminary antitumor activity in a phase I trial, when given as monotherapy in patients with several hematopoietic malignancy. Jak1 tyrosine kinase plays an important role in cytokine signaling. Jak1 functions to phosphorylate STAT3 transcription factor, which triggers their dimerization and nuclear translocation. In the present study, we investigated the combining effects of PF-04449913 and Jak1 inhibitor, PF-6667291 in terminal differentiation of MDS-derived induced potent stem cells (iPSC).
Methods:
We generated iPSCs from bone marrow mononuclear cells of two MDS patients (RAEB1 and RAEB2 by WHO classification) with chromosome 5 deletion and complex karyotypic abnormalities, respectively. Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. We also generated iPSCs from bone marrow mononuclear cells of normal volunteer as control. To investigate the effects of PF-04449913 on self-renewal and the relevance as a therapeutic target in MDS initiating cells, we examined the activity of PF-04449913 against MDS-derived iPSCs transferred NOD/SCID mice in vivo. NOD/SCID mice were injected subcutaneously with MDS-derived iPSCs or normal iPSCs then treated with PF-04449913 (100 mg/kg; p.o.) from day 10 for 28 days. We also used MDS-L, a myelodysplastic cell line established from MDS patient with del (5q) and complex karyotypic abnormalities for in vitro studies. 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 cytokines (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:
Both MDS-derived iPSCs transferred NOD/SCID mice and normal iPSCs transferred NOD/SCID mice demonstrated the engraftment of CD34+CD38- positive cells by flow cytometry. However, the treatment with PF-04449913 reduced the population of CD34+CD38- positive cells in MDS-derived iPSCs transferred NOD/SCID mice. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of CD45+ cells into secondary recipients. Following 50 days, all mice treated with vehicle engrafted with CD34+CD38- positive cells. In contrast, CD34+CD38- positive cells engraftment was not detected in recipient mice (n=3) from PF-04449913-treated donors. These results demonstrate the persistent effects of PF-0449913 on long term self-renewing MDS-initiating cells. Next we performed in vitro re-differentiation of MDS-iPSCs, which express CD34+CD38- population. CD34+CD38- cells from MDS-derived iPSCs were cultured with 2 μM of PF-04449913 and 1 μM of PF-6667291 in STEMdiff APEL medium for 14 days for CFC activities. Treatments with PF-04449913 and PF-6667291 significantly reduced the colony formations of mature erythroid, granulocyte-macrophage, and mixed of these hematopoietic cells. To identify the mechanisms that limit the terminal differentiation of MDS-derived iPSC by PF-04449913 and PF-6667291, MDS-L cells were cultured with PF-04449913 and PF-6667291 for 72 hrs. The treatments with PF-04449913 and PF-6667291 induced the expressions of p21Cip1, cleaved PARP and reduced the expression of BMI-1, c-Myc, Nanog, and phospho-Stat3.
Conclusion:
Our preclinical results indicate that the combination with PF-04449913 and PF-6667291 have potential as an important option for controlling the terminal differentiation of MDS-initiating cells. It is expected that the combination with PF-04449913 and PF-6667291 may become extremely useful therapeutic interventions in a number of hematological neoplasms, including MDS.
Disclosures
Tauchi: Pfizer Inc.: Research Funding. Ohyashiki:Bristol-Myers Squibb: Research Funding; Novartis International AG,: Honoraria, Research Funding.
Human rib bone marrow mononuclear cells spontaneously synthesize and secrete IgE in vitro
