Effects of SB559457, a Novel Small Molecule Thrombopoietin Receptor (TpoR) Agonist, on Human Hematopoietic Cell Growth and Differentiation.

Abstract

Abstract Treatment of thrombocytopenia resulting from chemotherapy, radiation, or stem cell transplantation remains problematic. Hopes that TPO, the major regulator of megakaryocyte maturation, might prove clinically useful for this purpose have not been fulfilled, in part because of untoward reactions to the molecule. To avoid undesirable side effects, small molecule TpoR agonists are being developed. We have evaluated the biological effects of one such molecule, SB559457, a hydrazone compound, on hematopoietic cells. We first examined SB559457’s ability to stimulate proliferation of the recombinant human TPO (rhTPO) dependent cell line UT-7/TPO. We found a dose dependent augmentation of cell proliferation at doses between 1 and 10 μM, with maximal effects, compared to 100ng/ml of rhTPO at 10μM. We then evaluated SB559457’s growth promoting effects on normal human CD34+ cells. We again found growth stimulation to be dose dependent between 1 and 10 μM and also noted enhancement when SB559457 was combined with IL-3 and SCF. Specifically, incubation of CD34 cells in 10 μM SB559457 for 7 days lead to a 2 fold increase in cell number; incubation of cells with IL3 and SCF in addition to SB559457 lead to a 12 fold increase in cell number after 7 days. Both results were comparable to those seen with 100 ng/ml of rhTPO. Curiously, the addition of IL-6 to cultures containing IL-3 and SCF compromised the ability of SB559457, but not Tpo, to stimulate CD34+ cell growth. To determine if SB559457 stimulates megakaryocyte differentiation directly, CD34+ cells were incubated in cytokines with rhTPO or SB559457 for periods of 7–10 days, after which time cells in culture were examined for degree of polyploidization, and expression of megakaryocyte lineage markers CD41 and CD61. 14% of cells grown in SB559457 showed greater than 4N DNA content compared to 8% in rhTPO. These same cells expressed the megakaryocyte markers CD41 and CD61on 35% of cells, comparable to the results with rhTPO. Preliminary results demonstrate that incubation of CD34 cells in SB559457 maintained stem cells numbers, as assayed by NOD/SCID engraftment, for at least 4 days at a level comparable to the levels maintained by rhTPO. These results demonstrate that SB559457 is a bona fide Tpo R agonist that likely acts through activation of c-mpl, the receptor for TPO. These experiments provide a rationale for the evaluation of SB559457, or SB559457 congeners, in patients with thrombocytopenia.