Thrombopoietin Affects the Behavior of Myelofibrosis Stem Cells

Recently the presence of myelofibrosis (MF) stem cells (MF-SC) in the spleens of MF patients which when transplanted into NOD/SCID/IL2R ^null (NSG) mice were capable of generating multiple hematopoietic lineages that belonged to the malignant clone has been observed. Although MF is characterized by marrow megakaryocyte (Mk) hyperplasia, limited numbers of human marrow Mks were, however, observed in these transplanted mice and evidence of both marrow and splenic fibrosis 9 months after the transplantation was lacking (Wang X, et al. J Clin Invest. 2012; 122:3888). Splenic MF CD34⁺ cells did retain the capacity to differentiate in vitro into CD41a⁺ and CD61⁺ Mks in the presence of thrombopoietin (TPO). We therefore investigated whether the administration of romiplostim, a novel human TPO peptide mimetic which lacks homology to endogenous TPO in MF humanized mice, might create appropriate environmental cues which affect the behavior of MF-SCs.

Elevated levels of TPO (345±114ng/ml) were detected in MF plasmas (n=13) as compared to levels detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF-SCs and MF hematopoietic progenitor cells (HPC). Following the culture of CB (n=3) or PB MF CD34⁺ cells (n=4) for 1 and 2 wks in serum free expansion media (SFEM) supplemented with SCF (50ng/ml) + romiplostim (100ng/ml), the numbers of total cells, CD34+Lin- cells and assayable HPCs, including CFU-Mk, CFU-GM and BFU-E, CD41a+CD34-CD15- cells and CD15+CD34-CD41a- cells generated were greater (CB) or similar (MF) to the number generated in the cultures containing SCF + TPO (100ng/ml). Moreover, similar proportions of colonies (CFU-GM+BFU-E) generated in cultures supplemented with SCF+TPO or SCF+ romiplostim (100ng/ml, 1000ng/ml) were JAK2V617F-positive. These findings suggest that both romiplostim and TPO are capable of promoting MF-SC and HPC proliferation in vitro.

To assess the effects of romiplostim on human (h) platelet production, CB CD34⁺ cells (5×10⁵) were transplanted via the tail vein into eight- to nine-week-old sublethally irradiated (240 cGy) NSG mice. One week after transplantation, mice were treated with water or 10, 100 or 1000 µg/kg of romiplostim. Both the percentage and number of hCD41a+ platelets in the PB increased 4 days following the treatment with 10ug/kg romiplostim, and peaked at day 8 with a 2.58±0.29 fold increase in the percentage (P<0.05) and 3.51±1.32 fold increase in the absolute number (P<0.05) of hCD41a+ platelets being observed. The number of human platelets in the PB, were reduced to the levels detected in mice not receiving romiplostim by day 15.

MF splenic CD34⁺ cells (5-10×10⁵) were next transplanted into romiplostim (10ug/kg or 30ug/kg) treated or control NSG mice. Two months after the transplantation, a 1.83±0.62 fold increase in the number of hCD41a+ cells was observed in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not treated with romiplostim. Moreover, a 7.13±2.13 fold elevation in the percentage of hCD45+ cells was detected in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not receiving romiplostim. Moreover, enhanced hCD45+ cell chimerism was achieved in both the marrow (55.6%, 7.2%) and spleen (47.7%, 1.5%) of mice receiving splenic CD34+ cells from 2 MF patients and romiplostim as compared with mice not receiving romiplostim (marrow: 30.4%, 5.6%; spleen: 23.6%, 0.9%), respectively. The splenic MF CD34+ cells receiving or not receiving romiplostim had a similar differentiation patterns in the marrow and spleen, however, the numbers of hCD33+, hCD19+, hCD3+, hCD41a+ and hCD15+ cells detected in mice treated with romiplostim were dramatically greater than those detected in mice not receiving romiplostim. Greater numbers of hCD34⁺ cells were detected in the BM (7.1%; 3.5%) of recipient mice receiving romiplostim as that detected in mice not receiving romiplostim (6.6%; 0.6%). Furthermore, a similar degree of hCD45+ cell and hCD34+ cell chimerism was observed in both the marrow and spleen of mice receiving splenic MF CD34+ cells and romiplostim 4 months after the transplantation. These findings suggest that the administration of thrombopietin agonist has a profound effect on the behavior of MF-SCs and that the elevated levels of TPO documented in MF patients likely has important effects on the biology of MF-SCs.