A Thrombopoietin Antagonist Is Capable of Affecting Stem and Progenitor Cells of Patients with Myelofibrosis

Aberrant thrombopoietin (TPO)/MPL signaling has been hypothesized to contribute to the pathogenesis of myelofibrosis (MF) (Kaushansky K. J Clin Invest. 2005; 115: 3339; Moliterno AR, et al. N Engl J Med.1998; 338:572). Agents that would be capable of inhibiting this signaling pathway are possible novel therapeutic agents that might be effective for MF treatment. A peptide antagonist of TPO, LCP4, has been created which is highly antagonistic to CB CD34⁺ cell proliferation and differentiation induced by TPO. In this report we examined the effect of LCP4 on the proliferation of MF CD34⁺ cells and their differentiation to megakaryocytes (Mk).

Elevated levels of TPO (345±114ng/ml, n=13) were detected in MF plasmas as compared to that detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF hematopoietic stem cells (HSC) and progenitor cells (HPC). MF splenic CD34⁺ cells (2.5×10⁴/mL) were incubated in serum free expansion media (SFEM) alone, with 50 ng/ml SCF+ varying doses of TPO (0, 10, 30,100 ng/mL) or 50 ng/ml SCF +varying doses of TPO+ varying concentrations of LCP4 (0, 10, 50, 100, 500, 1000nM) for 1 or 2 wks. Cells were then enumerated and stained with CD34, lineage cocktail, CD15 and CD41a mAbs. When splenic MF CD34⁺ cells were cultured in the presence of SCF and varying concentrations of TPO, 100ng/ml TPO resulted in the generation of the greatest numbers of CD34⁺Lin^-, CD41a⁺CD34^-CD15^- and CD15⁺CD34^-CD41a^- cells, suggesting that TPO promotes the proliferation of MF HSCs/HPCs and the production of MF MKs and myeloid cells. By contrast, after 1 wk of treatment of MF CD34⁺ cells (n=8; 5 MF spleens and 3 MF PB) with 100nM and 500nM LCP4, the numbers of MF total cells, CD34⁺Lin^- (MF HSCs/HPCs), CD34⁺CD41a⁺ (immature Mks), CD41a⁺CD34^-CD15^- (mature Mks), CD15⁺CD34^-CD41a^- cells (myeloid cells) as well as hematopoietic colonies (HC), including CFU-Mk, CFU-GM, BFU-E/CFU-E, CFU-GEMM, were all significantly reduced (P all <0.05), as compared with cells cultured with SCF and TPO alone (Table 1). Two days of treatment of MF splenic CD34⁺ with 100nM LCP4 led to a greater degree of apoptosis (15.4% ± 3.5%) as compared with cells treated with cytokines alone (6.75% ± 1.7%, P =0.05; n=4). These findings suggest that LCP4 is able to inhibit the proliferation of MF HSCs/HPCs and the generation of immature and mature Mks as well as myeloid cells in a dose dependent fashion. Furthermore, the treatment of JAK2V617F-positive MF CD34⁺ cells from 4 JAK2V617F-positive MF patients (JAK2V617F allele burden: 78-90%) with 100nM LCP4 for 7 days reduced the percentage of JAK2V617F-positive HCs by 9-20% and JAK2V617F homozygous HCs by 1-20%. Since LCP4 treatment of MF CD34⁺ cells resulted in a reduction in the number of HPCs, LCP4 treatment reduced the absolute numbers of JAK2V617F-positive HCs generated by 53.0±4.0% (P<0.0002) and the absolute number of JAK2V617F homozygous HCs by 50.3±4.3% (P<0.0003). These data suggest that LCP4 treatment is able to impair the in vitro generation of MF HPCs and thereby leads to a depletion but not elimination of the number of malignant HPCs.

We next examined the effect of LCP4 on MF HSCs by transplanting NSG mice with cells generated after splenic MF CD34⁺ cells were cultured in the presence of cytokines alone or cytokines plus LCP4 for 1 week. Two months after the transplantation, hCD45⁺ cells were detected in the BM of recipient mice receiving splenic MF CD34⁺ cells treated with cytokines alone and were reduced by 28% in mice receiving grafts treated with LCP4. MF CD34⁺ cells treated with or not treated with LCP4 had similar multilineage differentiation patterns (myeloid, lymphoid and erythroid). These data suggest that TPO antagonist therapy is capable of depleting MF HSCs and HPCs and that therapeutic strategy utilizing such strategies might serve as novel approaches to treating MF.