Introduction: OB756, a novel selective inhibitor of Janus kinase 2 (JAK2), has clinically significant activity and safety in patients with myeloproliferative neoplasm (MPN). The maturation and differentiation of megakaryocytes play an important role in treatment in patients with MPN. However, the effects and mechanism of OB756 on megakaryopoiesis remain unclear.
Methods: We investigated the effect and mechanism of OB756 induces thrombocytopenia using human megakaryoblastic and positive Janus kinase (JAK) mutation cell lines (SET-2, HEL and Meg-01). Megakaryocytes (MKs) were differentiated form human bone marrow CD34+ hematopoietic stem cells (HSCs) and cultured with SCF/IL-3/IL-11/TPO. MKs from CD34+ HSCs were treated with OB756 or DMSO for 11 days to determine the effect of OB756 on megakaryopoiesis. In vivo, C57BL/6 mice were with OB756 and observed MKs differentiation and platelet biogenesis. The platelet counts of patients with thrombocythemia (ET) and polycythemia vera (PV) were collected with treatment of OB756 16mg BID or 20mg BID at 17 sites in China. Flow cytometry were used to investigate MK ploidy, apoptosis, cell cycle and cell surface markers of MKs. RNA sequencing, western blot and quantitative polymerase chain reaction (QT-PCR) were carried out to detect the mechanism of OB756 on megakaryopoiesis.
Results: In vitro, our results showed that OB756 impaired the proliferation of SET-2, HEL and Meg-01 cells. The IC50 of SET-2, HEL and Meg-01 cells were 11.84μM, 1.88μM and 8.19μM, respectively. Compared with the control group in SET-2 cells, percentage of apoptosis increased 60% in OB756 group at dose of 3.125μM at day 4 (p value < 0.05), and OB756 inhibited cell cycle with high percentage of G0/G1 phase (3.125μM versus 0μM, 77% versus 57%, p value < 0.05, respectively). Similar results of apoptosis and cell cycle were also showed in Meg-01 and HEL cells (Figure 1A). Meg-01 cells were treated with 10 nM PMA to induced differentiation and were cultured in the presence or absence of OB756 (0.2μM, 0.78μM, 3.125μM, 6.25μM and 12.5μM) for four days. Compared with the control group, morphologic features of Meg-01 cells showed that Meg-01 cells decreased in size and exhibited fewer cytoplasmic extensions with increasing OB756 concentrations. Concurrently, the numbers of Meg-01 cells decreased with increasing OB756 concentrations (Figure 1B). The cell surface markers levels of CD41, CD42b and CD61 in PMA-induced Meg-01 cells were inhibited by OB756 in a dose dependent manner (Figure 1C). Therefore, OB756 inhibits Meg-01-derived MKs differentiation. Similarly, in the MKs differentiated form human CD34+ HSCs, flow cytometry results showed that OB756 reduced the expression of MKs in CD41 and CD61. Approximately 38%, 8%, and 3% of cells were positive for CD41 and CD61 after treatment with OB756 at different dose of 0μM, 0.78μM, and 6.25μM on days 11, respectively. These results indicated that OB756 inhibits CD34+ HSC-derived MK differentiation and functions (Figure 1D). 77 patients with ET and 66 patients with PV were enrolled at 17 sites in China. In clinical data, platelet counts in patients with ET reduced with the treatment of OB756 (Mean ± Std = -271.3 ± 296.8 at week 4, Mean ± Std = -254.0 ± 264.4 at week 24). Similarly, platelet counts in patients with PV also reduced with the treatment of OB756 (Mean ± Std = -78.6 ± 265 at week 4, Mean ± Std= -96.3 ± 227.3 at week 24) (Figure 1F).
Conclusion: These preclinical data suggested that OB756 could inhibit MK differentiation and platelet biogenesis in JAK mutation cells. OB756 also showed quick reduction of platelet counts in clinical data, which revealed the meaningful clinical benefits in patients with ET and PV.
This research was funded by the Key R&D Program of Zhejiang (No. 2022C03137) and the Zhejiang Medical Association Clinical Medical Research special fund project (No. 2022ZYC-D09). *Correspondence to: Prof Jian Huang, E-mail: househuang@zju.edu.cn.
No relevant conflicts of interest to declare.
This feature is available to Subscribers Only
Sign In or Create an Account Close Modal