Targeting TPO-independent strategy in the discovery of a novel megakaryocytopoiesis inducer DMAG for treatment of thrombocytopenia

Thrombocytopenia is a TPO-related life-threatening disorder with very limited treatment options. Typical thrombopoietic agents targeting TPO signaling encounter a huge challenge. Thus, it is urgent to discover a novel TPO-independent mechanism involving thrombopoiesis and its potential targeted medications. Here, we developed a drug screening model by the Multi-Grained Cascade Forest (gcForest) algorithm and identified that 3,8-Di-O-methylellagic acid 2-O-glucoside (DMAG) (10, 20 and 40 μM) promoted megakaryocyte differentiation in vitro. Subsequent investigations revealed that DMAG (40 μM) activated ERK1/2, HIF-1β and NF-E2. Inhibition of ERK1/2 blocked megakaryocyte differentiation and attenuated the upregulation of HIF-1β and NF-E2 induced by DMAG. Megakaryocyte differentiation induced by DMAG was inhibited via knockdown of NF-E2. In vivo studies showed that DMAG (5 mg/kg) accelerated platelet recovery and megakaryocyte differentiation in mice with thrombocytopenia. The platelet level of DMAG-treated group recovered to almost 72% and 96% of control group at day 10 and 14. The platelet counts in the DMAG-treated group exhibited almost 1.5 and 1.3 fold higher compared with the irradiation (IR) group at day 10 and 14. Moreover, DMAG (10, 25 and 50 μM) stimulated thrombopoiesis in zebrafish. DMAG (5 mg/kg) could also increase platelet levels in c-MPL knockout (c-MPL-/-) mice. In summary, we establish a drug screening model through gcForest and demonstrate DMAG promotes megakaryocyte differentiation via the ERK/HIF1/NF-E2 pathway, which is more importantly independent of the TPO/c-MPL classic pathway. The present study may provide new insights into drug discovery for thrombopoiesis, TPO-independent regulation of thrombopoiesis and a promising avenue for thrombocytopenia treatment.