S290: ATRA CAN CORRECT DEFECTIVE HIF-1Α/S1P AXIS-MEDIATED CYTOSKELETAL REORGANIZATION IN PROPLATELET FORMATION OF ITP

Abstract

Background: Bone marrow physiological hypoxia plays a crucial role in haematopoietic stem cell homeostasis. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. HIF-1α expression was decreased in the bone marrow of patients with immune thrombocytopenia (ITP), and HIF-1α activation was shown to enhance megakaryopoiesis in mice. Recent studies suggest that “inside-out” signalling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies (Blood, 2021; Lancet haematology, 2017; Lancet haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Aims: This study aimed to explore the role of hypoxia inducible factor-1α (HIF-1α) in proplatelet formation (PPF) and the underlying mechanisms of ATRA treatment in ITP patients. Methods: Thirty consecutive patients with newly diagnosed ITP and 30 healthy donors were included in our study. MKs were isolated from bone marrow samples. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Confocal microscopy and transmission electron microscopy were used to observe the PPF and cytoskeleton structure of ITP MKs. An ITP mouse model was established to observe the therapeutic effects of ATRA in the PPF. Results: In the present study, we observed that MKs displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF) in ITP patients. Targeted and untargeted metabolite profiling revealed a decreased sphingosine-1-phosphate (S1P) level in ITP. Downregulated sphingosine kinase 2 (SPHK2) expression in MKs accounted for the low level of S1P in ITP. S1P is essential for S1P receptor 1 (S1PR1) and Rac1 activation, Src family kinases (SFKs) activity, and subsequent cytoskeletal reorganization and PPF regulation. Moreover, we demonstrated that HIF-1α mediated SPHK2 activation and S1P production. Decreased HIF-1α levels were found in the MKs of patients with ITP, contributing to impaired PPF. We then investigated the effect of ATRA on PPF in ITP patients. ATRA upregulated HIF-1α and SPHK2 expression, increased S1P production and corrected impaired PPF in vitro. In an ITP mouse model, ATRA alleviated thrombocytopenia and restored cytoskeletal reorganization. ATRA corrected impaired PPF by upregulating HIF-1α expression. The exposure of ITP MKs to selective RARα (AM580) or RARγ (BMS961) agonists did not change PPF. However, the treatment of ITP MKs with a selective RARβ agonist (CD2314) significantly increased PPF. Furthermore, we found that the effect of ATRA on enhancing PPF in ITP MKs was reversed in the presence of an RARβ antagonist (CD2665). These data suggest that impaired PPF in ITP MKs is corrected by ATRA in a RARβ-dependent manner. Summary/Conclusion: Together, our data show that the HIF-1α/S1P axis mediates altered cytoskeletal reorganization and impaired PPF in ITP and suggest that ATRA correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.