Session: 506. Bone Marrow Microenvironment: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
Methods: BM MΦ and CD34+ cells were isolated from ITP patients and healthy control subjects. Excessive autophagy in bone marrow macrophages from ITP patients was confirmed by Western blotting and transmission electron microscopy. PCR and Western blotting were used to assess the expression of A20 and autophagy-related AMPK/mTOR/ULK1 pathway components in ITP-MΦ. Vectors carrying A20 cDNA/TNFAIP3-specific shRNA were transduced into the BM MΦ from either healthy control subjects or ITP patients. The different BM MΦ populations were separately cocultured with CD34+ cells. We compared the ability of BM MΦ to support megakaryocytopoiesis by determining the relative percentages of MKs at different stages using flow cytometry as well as CFU counts of megakaryocytes and platelets in culture. The mechanism by which sitagliptin normalized the impaired function of ITP-MΦ was evaluated in vitro. A prospective single-arm clinical cohort study was designed to explore the efficacy and safety of sitagliptin in the treatment of corticosteroid-resistant or relapsed ITP.
Results: The expression of LC3-II/LC3-I and Beclin1 increased in ITP-MΦ, and the expression of P62 decreased. The number of autophagic vesicles also increased, indicating enhanced autophagy in ITP-MΦ. Transcriptome sequencing and untargeted metabolomics revealed that macrophage autophagy-related pathways were involved in the pathogenesis of ITP. Further studies confirmed that ITP-MΦ regulated megakaryopoiesis, which was dependent on the A20-mediated autophagy-related AMPK/mTOR/ULK1 pathway. Inhibition of A20 expression with A20-shRNA inhibited the AMPK/mTOR/ULK1 pathway and alleviated excessive autophagy in ITP-MΦ, thus increasing the number of megakaryocytes, the proportion of hyperploid megakaryocytes and the number of platelets produced in the ITP-MΦ coculture system. Inhibition of autophagy with the autophagy inhibitor 3-MA reversed the A20 overexpression-induced excessive autophagy in MΦ and normalized the differentiation and maturation of megakaryocytes. Inhibiting the AMPK/mTOR/ULK1 signaling pathway with the AMPK inhibitor Compound C reversed the A20 overexpression-induced increase in the expression of AMPK/mTOR/ULK1 autophagy pathway components in MΦ and normalized megakaryopoiesis. Sitagliptin was found to inhibit the expression of A20 and AMPK/mTOR/ULK1 autophagy pathway components in ITP-MΦ in vitro. Sitagliptin improved the ability of ITP-MΦ to promote the differentiation and development of megakaryocytes and platelet production. Sitagliptin was administered to a total of 56 patients with corticosteroid-resistant or relapsed ITP. Complete response and overall response were observed in 11 (19.6%) and 21 patients (37.5%), respectively.
Conclusions: A20 mediated the AMPK/mTOR/ULK1 signaling pathway to regulate autophagy in ITP-MΦ and affected megakaryopoiesis and platelet formation. Sitagliptin could restore the proper function of the A20-mediated AMPK/mTOR/ULK1 signaling pathway in ITP-MΦ, thus promoting megakaryopoiesis. Sitagliptin resulted in a sustained response in patients with corticosteroid-resistant/recurrent ITP and exhibited good safety.
Disclosures: No relevant conflicts of interest to declare.
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