Paquinimod regulate megakaryocyte function by restoring mitochondrial oxidative phosphorylation in acute myeloid leukemia

Abstract

Abstract Introduction A malignant inflammatory microenvironment exists in acute myeloid leukemia (AML), and many inflammatory molecules affect the differentiation and development of hematopoietic lineages. One of them is calprotectin, a heterodimer of S100a8 and S100a9. Previous studies have reported that S100a8/a9 can affect cell survival by influencing the function and content of mitochondria. Our previous in vitro experiments revealed that S100a8/a9 can inhibit mitochondrial function and promote megakaryocyte apoptosis. Therefore, the aim of this study is to investigate the mechanism by which S100a8/a9 in AML leads to impaired platelet production by megakaryocytes and results in thrombocytopenia. Methods The megakaryocytes of AML patients and donors were isolated for RNA-seq detection. C57BL/6J female mice aged 6–8 weeks were injected with the C1498 mouse AML cell line via the tail vein to establish the model. The levels of S100a8/a9 in the mice were detected by ELISA. Flow cytometry was used to analyze the proportion of megakaryocytes in the bone marrow of mice, the ploidy of megakaryocytes, the development of megakaryocyte lineages, and the apoptosis of megakaryocytes. The content and quality of the mitochondria in the megakaryocytes were detected by electron microscopy, and the size of the megakaryocytes and the distance from the sinusoids in the bone marrow of the mice were observed by HE staining. Changes in the blood parameters and survival time of the mice were compared between the combination treatment with cytarabine and Paquinimod and treatment with cytarabine alone. Results RNA-seq of megakaryocytes from AML patients and healthy donors revealed that MAPK signaling pathway and Toll-like receptor signaling pathway were upregulated in AML patients, whereas apelin signaling pathway was upregulated in healthy controls. Mouse bone marrow cells were cultured in vitro using recombinant murine S100a8/a9. The proportion of megakaryocytes decreased, the apoptosis of megakaryocytes increased, and the content of mitochondria and the release of mitochondrial ROS decreased. The AML mouse model showed a continuous increase in white blood cells, a continuous decrease in hemoglobin and platelets, and a significant increase in spleen size and ovarian metastasis in the later stages of the disease. Compared with that in control WT mice, the expression of S100a8/a9 in AML mice was increased, the apoptosis of bone marrow MK cells was increased, the proportion of bone marrow MK cells with ploidy greater than 8n was decreased, and the proportion of 2n cells was increased. However, the proportion of polyploidy in the AML spleen increased while the proportion of hypoploidy decreased, which is considered as a compensatory response to the bone marrow. In addition, the development of MKP and MEP lineages of bone marrow in AML mice was inhibited. Electron microscopy revealed a reduction in the mitochondrial content and morphological changes in AML mice, and HE staining revealed a decrease in the number of megakaryocytes and an increase in the distance from the sinusoids. The expression of MK mitochondrial genes such as mitochondrial complex I, NRF1, and PGC1α has decreased, while the expression of c-caspase3, a protein related to cell apoptosis, has increased, and the expression of BCL-2, a protein related to anti-cell apoptosis, has decreased. Mechanistic verification revealed that S100a8/a9 in AML inhibited the oxidative phosphorylation of megakaryocytes through the TLR4/ERK-NRF1 pathway. The combination of cytarabine chemotherapy and Paquinimod increased the proportion of MK in the bone marrow, increased the MKP and MEP populations, reduced the level of MK apoptosis in the bone marrow, improved the expression of genes related to oxidative phosphorylation, and increased the levels of platelets and hemoglobin. Conclusions In AML, the inflammatory molecules S100a8/a9 mediate mitochondrial dysfunction and MK apoptosis through the TLR4/ERK-NRF1 signaling pathway, thereby leading to thrombocytopenia. Paquinimod, when used in combination with chemotherapy, improves the mitochondrial function of megakaryocytes to alleviate thrombocytopenia in AML patients.