Anagrelide Inhibits Proliferation and Platelet Generation in Immortalized Megakaryocyte Progenitor Cell Lines Established from Human Induced Pluripotent Stem Cells
Authors: Takaishi Koji , Takeuchi Masahiro , Tsukamoto Shokichi , Takayama Naoya , Oshima Motohiko , Isshiki Yusuke , Kayamori Kensuke , Kimura Kenji , Hino Yutaro , Oshima-Hasegawa Nagisa , Mitsukawa Shio , Takeda Yusuke , Mimura Naoya , Ohwada Chikako , Iseki Tohru , Nakamura Sou , Eto Koji , Iwama Atsushi , Yokote Koutaro , Nakaseko Chiaki , Sakaida Emiko 
Published: October 2019

Abstract
Background : Anagrelide is a widely used therapeutic agent for patients with essential thrombocythemia. While other cytoreductive agents, such as hydroxyurea, influence multi-lineage blood cells, anagrelide exerts less effect on the white and red blood cell lineages. Although the clinical efficacy of anagrelide has been reported, the exact mechanism of action is unclear. Recently, immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from human induced pluripotent stem (iPS) cells by the introduction of doxycycline-inducible lentiviral vectors harboring c-MYC, BMI1, and BCL-XL for the clinical application of artificially generated platelets. In this study, we aimed to elucidate the molecular mechanism of anagrelide on the inhibition of platelet production using imMKCLs as an ideal model for human megakaryogenesis and platelet formation.
Materials and Methods : imMKCLs, established at Center for iPS Cell Research and Application, Kyoto University, Japan, were cultured in Iscove's modified Dulbecco's medium with thrombopoietin (TPO), stem cell factor (SCF), and doxycycline. The differentiation of imMKCLs and platelet generation were induced by doxycycline removal. The generation of mature platelets was observed approximately 7 days after the differentiation was initiated. Both undifferentiated and differentiated imMKCLs were treated with several different concentrations of anagrelide. The cell proliferation and number of generated platelets following anagrelide treatment were analyzed by BrdU cell proliferation assay and flow cytometry, respectively. To explore the molecular mechanism of anagrelide treatment in imMKCLs, we performed mRNA sequencing in imMKCLs treated with or without anagrelide followed by gene ontology (GO) analysis and gene set enrichment analysis (GSEA). The expression of genes related to megakaryogenesis and platelet formation was also analyzed utilizing quantitative real-time PCR.
Results : Anagrelide exposure caused morphologically suppressive changes in the differentiation of imMKCLs. Anagrelide treatment also suppressed the mRNA expression of the megakaryocytic surface markers CD41 and CD61 in both undifferentiated (P < 0.01 and P < 0.001, respectively) and differentiated (P < 0.01 and P < 0.001, respectively) settings. The BrdU incorporation rate in differentiated imMKCLs decreased significantly following anagrelide treatment (P < 0.001, anagrelide 0 vs. 1 or 10 µM). The resultant generation of mature platelets (double positive for CD41 and CD42b) was significantly decreased by exposure to anagrelide, as analyzed by flow cytometry (P < 0.001). Regarding the molecular mechanism of anagrelide treatment on imMKCLs, GO analysis following RNA sequencing demonstrated that gene sets related to platelet activation and degranulation were significantly downregulated in both undifferentiated and differentiated conditions. Moreover, GSEA revealed that gene sets related to the cell cycle, such as mitosis and DNA replication, were decreased as well as platelet-specific genes. The mRNA expression levels of genes related to megakaryogenesis and platelet-formation, such as FLI1, TAL1, GATA1, and PF4, were significantly downregulated, especially in differentiated imMKCLs, by anagrelide treatment (P < 0.001, P = 0.013, P < 0.01, and P < 0.01, respectively).
Conclusions : We successfully reproduced the platelet-lowering effect of anagrelide by using imMKCLs from human iPS cells that could generate functional platelets in culture. Our RNA sequencing results revealed that anagrelide specifically suppressed megakaryogenesis and platelet formation-related genes. Additional studies including an apoptosis assay and cell cycle analysis of imMKCLs following anagrelide exposure are ongoing to elucidate further molecular mechanisms of anagrelide treatment.

Disclosures
Takayama: Megakaryon co. Ltd.: Research Funding. Eto:Megakaryon co. Ltd.: Research Funding.