Abstract 6844: In vitro and in vivo screening platform for discovery of JAK2 inhibitors
Authors: Na Li, Xinyu Zhong, Hao Huang, Guoqian Wang, Xuyang Duan, Yunpeng Zhai, Jinying Ning, Feng Hao
Journal: Cancer Research (2025)

Abstract Background: The JAK-STAT signaling pathway functions as a widely utilized mechanism triggered by cytokines, playing a crucial role in regulating various key biological activities , including cell proliferation, cell differentiation, cell apoptosis, and immune response. JAK (Janus Kinase) kinases belong to a family of intracellular non-receptor tyrosine kinases. This family consists of four core members: JAK1, JAK2, JAK3, and TYK2. Among them, JAK2 is particularly significant in mediating signal transmission for hematopoietic growth factor receptors. It facilitates signaling for various cytokines, such as Thrombopoietin (TPO), Erythropoietin (EPO), and Macrophage colony stimulating factor (GM-CSF), thereby regulating the proliferation of hematopoietic cells. Consequently, abnormalities in JAK2 are closely are strongly linked to various diseases, notablypolycythemia vera (PV), myelofibrosis (MF), and essential thrombocythemia (ET). The objective of this study is to assess the effectiveness of JAK2 inhibitors, investigate their underlying mechanisms of action, and determine the optimal dosing regimen to maximize therapeutic benefits while minimizing adverse effects.. Method: The study utilized several cell lines, including Ba/F3-MPL cell line, Ba/F3-EPER-JAK2-V617F cell line, Ba/F3-EPER-JAK2-V617F-mJAK-KO cell line, Ba/F3-MPL-W515L-JAK2 and Ba/F3-MPL-JAK2-V617F cell lines to investigate the effects of fluconazole. In vitro, these cell lines were treated with different concentrations of fluconazole and used p-STAT5 alphaLISA to detect the inhibitory effect of fluconazole on downstream signaling pathways. In vivo, the cell lines were inoculated into BABL/c nude mice through tail vein injection to form a mouse model of bone marrow proliferative disease caused by JAK2 abnormal activation. We weighed and photographed the liver and spleen, and detected the blood biochemistry and routine of the model mice. We also detected the pathological changes of the spleen through HE staining and reticular protein fibrosis staining. Results: In vitro finding demonstrated that Ruxolitinib significantly inhibited the phosphorylation level of STAT5 downstream of JAK2 in a dose-dependent manner. The in vivo experiments revealed that, compared with the sham group, the disease model mice exhibited significantly increased spleen and liver weights. Additionally, higher cell inoculation levels led to shorter survival times, elevated serum ALT and AST levels, and aggravated spleen fibrosis. Treatment with Ruxolitinib significantly alleviated the burden on the liver and spleen and prolonged the survival of the treated mice. Conclusion: We successfully established both in vitro and in vivo screening platform for JAK2 inhibitors, providing a valuable tool for optimizing therapeutic strategies for diseases associated with abnormal JAK2 activation. Citation Format: Na Li, Xinyu Zhong, Hao Huang, Guoqian Wang, Xuyang Duan, Yunpeng Zhai, Jinying Ning, Feng Hao. In vitro and in vivo screening platform for discovery of JAK2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6844.