Abstract 7536: In vitro and in vivo screening platform for discovery of JAK2 inhibitors

Abstract Background: The JAK-STAT signaling pathway, activated by cytokine receptors, plays a central role in regulating cell proliferation, differentiation, apoptosis, and immune responses. JAK2, a key kinase in this pathway, transduces signals from hematopoietic growth factor receptors such as thrombopoietin (TPO), erythropoietin (EPO), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Dysregulation of JAK2 is implicated in the pathogenesis of myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), myelofibrosis (MF), and essential thrombocythemia (ET). Objectives: This study aimed to evaluate the efficacy of JAK2 inhibitors, elucidate their mechanisms of action, and identify optimal dosing strategies to maximize therapeutic benefit while minimizing adverse effects. Method: An In vivo, a murine model of JAK2-driven myeloproliferative disease was established by intravenous transplantation of JAK2-activated cell lines into BALB/c nude mice. Disease progression was monitored using bioluminescent imaging. Terminal analyses included evaluation of splenomegaly, serum biochemistry (ALT and AST), and splenic histopathology (hematoxylin and eosin staining).For in vitro analysis, a panel of cell lines was treated with increasing concentrations of JAK inhibitors (Ruxolitinib, Fedratinib, and Tofacitinib) to evaluate compound efficacy. Inhibition of downstream JAK2-STAT5 signaling was quantified using an α-LISA for phosphorylated STAT5 (p-STAT5). Dose-response profiles were generated, and IC50 values were calculated to determine the relative sensitivity of the cells to each agent. Results: In vivo, our model mice displayed progressive increases in bioluminescent signal, marked splenomegaly and hepatomegaly, decreased survival, and elevated serum ALT/AST levels. Ruxolitinib treatment significantly suppressed bioluminescent signal progression, reduced hepatic and splenic tumor burden, and extended survival.In vitro, Ruxolitinib potently and dose-dependently inhibited JAK2-STAT5 signaling, with IC50 values confirming target sensitivity. The cell lines exhibited differential sensitivity to the JAK2 inhibitors Ruxolitinib, Fedratinib, and Tofacitinib. Conclusion: We established integrated in vivo and in vitro screening platforms for the discovery and evaluation JAK2 inhibitors. This model provides a valuable tool for optimizing treatment regimens against JAK2-driven pathologies. Citation Format: Na Li, Hao Huang, Xinyu Zhong, Guoqian Wang, Xuyang Duan, Yue Huang, 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 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7536.