Abstract C115: <i>In vitro</i> and <i>in vivo</i> screening platform for discovery of JAK2 inhibitors

Abstract Background: The JAK-STAT signaling pathway, activated by cytokine receptors, crucially regulates cell proliferation, differentiation, apoptosis, and immune responses. JAK2, a core kinase within this pathway, transduces signals from hematopoietic growth factor receptors (e.g., thrombopoietin [TPO], erythropoietin [EPO], granulocyte-macrophage colony-stimulating factor [GM-CSF]). Dysregulation of JAK2 is pathologically implicated in myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), myelofibrosis (MF), and essential thrombocythemia (ET). Objectives: To assess the efficacy of JAK2 inhibitors, elucidate their mechanism of action, and identify optimal dosing strategies to maximize therapeutic efficacy while minimizing adverse effects. Method: In vivo: A murine model of JAK2-driven myeloproliferative disease was established via intravenous transplantation of JAK2-activated cell lines into BALB/c nude mice. Disease progression was monitored using bioluminescent imaging. Terminal analyses included assessment of splenomegaly, serum biochemistry (ALT, AST), splenic histopathology (hematoxylin and eosin staining), and reticulin fibrosis.In vitro: Cell lines were treated with titrated concentrations of Ruxolitinib. Inhibition of downstream signaling was quantified via phosphorylated STAT5 (p-STAT5) αLISA. Inhibitory concentration 50% (IC50) values were determined to evaluate cellular sensitivity. Results: In vivo: Model mice exhibited progressive increases in bioluminescent signal, significant splenomegaly and hepatomegaly, reduced survival, and elevated serum ALT/AST levels. Ruxolitinib treatment significantly attenuated bioluminescent signal progression, reduced hepatic and splenic tumor burden, and prolonged survival.In vitro: Ruxolitinib demonstrated potent, concentration-dependent inhibition of JAK2-STAT5 signaling, with IC50 values confirming target sensitivity. Conclusion: We established a in vivo and in vitro screening platform for JAK2 inhibitors. This model provides a valuable tool for optimizing therapeutic regimens targeting JAK2-driven pathologies. Citation Format: Na Li, Hao Huang, Xinyu Zhong, Guoqian Wang, Xuyang Duan, Siyu Li, Jinying Ning, Feng Hao. In vitro and in vivo screening platform for discovery of JAK2 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C115.