Abstract Background Ponatinib, a third-generation tyrosine kinase inhibitor (TKI), is the only approved TKI that is effective against T315I mutations in patients with chronic myeloid leukemia (CML). Specific activation of Notch signaling in CML cells by ponatinib can be considered as the “on-target effect” on the tumor and represents a therapeutic approach for CML. Nevertheless, ponatinib-induced vascular toxicity remains a serious concern, with underlying mechanisms poorly understood. Aims We aimed at determining mechanisms of ponatinib-induced vascular toxicity, defining associated signaling pathways and identifying potential rescue strategies. Methods and results We exposed human umbilical endothelial cells (HUVECs) to ponatinib or vehicle in the presence or absence of the neutralizing factor anti-Notch-1 antibody for exposure times of 0–72 hours. Label-free proteomics and network analysis showed that protein cargo of HUVECs treated with ponatinib triggered apoptosis, and inhibited vasculature development (Fig. 1). We validated the proteomic data showing the inhibition of matrigel tube formation, an upregulation of cleaved caspase-3 and a downregulation of phosphorylated AKT and phosphorylated eNOS. We delineated the signaling of ponatinib-induced vascular toxicity demonstrating that ponatinib inhibits endothelial survival, reduces angiogenesis and induces endothelial senescence and apoptosis via Notch-1 pathway. Conclusion Ponatinib induced endothelial toxicity in vitro. Hyperactivation of Notch-1 in the vessels can lead to abnormal vascular development and vascular dysfunction. By hyperactivating Notch-1 in the vessels, ponatinib exerts an “on-target off turmor effect”, which leads to deleterious effects and may explain the drug's vasculotoxicity. Selective blockade of Notch-1 prevented ponatinib-induced vascular toxicity. Figure 1 Funding Acknowledgement Type of funding source: None