The inhibition of endothelial DLL4-NOTCH1 signaling by 2'-hydroxyflavanone enhances anti-PD-1 therapy in melanoma.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapeutics; however limited patient responses necessitate combination strategies to improve therapeutic efficacy. Among potential combination partners, drugs targeting DLL4-NOTCH1 signaling pathway-a critical regulator of vascular function-show promise as angiogenesis modulators, but their clinical development have been hindered by safety concerns. To address this challenge, we adopted a novel approach by screening natural compounds with a long history of human consumption. Building upon our earlier findings, we identified three inhibitors of DLL4-NOTCH1 signaling: steppogenin, sanggenon F, and dehydrovomifoliol. Steppogenin inhibited both DLL4 and NOTCH1 activities, while sanggenon F and dehydrovomifoliol selectively suppressed DLL4 and NOTCH1 activity, respectively. We assessed their impact on key angiogenic processes, including endothelial cell migration, sprouting, and proliferation, and elucidated the relative contributions of selective DLL4 or NOTCH1 inhibition to the anti-angiogenic effect. By comparing structurally similar compounds, we identified the 2'-hydroxyflavanone moiety as a key element for DLL4 inhibition. Notably, combining steppogenin with an ICI demonstrated that a nature-derived angiogenesis inhibitor can boost the anti-cancer effect of ICI in a mouse melanoma allograft model. This comprehensive analysis of structure-activity relationships and in vivo therapeutic evaluation provides valuable insights into the development of novel anti-angiogenic compounds for combination therapy with ICIs in cancer treatment.