Targeting endothelial MYC using siRNA or miR-218 nanoparticles sensitizes chemo- and immuno-therapies by recapitulating the Notch activation-induced tumor vessel normalization

<b>Background:</b> The chaotic, over-activated tumor vasculature promotes tumor growth and erodes most current therapies. Although Notch activation critically regulates angiogenesis, the broad roles of Notch has dampened its druggability. <b>Methods:</b> Gene-modified mice with a Cdh5-Cre^ERT transgene were employed to activate/block Notch signaling in endothelial cells (ECs). Multiple transcriptome analyses were conducted to compare gene expression profiles. qRT-PCR and western blotting were used to determine gene expression level. Immunofluorescence and flow cytometry were used to observe morphological alterations and immune microenvironment in tumors. Nanoparticles (PEI-PEG-cRGD) were used to deliver siRNA into tumor ECs (TECs) <i>in vivo</i>. <b>Results:</b> Genetic Notch activation or blockade in TECs normalizes or deteriorates tumor vessels, respectively. Single-cell RNA sequencing showed that Notch activation selectively reduced the proliferating TEC subset, which accounted for about 30% of TECs and gave rise to other TEC subsets. Notch activation or blockade downregulated or upregulated MYC, respectively. MYC overexpression canceled Notch activation-induced proliferation arrest of TECs <i>in vitro</i>, and a MYC inhibitor normalized tumor vessels in RBPj deficient mice, suggesting that MYC is the authentic Notch target in normalizing tumor vessels. Nanoparticles encapsulated with MYC siRNA (EC-siMYC) or miR-218 (EC-miR-218), a Notch-downstream miRNA suppressing MYC, were able to mitigate Notch inhibition-induced tumor vessel defects. Combination of cisplatin with MYC blockade exhibited improved therapeutic effects. Moreover, MYC blockade promoted T cell infiltration and enhanced anti-PD1 immunotherapy. <b>Conclusions:</b> Together, our data have demonstrated that Notch activation normalizes tumor vessels by repressing the proliferating TEC subset via MYC, and targeting endothelial MYC using nanoparticles bearing siRNA or miRNA is an efficient strategy for tumor anti-angiogenic therapy.