Endothelial RNA polymerase I, which is regulated by SPEN, is a target for improving anti-PD-1 immunotherapy of cancer.

Dysfunctional tumor vasculature limits immune checkpoint blockade efficacy. We have recently established that endothelial SPEN deficiency promotes tumor vessel normalization by suppressing RNA polymerase I (RNAPI)-mediated rDNA transcription via inducing disrupted ribosome biogenesis and nucleolar stress, but whether this pathway could be practically employed in immunotherapy has been unclear. In the current study, analysis of publicly available single‑cell RNA‑seq datasets revealed that endothelial SPEN expression was downregulated in clinical responders, including colorectal cancer patients achieving pathological complete response (pCR) after PD‑1 blockade and lung adenocarcinoma patients showing major pathological response (MPR) after neoadjuvant PD‑1 blockade plus chemotherapy. Using endothelial-specific SPEN knockout mice (eSPEN-/-), we demonstrate that SPEN loss in established tumors remodels the immune microenvironment by enhancing the infiltration and perivascular accumulation of both CD8+ and CD4+ T-cells, and in addition, boosting the effector function of CD8+ T-cells. A combined therapy of anti-PD-1 with CX-5461, a small-molecule inhibitor of RNAPI yielded better tumor inhibition than monotherapy with anti-PD-1 alone. This was attributable to increased T-cell infiltration and a marked expansion of polyfunctional CD8⁺ T-cells capable of producing both IFN-γ and TNF-α. In summary, our study identifies endothelial RNAPI, which is regulated by SPEN, as a target of normalizing tumor-immune microenvironment and that the RNAPI inhibitor, which is under clinical trial, is potentially useful to improve PD-1 blocker in cancer therapy.