Synergistic antitumor activity of METTL3 peptide degrader and immune checkpoint inhibitor in advanced urothelial cancer.

e14623 Background: In patients afflicted with advanced urothelial carcinoma (aUC), the durable response rate to immune checkpoint inhibitor (ICI) is approximately 20%. Recent study showed the pivotal role of N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) in immune escape and the progression of aUC. This study aimed to develop a novel METTL3 peptide degrader RKL and investigate its synergistic effects and underlying mechanisms in combination with ICI for aUC. Methods: Methyltransferase expression profiles were scrutinized using The Cancer Genome Atlas database; while the prognostic implications of METTL3 expression in response to ICI were analyzed in the IMvigor210 cohort study. Single-cell RNA sequencing (scRNA-seq) was employed to elucidate the mechanistic insights. The METTL3 peptide degrader RKL was synthesized using Fmoc solid-phase peptide synthesis and purified with high-performance liquid chromatography. In vivo and in vitro experiments were conducted to assess the impact of RKL, ICI, and their combination on aUC inhibition. Results: In aUC, METTL3 exhibited the most significant up-regulation among m6A methyltransferases, while METTL14 and WTAP showed down-regulated expression. In the IMvigor210 cohort study, aUC patients with elevated METTL3 expression and treated with ICI demonstrated a poorer prognosis. scRNA-seq revealed that METTL3 upregulation promoted the expression of genes regulating cancer-associated fibroblast proliferation, contributing to enhanced tumor resistance to immunotherapy. RKL demonstrated high toxicity to urothelial carcinoma cell lines, inhibiting their proliferative, migratory, and invasive capacities in vitro. The combined treatment of RKL and ICI significantly reduced tumor volume (P<0.001), with a complete response achieved in 87.5% (7/8) of mice in the combination group, 12.5% (1/8) in the RKL group, and 25.0% (2/8) in the ICI group. Conclusions: METTL3 overexpression in aUC is associated with immune escape and resistance to ICI therapy. The METTL3 peptide degrader RKL, when combined with ICI, demonstrates synergistic antitumor effects in aUC, warranting further exploration in clinical research.