Dual Aptamers‐Based SETDB1 PROTACs as Effective Anti‐Tumor Strategies for Breast Cancer

PROteolysis TArgeting Chimeras (PROTACs) have emerged as a promising strategy for drug development targeting oncogenic proteins. Here, we report the development and characterization of dual aptamer-based PROTACs targeting SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), a key epigenetic regulator implicated in breast cancer progression, drug resistance, and tumor immune evasion. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process, we identified a high-affinity single-stranded DNA (ssDNA) aptamer against SETDB1. This aptamer was conjugated to the nucleolin-targeting aptamer AS1411, generating a single-strand PROTAC (AP-SETDB1-S6A) and a partial double-strand PROTAC (AP-SETDB1-D2), both of which exhibit good serum stability. These two PROTACs directly penetrate breast cancer cells and effectively recruit the E3 ligase mouse double minute 2 homolog (MDM2) to SETDB1, inducing proteasome-dependent degradation of SETDB1. Functional assays demonstrated that both AP-SETDB1-S6A and AP-SETDB1-D2 significantly inhibit breast cancer cell proliferation and migration, and resensitize drug-resistant breast cancer cells to tamoxifen. Notably, they further enhance the cytotoxic activity of CD8⁺ T cells against breast cancer cells and directly target breast cancer cells to suppress tumor growth in vivo. This study establishes dual aptamers-based PROTACs targeting SETDB1, offering effective therapeutic strategies for breast cancer treatment.