Cancer stem cells (CSCs), functionally characterized by self-renewal and tumor-initiating activity, contribute to decreased tumor immunogenicity, while fostering tumor growth and metastasis. Targeting G9a histone methyltransferase (HMTase) effectively blocks CSC functions in colorectal tumors by altering pluripotent-like molecular networks; however, existing molecules directly targeting G9a HMTase activity failed to reach clinical stages due to safety concerns. Using a stem cell-based phenotypic drug-screening pipeline, we identified the dopamine transporter (DAT) antagonist vanoxerine, a compound with previously demonstrated clinical safety, as a cancer-specific downregulator of G9a expression. Here we show that gene silencing and chemical antagonism of DAT impede colorectal CSC functions by repressing G9a expression. Antagonizing DAT also enhanced tumor lymphocytic infiltration by activating endogenous transposable elements and type-I interferon response. Our study unveils the direct implication of the DAT–G9a axis in the maintenance of CSC populations and an approach to improve antitumor immune response in colon tumors.

癌症干细胞（CSC）的功能特征是自我更新和肿瘤启动活性，有助于降低肿瘤免疫原性，同时促进肿瘤生长和转移。靶向 G9a 组蛋白甲基转移酶 (HMTase) 通过改变多能样分子网络，有效阻断结直肠肿瘤中的 CSC 功能；然而，由于安全问题，直接靶向 G9a HMTase 活性的现有分子未能达到临床阶段。使用基于干细胞的表型药物筛选流程，我们确定了多巴胺转运蛋白 (DAT) 拮抗剂凡诺司林（一种先前已证明具有临床安全性的化合物）作为癌症特异性 G9a 表达下调剂。在这里，我们发现 DAT 的基因沉默和化学拮抗作用通过抑制 G9a 表达来阻碍结直肠 CSC 功能。拮抗 DAT 还可以通过激活内源性转座元件和 I 型干扰素反应来增强肿瘤淋巴细胞浸润。我们的研究揭示了 DAT-G9a 轴在维持 CSC 群体中的直接影响以及改善结肠肿瘤抗肿瘤免疫反应的方法。