Mouse methylation array of glioblastoma mGB2 cells. Mouse methylation array of glioblastoma mGB2 cells

Glioblastoma is the most common malignant brain tumor in adults. Cellular plasticity and the poorly differentiated features result in a fast relapse of the tumors following treatment. Moreover, the immunosuppressive microenvironment proved to be a major obstacle to immunotherapeutic approaches. Branched-chain amino acid transaminase 1 (BCAT1) is a metabolic enzyme that converts branched-chain amino acids into branched-chain keto acids, depleting cellular α-ketoglutarate and producing glutamate. BCAT1 was shown to drive the growth of glioblastoma and other cancers; however, its oncogenic mechanism remains poorly understood. Here, we show that BCAT1 is crucial for maintaining the poorly differentiated state of glioblastoma cells and that its low expression correlates with a more differentiated glioblastoma phenotype. Furthermore, orthotopic tumor injection into immunocompetent mice demonstrated that the brain microenvironment is sufficient to induce differentiation of Bcat1-KO tumors in vivo. We link the transition to a differentiated cell state to the increased activity of TET demethylases and the hypomethylation and activation of neuronal differentiation genes. In addition, the knockout of Bcat1 attenuated immunosuppression, allowing for an extensive infiltration of CD8 + cytotoxic T-cells and complete abrogation of tumor growth. Further analysis in immunodeficient mice revealed that both tumor cell differentiation and immunomodulation following BCAT1-KO contribute to the long-term suppression of tumor growth. In summary, our study unveils BCAT1's pivotal role in promoting glioblastoma growth by inhibiting tumor cell differentiation and sustaining an immunosuppressive milieu. These findings offer a novel therapeutic avenue for targeting glioblastoma through the inhibition of BCAT1. Overall design: DNA was extracted from cultured mGB2 cells. Samples unclude the control cells (NT), Bcat1-knockout cells (BKO) and Bcat1-KO cells expressing shRNA targeting Tet1 (shTet1) or Tet2 (shTet2). Each sample was present in triplicates

胶质母细胞瘤（Glioblastoma）是成年人最常见的恶性脑肿瘤。细胞可塑性与低分化特征会导致治疗后肿瘤快速复发。此外，免疫抑制微环境被证实是免疫治疗方法面临的主要障碍。支链氨基酸转氨酶1（BCAT1）是一类可将支链氨基酸转化为支链酮酸的代谢酶，其会消耗细胞内的α-酮戊二酸并产生谷氨酸。已有研究表明BCAT1可驱动胶质母细胞瘤及其他癌症的生长，但其致癌机制仍不甚明晰。本研究发现，BCAT1对维持胶质母细胞瘤细胞的低分化状态至关重要，且其低表达与更趋于分化的胶质母细胞瘤表型相关。进一步实验显示，向免疫健全小鼠进行原位肿瘤注射表明，脑微环境足以在体内诱导Bcat1敲除（Bcat1-KO）肿瘤发生分化。本研究将这种向分化细胞状态的转变与TET去甲基化酶活性增强、神经元分化基因的低甲基化及激活建立了关联。此外，Bcat1敲除可减弱肿瘤的免疫抑制状态，使得CD8+细胞毒性T细胞大量浸润，并完全阻断肿瘤生长。在免疫缺陷小鼠中的进一步分析表明，BCAT1敲除后引发的肿瘤细胞分化与免疫调节过程，均有助于长期抑制肿瘤生长。综上，本研究揭示了BCAT1通过抑制肿瘤细胞分化并维持免疫抑制微环境，从而促进胶质母细胞瘤生长的关键作用。上述发现为通过靶向BCAT1治疗胶质母细胞瘤提供了全新的治疗途径。实验设计概述：从培养的mGB2细胞中提取DNA。样本包括对照组细胞（NT）、Bcat1敲除细胞（BKO）以及表达靶向Tet1（shTet1）或Tet2（shTet2）的短发夹RNA（shRNA）的Bcat1-KO细胞。每个样本设置三次生物学重复。