Data_Sheet_1_The Inhibition of B7H3 by 2-HG Accumulation Is Associated With Downregulation of VEGFA in IDH Mutated Gliomas.pdf

B7H3 (also known as CD276) is a co-stimulator checkpoint protein of the cell surface B7 superfamily. Recently, the function beyond immune regulation of B7H3 has been widely studied. However, the expression preference and the regulation mechanism underlying B7H3 in different subtypes of gliomas is rarely understood. We show here that B7H3 expression is significantly decreased in IDH-mutated gliomas and in cultured IDH1-R132H glioma cells. Accumulation of 2-HG leads to a remarkable downregulation of B7H3 protein and the activity of IDH1-R132H mutant is responsible for B7H3 reduction in glioma cells. Inhibition of autophagy by inhibitors like leupeptin, chloroquine (CQ), and Bafilomycin A1 (Baf-A1) blocks the degradation of B7H3 in glioma cells. In the meantime, the autophagy flux is more active with higher LC3B-II and lower p62 in IDH1-R132H glioma cells than in IDH1-WT cells. Furthermore, sequence alignment analysis reveals potential LC3-interacting region (LIR) motifs “F-V-S/N-I/V” in B7H3. Moreover, B7H3 interacts with p62 and CQ treatment significantly enhances this interaction. Additionally, we find that B7H3 is positively correlated with VEGFA and MMP2 by bioinformatics analysis in gliomas. B7H3 and VEGFA are decreased in IDH-mutated gliomas and further reduced in 2-HGhigh gliomas compared to 2-HGlow glioma sections by IHC staining. Our study demonstrates that B7H3 is preferentially overexpressed in IDH wild-type gliomas and could serve as a potential theranostic target for the precise treatment of glioma patients with wild-type IDH.

B7H3（又称CD276）是细胞表面B7超家族的共刺激检查点蛋白。近年来，学界对B7H3超越免疫调控的功能开展了广泛研究。然而，目前关于B7H3在不同胶质瘤亚型中的表达偏好及其潜在调控机制仍知之甚少。本研究发现，B7H3在IDH突变型胶质瘤及体外培养的IDH1-R132H胶质瘤细胞中表达显著下调。2-羟基戊二酸（2-HG）的积累可显著下调B7H3蛋白水平，而IDH1-R132H突变体的活性是胶质瘤细胞中B7H3表达降低的原因。使用亮抑酶肽、氯喹（CQ）、巴弗洛霉素A1（Baf-A1）等自噬抑制剂可阻断胶质瘤细胞中B7H3的降解。与此同时，与IDH1野生型（IDH1-WT）胶质瘤细胞相比，IDH1-R132H胶质瘤细胞的自噬流更为活跃，表现为LC3B-II水平更高、p62水平更低。进一步的序列比对分析显示，B7H3中存在潜在的LC3相互作用区域（LIR）基序"F-V-S/N-I/V"。此外，B7H3可与p62发生相互作用，而氯喹（CQ）处理可显著增强这一相互作用。通过胶质瘤生物信息学分析，本研究还发现B7H3与血管内皮生长因子A（VEGFA）、基质金属蛋白酶2（MMP2）呈正相关。免疫组化（IHC）染色结果显示，相较于2-HG低表达（2-HGlow）胶质瘤组织，IDH突变型胶质瘤中B7H3与VEGFA的表达均显著降低，而在2-HG高表达（2-HGhigh）胶质瘤组织中二者的表达进一步下调。本研究证实，B7H3在IDH野生型胶质瘤中呈优先高表达态势，可作为IDH野生型胶质瘤患者精准治疗的潜在诊疗一体化靶点。