Rozovski Uri, Harris David M, Li Ping, Liu Zhiming, Jain Preetesh, Manshouri Taghi, Veletic Ivo, Ferrajoli Alessandra, Bose Prithviraj, Thompson Phillip, Jain Nitin, Verstovsek Srdan, Wierda William, Keating Michael J, Estrov Zeev
Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Division of Hematology, Davidoff Cancer Center, Rabin Medical Center, Petach Tiqva, and The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Oncotarget. 2021 Mar 2;12(5):401-411. doi: 10.18632/oncotarget.27884.
The glioma associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, was detected in chronic lymphocytic leukemia (CLL), but not normal adult cells. GLI1 activating mutations were identified in 10% of patients with CLL. However, what induces GLI1 expression in GLI1-unmutated CLL cells is unknown. Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in CLL cells and sequence analysis detected putative STAT3-binding sites in the GLI1 gene promoter, we hypothesized that STAT3 induces the expression of GLI1. Western immunoblotting detected GLI1 in CLL cells from 7 of 7 patients, flow cytometry analysis confirmed that CD19+/CD5+ CLL cells co-express GLI1 and confocal microscopy showed co-localization of GLI1 and phosphorylated STAT3. Chromatin immunoprecipitation showed that STAT3 protein co-immunoprecipitated GLI1 as well as other STAT3-regulated genes. Transfection of CLL cells with STAT3-shRNA induced a mark decrease in GLI1 levels, suggesting that STAT3 binds to and induces the expression of GLI1 in CLL cells. An electromobility shift assay confirmed that STAT3 binds, and a luciferase assay showed that STAT3 activates the GLI1 gene. Transfection with GLI1-siRNA significantly increased the spontaneous apoptosis rate of CLL cells, suggesting that GLI1 inhibitors might provide therapeutic benefit to patients with CLL.
胶质瘤相关致癌基因1（GLI1）是胚胎期Hedgehog信号通路的下游效应因子，在慢性淋巴细胞白血病（CLL）细胞中可检测到，但在正常成人细胞中未检测到。在10%的CLL患者中发现了GLI1激活突变。然而，在GLI1未突变的CLL细胞中，是什么诱导GLI1表达尚不清楚。由于信号转导和转录激活因子3（STAT3）在CLL细胞中持续激活，且序列分析在GLI1基因启动子中检测到假定的STAT3结合位点，我们推测STAT3诱导GLI1的表达。蛋白质免疫印迹法在7例患者的CLL细胞中均检测到GLI1，流式细胞术分析证实CD19+/CD5+CLL细胞共表达GLI1，共聚焦显微镜显示GLI1与磷酸化STAT3共定位。染色质免疫沉淀显示，STAT3蛋白与GLI1以及其他STAT3调控基因共免疫沉淀。用STAT3-shRNA转染CLL细胞可导致GLI1水平显著降低，表明STAT3在CLL细胞中与GLI1结合并诱导其表达。电泳迁移率变动分析证实STAT3结合，荧光素酶分析显示STAT3激活GLI1基因。用GLI1-siRNA转染可显著提高CLL细胞的自发凋亡率，提示GLI1抑制剂可能对CLL患者有治疗益处。
