DataSheet_1_High Expression of BCL11A Predicts Poor Prognosis for Childhood MLL-r ALL.docx

BackgroundDespite much improvement in the treatment for acute lymphoblastic leukemia (ALL), childhood ALLs with MLL-rearrangement (MLL-r) still have inferior dismal prognosis. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. MethodsGSE13159 and GSE28497 were selected via the Oncomine website. Differentially expressed genes (DEGs) between MLL-r ALLs and normal samples were identified by R software. Next, functional enrichment analysis of these DEGs were carried out by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Then, the key hub genes and modules were identified by Weighted Gene Co-expression Network Analysis (WGCNA). Therapeutically Applicable Research to Generate Effective Treatments (TARGET) ALL (Phase I) of UCSC Xena analysis, qPCR, and Kaplan-Meier analysis were conducted for validating the expression of key hub genes from bone marrow cells of childhood ALL patients or ALL cell lines. ResultsA total of 1,045 DEGs were identified from GSE13159 and GSE28497. Through GO, KEGG, GSEA, and STRING analysis, we demonstrated that MLL-r ALLs were upregulating “nucleosome assembly” and “B cell receptor signal pathway” genes or proteins. WGCNA analysis found 18 gene modules using hierarchical clustering between MLL-r ALLs and normal. The Venn diagram was used to filter the 98 hub genes found in the key module with the 1,045 DEGs. We identified 18 hub genes from this process, 9 of which were found to be correlated with MLL-r status, using the UCSC Xena analysis. By using qPCR, we validated these 9 hub key genes to be upregulated in the MLL-r ALLs (RS4;11 and SEM) compared to the non-MLL-r ALL (RCH-ACV) cell lines. Three of these genes, BCL11A, GLT8D1 and NCBP2, were shown to be increased in MLL-r ALL patient bone marrows compared to the non-MLL-r ALL patient. Finally, Kaplan–Meier analysis indicated that childhood ALL patients with high BCL11A expression had significantly poor overall survival. ConclusionThese findings suggest that upregulated BCL11A gene expression in childhood ALLs may lead to MLL-r ALL development and BCL11A represents a new potential therapeutic target for childhood MLL-r ALL.背景：尽管急性淋巴细胞白血病（acute lymphoblastic leukemia, ALL）的治疗手段已取得诸多进展，但携带混合谱系白血病重排（MLL-rearrangement, MLL-r）的儿童急性淋巴细胞白血病患者预后仍极差。因此，阐明MLL-r ALL的维持机制，对开发高效治疗策略至关重要。 研究方法：本研究通过Oncomine数据库筛选获取GSE13159与GSE28497数据集。利用R软件鉴定MLL-r ALL样本与正常对照样本间的差异表达基因（differentially expressed genes, DEGs）。随后，通过基因本体论（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）、基因集富集分析（Gene Set Enrichment Analysis, GSEA）以及搜索相互作用基因/蛋白质工具（Search Tool for the Retrieval of Interacting Genes/Proteins, STRING）对上述DEGs开展功能富集分析。进一步通过加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA）筛选关键核心基因与基因模块。最后，借助UCSC Xena平台的治疗性应用研究以生成有效治疗（Therapeutically Applicable Research to Generate Effective Treatments, TARGET）儿童ALL队列（第一阶段）、实时定量PCR（qPCR）以及Kaplan-Meier生存分析，验证儿童ALL患者骨髓细胞或ALL细胞系中核心基因的表达水平。 研究结果：从GSE13159与GSE28497数据集中共鉴定得到1045个DEGs。通过GO、KEGG、GSEA及STRING分析，本研究发现MLL-r ALL中“核小体组装”与“B细胞受体信号通路”相关基因/蛋白质呈上调表达。WGCNA分析通过层级聚类在MLL-r ALL与正常对照样本间筛选得到18个基因模块。通过韦恩图，将关键模块中的核心基因与1045个DEGs取交集，共获得98个候选核心基因。经UCSC Xena分析进一步筛选后，共鉴定得到18个与MLL-r状态显著相关的核心基因，其中9个基因的表达水平经qPCR验证：在MLL-r ALL细胞系（RS4;11与SEM）中，这9个基因的表达量显著高于非MLL-r ALL细胞系（RCH-ACV）。进一步分析显示，BCL11A、GLT8D1与NCBP2这3个基因在MLL-r ALL患者骨髓样本中的表达量显著高于非MLL-r ALL患者。最终Kaplan-Meier生存分析结果表明，BCL11A高表达的儿童ALL患者总体生存期显著更差。 研究结论：本研究结果提示，儿童ALL中BCL11A基因的上调表达可能参与MLL-r ALL的发生发展，BCL11A有望成为儿童MLL-r ALL全新的潜在治疗靶点。