Anaplastic Lymphoma Kinase signature. Homo sapiens

Anaplastic Large Cell Lymphomas (ALCL) represent a subset of lymphomas in which the Anaplastic Lymphoma Kinase (ALK) gene is frequently fused to the NPM gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo, and that ALK activity is strictly required for the survival of ALK positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK positive ALCL cell lines abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPb and the anti-apoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions. Keywords: other Overall design: This series of microarray experiments contains the gene expression profiles of Anaplastic Large Cell Lymphoma (ALCL) cell lines (TS [a subclone of Sup-M2] and Su-DHL1) engineered to express ALK-A5 shRNA under a doxycycline-inducible promoter or treated with cell permeable pyrrolocarbazole-derived ALK inhibitors. A mutated ALK-A5M shRNA was used as control. Briefly, cells were transduced with pLV-DsRed-tTRKRAB, expanded, and used for transduction with pLVTH-GFP-shRNA lentiviral particles. Cells were induced with doxycycline (1 microg/ml) for 12 hours, double GFP+ DsRed+ cells selected by fluorescence-activated cell sorting. Cells expressing GFP in the absence of the inducer were removed by a second flow cytometry sorting, and expanded. shRNA expression was induced by doxycycline treatment for 72 or 84 hours. Drug treatments (300 nM), were performed in TS cells with ALK inhibitors (A2 or A3), mock compound (A1), or control diluent for 6 hours. 5 micrograms of total RNA was processed and hybridized to the Affymetrix HG-U133A chip following the manufacturer's instructions.

间变性大细胞淋巴瘤（Anaplastic Large Cell Lymphomas, ALCL）是淋巴瘤的一个亚型，其间变性淋巴瘤激酶（Anaplastic Lymphoma Kinase, ALK）基因常与NPM基因发生融合。本团队既往研究证实，ALK嵌合蛋白的组成型磷酸化足以在体外和体内诱导细胞转化，且ALK活性是ALK阳性ALCL细胞存活的绝对必需条件。为阐明ALK介导的细胞转化及肿瘤维持所需的信号通路，我们通过诱导型ALK RNA干扰（RNA interference, RNAi）或强效可细胞渗透的ALK抑制剂，阻断多种ALK阳性ALCL细胞系的ALK介导信号通路，并对其转录组进行分析。基因表达谱（gene expression profiling, GEP）分析得到的转录本揭示了一组可重复的特征基因，其中包含一类新的ALK调控基因。对这些ALK转录靶标进行功能RNAi筛选后发现，转录因子C/EBPb及抗凋亡蛋白BCL2A1是诱导细胞转化，以及维持ALK阳性ALCL细胞生长与存活的绝对必需因子。由此证实，经实验控制且功能验证的GEP分析是识别新型致病网络、验证适合治疗干预的生物学靶基因的有力工具。

关键词：其他
整体设计：本系列微阵列实验包含ALK阳性间变性大细胞淋巴瘤细胞系（TS为Sup-M2的亚克隆，以及Su-DHL1）的基因表达谱，这些细胞系经工程改造，可在多西环素诱导型启动子下表达ALK-A5短发夹RNA（short hairpin RNA, shRNA），或经可细胞渗透的吡咯并咔唑类ALK抑制剂处理。本实验以突变型ALK-A5M shRNA作为对照。
简要实验流程：首先使用pLV-DsRed-tTRKRAB慢病毒载体转导细胞，扩增后再以pLVTH-GFP-shRNA慢病毒颗粒进行转导。以1μg/ml多西环素诱导细胞12小时，通过荧光激活细胞分选筛选GFP阳性且DsRed阳性的双阳性细胞。通过第二轮流式细胞分选移除未诱导状态下表达GFP的细胞，随后扩增细胞。通过多西环素处理72或84小时以诱导shRNA表达。针对TS细胞，分别使用300nM的ALK抑制剂（A2或A3）、阴性对照化合物（A1）或溶剂对照进行6小时药物处理。按照制造商说明书，将5μg总RNA进行样本处理，并杂交至Affymetrix HG-U133A芯片。