BCR/ABL1-Dependent Transcriptional Response Reveals Enrichment for Genes Involved in Negative Feedback Regulation

Philadelphia (Ph) chromosome-positive leukemia is characterized by the BCR/ABL1 fusion protein that affects a wide range of signal transduction pathways. The knowledge about its downstream target genes is, however, still quite limited. To identify novel BCR/ABL1-regulated genes we used global gene expression profiling of several Ph-positive and Ph-negative cell lines treated with imatinib. Following imatinib treatment, the Ph-positive cells showed decreased growth, viability, and reduced phosphorylation of BCR/ABL1 and STAT5. In total, 142 genes were identified as being dependent on BCR/ABL1-mediated signaling, mainly including genes involved in signal transduction, e.g. the JAK/STAT, MAPK, TGFB and insulin signaling pathways, and in regulation of metabolism. Interestingly, BCR/ABL1 was found to activate several genes involved in negative feedback regulation (CISH, SOCS2, SOCS3, PIM1, DUSP6, and TNFAIP3), which may act to indirectly suppress the tumor promoting effects exerted by BCR/ABL1. In addition, several genes identified as deregulated upon BCR/ABL1 expression could be assigned to the TGFB and NFkB signaling pathways, as well as to reflect the metabolic adjustments needed for rapidly growing cells. Apart from providing important pathogenetic insights into BCR/ABL1-mediated leukemogenesis, the present study also provides a number of pathways/individual genes that may provide attractive targets for future development of targeted therapies. Keywords: global gene expression profiling, Ph chromosome, BCR/ABL1, imatinib mesylate Five Philadelphia-positive (Ph+) and five Philadelphia-negative (Ph-) cell lines were cultured in the absence or presence of 1 microM imatinib mesylate for 3 and 12 hours. Total RNA was extracted from untreated and treated cell cultures at both time points, resulting in 4 samples per cell line. RNA extraction, labeling, hybridization, washing, scanning, and feature analysis were performed as described (Håkansson et al., 2008, Genes Chromosomes Cancer). Dye-swap labeling was performed in 32 out of 40 samples. In total, 72 slides were hybridized and scanned. The transcriptional response was studied using the mean value of the 3 and 12 hour microarray measurements.

费城染色体（Philadelphia chromosome，Ph）阳性白血病以影响广泛信号转导通路的BCR/ABL1融合蛋白为特征。然而，目前对其下游靶基因的认知仍相当有限。为鉴定新型BCR/ABL1调控基因，我们对经伊马替尼（imatinib）处理的多株Ph阳性与Ph阴性细胞系开展了全基因表达谱分析（global gene expression profiling）。伊马替尼处理后，Ph阳性细胞的增殖能力、细胞活力均出现下降，且BCR/ABL1与STAT5的磷酸化水平显著降低。本研究共鉴定出142个依赖BCR/ABL1介导信号通路的基因，其中主要包括参与信号转导的基因（如JAK/STAT、MAPK、TGFB及胰岛素信号通路相关基因）以及代谢调控相关基因。值得注意的是，BCR/ABL1可激活若干参与负反馈调控的基因（CISH、SOCS2、SOCS3、PIM1、DUSP6及TNFAIP3），这些基因或可间接抑制BCR/ABL1的促肿瘤效应。此外，若干经鉴定存在BCR/ABL1表达失调的基因可归类至TGFB与核因子κB（NFκB）信号通路，同时也反映了快速增殖细胞所需的代谢适应机制。本研究不仅为BCR/ABL1介导的白血病发生机制提供了重要的致病学见解，还筛选出多个通路与单个基因靶点，可为未来靶向治疗的开发提供极具吸引力的方向。关键词：全基因表达谱分析（global gene expression profiling），费城染色体（Philadelphia chromosome），BCR/ABL1，甲磺酸伊马替尼（imatinib mesylate）。本研究培养了5株Ph阳性（Ph+）与5株Ph阴性（Ph-）细胞系，分别在添加1μM甲磺酸伊马替尼（imatinib mesylate）或不含该药物的培养基中培养3小时与12小时。于两个时间点分别收集处理组与未处理组的细胞培养物，提取总RNA，每株细胞系共获得4份样本。RNA提取、标记、杂交、洗涤、扫描及特征分析均按照已发表方法完成（Håkansson等，2008，Genes Chromosomes Cancer）。40份样本中的32份进行了染料交换标记。最终共完成72张芯片的杂交与扫描。本研究采用3小时与12小时微阵列（microarray）检测结果的平均值来分析转录应答反应。