Role of C/EBPalpha in MDS and MDS Progression

描述(由申请人提供)： C/EBPA是髓系发育的关键调节因子。在10%的急性心肌梗死患者中，CEBPA基因存在点突变。AML1-ETO、Cbfb-SMMHC和FI13ITD抑制CEBPA转录，BCR-ABL和AML1-MDS1-EVI1抑制CEBPA翻译。在8%的MDS病例中也检测到CEBPA基因突变，并预测即将进展为急性心肌梗死。65%的CEBPA突变是N端的，导致内部ATG的P30蛋白被截断表达，35%是亮氨酸拉链(LZ)的亚基突变，阻止DNA结合。我们有了新的发现，C/EBPap30和C/EBPaLZ癌蛋白通过诱导bcl-2抑制细胞凋亡，这依赖于功能上的和可能直接与NF-kB的p50亚基的相互作用。抑制细胞凋亡可能是MDS向AML进展的关键步骤。为了进一步了解该通路在MDS进展中的作用，我们提出了以下目标：目的1：确定与NF-kappaB p50的直接相互作用是否在C/EBPA诱导bcl2的过程中起关键作用。目的2：确定C/EBPA和核因子-kappaB p50中调节其生化和功能相互作用的关键残基。目的：建立C/EBPA相关MDS小鼠模型，探讨NFkappaB和LZ的相互作用在此过程中的作用。目的：利用基因芯片技术，在依赖于C/EBPA与核因子-kappaB相互作用的MDS和AML细胞以及依赖于DNA结合的细胞中鉴定C/EBPA的遗传靶点。免疫共沉淀将用于评估生化相互作用，启动子研究，芯片，p50抑制剂的表达，以及使用核转录因子-kappaB基因敲除细胞系将评估功能相互作用。在p15(-/-)骨髓中逆转录病毒转导C/EBPap30或C/EBPaLZ，单独或与激活的N-RAS一起，将被用于建立MDS(早期时间点)和AML(随着小鼠年龄的增长)的模型。不结合核因子-kB的C/EBPap30和C/EBPaLZ的变体将进行类似的评估以进行比较。将利用小鼠MDS和AML细胞来评估bcl2的诱导以及对自发和化疗诱导的细胞凋亡的敏感性，并将对它们的mRNA进行微阵列分析，以确定直接的C/EBPA遗传靶点和那些通过拴系于NF-kappaB而间接反映基因激活的靶点。这些数据将有助于解释来自人类MDS患者的表达数据，并可能揭示导致抑制细胞凋亡和进展为AML的共同途径。

DESCRIPTION (provided by applicant): C/EBPa is a key mediator of myeloid development. The CEBPA gene harbors point mutations in 10% of AMI cases. Also, AML1-ETO, CBFb-SMMHC, and FI13ITD inhibit CEBPA transcription, and bcr-abl and AML1-MDS1-EVI1 inhibit CEBPA translation. CEBPA gene mutations have also been detected in 8% of MDS cases and predict imminent progression to AMI. 65% of CEBPA mutations are N-terminal and lead to expression of a truncated p30 protein from an internal ATG, and 35% are inframe mutations in the leucine zipper (LZ) which prevent DNA-binding. We have made the novel finding that the C/EBPap30 and C/EBPaLZ oncoproteins inhibit apoptosis via induction of bcl-2, dependent upon functional and likely direct interaction with the p50 subunit of NF-kB. Inhibition of apoptosis may be a key step in progression of MDS to AML. To further our understanding of the role of this pathway in MDS progression, we propose the following aims: AIM 1: To determine whether direct interaction with NF-kappaB p50 is critical for induction of bcl-2 by C/EBPa. AIM 2: To identify the critical residues within C/EBPa and NF-kappaB p50 which mediate their biochemical and functional interaction. AIM 3: To develop murine models of C/EBPa associated MDS and to determine the role of interaction with NFkappaB and the LZ in these processes. AIM 4: To use microarray analysis to identify genetic targets of C/EBPa in murine MDS and AML cells dependent upon its interaction with NF-kappaB and those dependent upon DNA-binding. Co-immunoprecipitation will be used to assess biochemical interaction, promoter studies, ChIP, expression of a p50 inhibitor, and use of NF-kappaB knockout cell line lines will evaluate functional interactions. Retroviral transduction of C/EBPap30 or C/EBPaLZ in p15(-/-) marrow, alone or with activated N-Ras, will be employed to develop models of MDS (at early time points) and AML, (as the mice age). Variants of C/EBPap30 and C/EBPaLZ which do not bind NF-kB will be evaluated similarly for comparison. Bcl-2 induction and susceptibility to spontaneous and chemotherapy induced apoptosis will be evaluated using murine MDS and AML cells, and their mRNA will be subjected to microarray analysis to identify direct C/EBPa genetic targets and those reflecting activation of genes indirectly, via tethering to NF-kappaB. These data will be useful for interpreting expression data from human MDS patients and may uncover a common pathway leading to inhibition of apoptosis and progression to AML.