Role of C/EBPalpha in MDS and MDS Progression

描述（由申请人提供）： C/EBPa是骨髓发育的关键介质。CEBPA基因在10%的AMI病例中存在点突变。此外，AML 1-ETO、CBFb-SMMHC和FI 13 ITD抑制CEBPA转录，bcr-abl和AML 1-MDS 1-EVI 1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AMI。65%的CEBPA突变是N-末端的，导致从内部ATG表达截短的p30蛋白，35%是亮氨酸拉链（LZ）中的框内突变，阻止DNA结合。我们发现C/EBPap 30和C/EBPaLZ癌蛋白通过诱导bcl-2抑制细胞凋亡，依赖于与NF-κ B的p50亚基的功能性和可能的直接相互作用。细胞凋亡的抑制可能是MDS向AML进展的关键步骤。为了进一步理解该通路在MDS进展中的作用，我们提出了以下目标：目的1：确定与NF-κ B p50的直接相互作用是否是C/EBPa诱导bcl-2的关键。目的2：鉴定C/EBP α和NF-κ B p50中介导其生化和功能相互作用的关键残基。目的3：建立C/EBPa相关MDS小鼠模型，并确定与NF κ B和LZ相互作用在这些过程中的作用。目标4：使用微阵列分析来鉴定小鼠MDS和AML细胞中依赖于其与NF-κ B相互作用的C/EBPa的遗传靶点以及依赖于DNA结合的靶点。免疫共沉淀将用于评估生物化学相互作用、启动子研究、ChIP、p50抑制剂的表达，并使用NF-κ B敲除细胞系评价功能相互作用。将单独或与活化的N-Ras一起使用p15（-/-）骨髓中的C/EBPap 30或C/EBPaLZ的逆转录病毒转导来开发MDS（在早期时间点）和AML（随着小鼠年龄增长）的模型。将类似地评估不结合NF-κ B的C/EBPap 30和C/EBPaLZ的变体以进行比较。将使用鼠MDS和AML细胞评价Bcl-2诱导和对自发性和化疗诱导的细胞凋亡的易感性，并且将对它们的mRNA进行微阵列分析以鉴定直接C/EBPa遗传靶标和通过与NF-κ B的连接间接反映基因活化的那些。这些数据将有助于解释人类MDS患者的表达数据，并可能揭示导致细胞凋亡抑制和AML进展的共同途径。

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.