Identifying Regulators of Oncogenic CARD11 in Diffuse Large B Cell Lymphoma

鉴定弥漫性大 B 细胞淋巴瘤中致癌 CARD11 的调节因子

• 批准号: 8525889
• 负责人: deMauri Mackie
• 金额: $ 4.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525889
• 关键词: Accounting; Activities of Daily Living; Adaptor Signaling Protein; Affect; Alleles; Antibodies; Antigens; Autoimmunity; B-Lymphocytes; Binding; Biological Assay; Bioluminescence; CD28 gene; CD3 Antigens; Cell Death; Cell Line; Cell Nucleus; Cells; Co-Immunoprecipitations; Complementary DNA; Complex; Deletion Mutation; Dependence; Drug Targeting; Energy Transfer; Hyperactive behavior; Immunoprecipitation; Jurkat Cells; Kinetics; Location; Luciferases; Lymphocyte Function; MAP3K7 gene; Malignant Neoplasms; Measures; Mediating; Methods; Mutate; Mutation; NFAT Pathway; Non-Hodgkin' s Lymphoma; Oncogenic; Outcome; Pathway interactions; Patients; Phenotype; Phosphotransferases; Play; Property; Proteins; RNA Interference; Reagent; Regulation; Reporter; Research; Resistance; Role; Sampling; Scaffolding Protein; Sequence Analysis; Series; Signal Transduction; Specificity; Stimulus; T-Lymphocyte; TNF gene; TRAF6 gene; Testing; Time; Tissue Sample; Western Blotting; cancer cell; caspase-8; cofactor; knock-down; large cell Diffuse non-Hodgkin' s lymphoma; member; mutant; novel; novel therapeutics; protein protein interaction; public health relevance; research study; response; small hairpin RNA

DESCRIPTION (provided by applicant): The Activated B-Cell (ABC) Like subtype of Diffuse Large B-Cell Lymphoma (DLBCL) is known for its dependence on constitutive NF- B activity. Mutated forms of the scaffolding protein CARD11 have been identified from ABC DLBCL patient samples. A high-throughput protein-protein interaction screen identified the protein C9orf9 as a potential modulator of oncogenic CARD11 activity. The proposed studies seek to characterize the mechanism of action of C9orf9 in the TCR-to-NF- B pathway, including characterizing the phenotype when C9orf9 is knocked down in Jurkat cells, confirming the location of C9orf9 in the TCR-to-NF- B pathway, and identifying critical regions or residues of C9orf9. Furthermore, the role and mechanism of C9orf9 in the DLBCL line OCI-Ly3 will be elucidated. To achieve these aims, short hairpin RNAs will be used to knock down C9orf9 in Jurkat and an NF- B luciferase reporter will be used to compare TCR-to-NF- B signaling in C9orf9 knockdown Jurkats and in a control Jurkat line. Hairpin resistant C9orf9 cDNAs will be used to rescue any phenotype in the C9orf9 knockdown lines and demonstrate TCR-to-NF- B pathway specificity. C9orf9 is hypothesized to act downstream of PKC / and upstream of the IKK complex. This location will be confirmed by stimulating C9orf9 Jurkat knockdown lines and a control line using CD3/ CD28 antibodies. Samples will be collected over a time course and Western blots probed for phospho- PKC and the I B substrate of the IKK complex. If the hypothesized location of C9orf9 is correct, phospho- PKC levels will be consistent between the C9orf9 knockdown lines and the control Jurkat line. Conversely, if C9orf9 acts above the IKK complex as predicted, I B will fail to be degraded in C9orf9 knockdown lines and will be detectable at higher levels than in control Jurkat cells. The functional region of C9orf9 will be identified by testing a series of deletion an mutation constructs in HEK293T cells for their ability to activate the NF- B luciferase reporter, where constructs that lack the functional region will not activate the reporter. Kinetic associatio of C9orf9 and CARD11 will be measured using Bioluminescence Resonance Energy Transfer (BRET). If C9orf9 and CARD11 associate in a stimulation dependent manner, this interaction will be measured as an increase in BRET signal in Jurkat cells. Stimulation-dependent associations of CARD11 and its cofactors will be measured by immunoprecipitation and Western blot in C9orf9 Jurkat knockdown lines and in a control Jurkat line. If C9orf9 is required for binding of a known cofactor with CARD11, less of that co-factor will be detectable in Western blots of co-immunoprecipitation assays using C9orf9-deficient cells. Finally, the role of C9orf9 in the OCI-Ly3 DLBCL cell line will be elucidated. If C9orf9 is required for OCI-Ly3 survival, shRNA infected OCI-Ly3 cells will show rapid cell death as compared to OCI-Ly3 cells infected with the non-target hairpin. The proposed studies seek to confirm C9orf9 as a critical part of the CARD11-to-NF- B pathway in ABC DLBCL and as a novel therapeutic drug target for DLBCL treatment.

描述(由申请人提供)：弥漫性大B细胞淋巴瘤(DLBCL)的激活B细胞(ABC)样亚型以其对结构性核因子-B活性的依赖而闻名。从ABC DLBCL患者样本中发现了支架蛋白CARD11的突变形式。高通量蛋白质-蛋白质相互作用筛选确定C9orf9蛋白是致癌CARD11活性的潜在调节器。这些研究试图确定C9orf9在TCR-to-NF-B途径中的作用机制，包括确定C9orf9在Jurkat细胞中被敲除时的表型，确定C9orf9在TCR-to-NF-B途径中的位置，以及确定C9orf9的关键区域或残基。此外，还将阐明C9orf9在DLBCL OCI-Ly3中的作用和机制。为了实现这些目标，将使用短发夹状RNA在Jurkat中敲除C9orf9，并将使用NF-B荧光素酶报告程序来比较C9orf9敲除Jurkat和对照Jurkat中TCR和NF-B信号的差异。抗发夹的C9orf9 cDNA将被用来挽救C9orf9基因敲除系中的任何表型，并展示TCR-to-NF-B途径的特异性。假设C9orf9作用于PKC/下游和IKK复合体的上游。这一位置将通过刺激C9orf9 Jurkat基因敲除株和使用CD3/CD28抗体的对照株来确认。将在一段时间内收集样本，并通过Western blotting检测磷酸化PKC和IKK复合体的I B底物。如果C9orf9的假设位置正确，则C9orf9基因敲除系和对照Jurkat系之间的磷酸化PKC水平将保持一致。相反，如果C9orf9如预测的那样作用于ikk复合体之上，I B将无法在C9orf9基因敲除系中被降解，并将在比对照Jurkat细胞更高的水平上被检测到。C9orf9的功能区将通过测试HEK293T细胞中一系列缺失和突变结构的激活核因子-B荧光素酶报告基因的能力来确定，其中缺乏功能区的结构将不会激活该报告基因。C9orf9和CARD11的动力学关联将用生物发光共振能量转移(BRET)来测量。如果C9orf9和CARD11以刺激依赖的方式结合，这种相互作用将被测量为Jurkat细胞中Bret信号的增加。在C9orf9 Jurkat基因敲除系和对照Jurkat系中，将通过免疫沉淀和Western印迹来测量CARD11及其辅助因子的刺激依赖关系。如果已知的辅因子与CARD11的结合需要C9orf9，则在使用C9orf9缺陷细胞的免疫共沉淀分析的Western blotts中可以检测到较少的该辅因子。最后，C9orf9在 OCI-Ly3 DLBCL细胞系将被阐明。如果C9orf9是OCI-Ly3生存所必需的，那么与感染非靶标发夹的OCI-Ly3细胞相比，shRNA感染的OCI-Ly3细胞将显示出快速的细胞死亡。这些研究试图证实C9orf9是ABC DLBCL中CARD11-to-NF-B通路的关键部分，并作为治疗DLBCL的新的治疗药物靶点。