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: 8827291
• 负责人: deMauri Mackie
• 金额: $ 4.31万
• 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/8827291
• 关键词: Accounting; Activities of Daily Living; Adaptor Signaling Protein; Affect; Alleles; Antibodies; Antigens; Autoimmunity; B-Lymphocytes; Binding; Biological Assay; Bioluminescence; Cell Death; Cell Line; Cell Nucleus; Cells; Co-Immunoprecipitations; Complementary DNA; Complex; Deletion Mutation; Dependence; Drug Targeting; Energy Transfer; Health; 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; 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-κB 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-κB pathway, including characterizing the phenotype when C9orf9 is knocked down in Jurkat cells, confirming the location of C9orf9 in the TCR-to-NF-κB 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-κB luciferase reporter will be used to compare TCR-to-NF-κB 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-κB pathway specificity. C9orf9 is hypothesized to act downstream of PKCθ/ß and upstream of the IKK complex. This location will be confirmed by stimulating C9orf9 Jurkat knockdown lines and a control line using αCD3/αCD28 antibodies. Samples will be collected over a time course and Western blots probed for phospho- PKC and the IκBα 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 NFκB 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-κB 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）的活化B细胞（ABC）样亚型因其对组成型NF-κB活性的依赖性而闻名。已经从ABC DLBCL患者样品中鉴定了支架蛋白CARD 11的突变形式。高通量蛋白质-蛋白质相互作用筛选鉴定了蛋白质C9 orf 9作为致癌CARD 11活性的潜在调节剂。所提出的研究试图表征C9 orf 9在TCR至NF-κB途径中的作用机制，包括表征C9 orf 9在Jurkat细胞中被敲低时的表型，确认C9 orf 9在TCR至NF-κB途径中的位置，以及鉴定C9 orf 9的关键区域或残基。此外，将阐明C9 orf 9在DLBCL细胞系OCI-Ly 3中的作用和机制。为了实现这些目标，短发夹RNA将用于敲低Jurkat中的C9 orf 9，并且NF-κB荧光素酶报告基因将用于比较C9 orf 9敲低Jurkat和对照Jurkat系中的TCR至NF-κB信号传导。发夹抗性C9 orf 9 cDNA将用于挽救C9 orf 9敲低系中的任何表型，并证明TCR至NF-κB途径特异性。假设C9 orf 9作用于 PKCθ/PKC β和IKK复合物上游。将通过使用α CD 3/α CD 28抗体刺激C9 orf 9 Jurkat敲低细胞系和对照细胞系来确认该位置。将在一段时间内采集样本，并使用蛋白质印迹法检测磷酸化PKC和IKK复合物的IκBα底物。如果C9 orf 9的假设位置是正确的，那么C9 orf 9敲低品系和对照Jurkat品系之间的磷酸化PKC水平将是一致的。相反，如果C9 orf 9如预测的那样作用于IKK复合物之上，则I B将不能在C9 orf 9敲低细胞系中降解，并且将在比对照Jurkat细胞中更高的水平下可检测到。将通过在HEK 293 T细胞中测试一系列缺失和突变构建体激活NFκB荧光素酶报告基因的能力来鉴定C9 orf 9的功能区，其中缺乏功能区的构建体将不会激活报告基因。将使用生物发光共振能量转移（BRET）测量C9 orf 9和CARD 11的动力学缔合。如果C9 orf 9和CARD 11以刺激依赖性方式缔合，则这种相互作用将被测量为Jurkat细胞中BRET信号的增加。CARD 11及其辅因子的刺激依赖性关联将通过免疫沉淀和蛋白质印迹在C9 orf 9 Jurkat敲低系和对照Jurkat系中测量。如果C9 orf 9是已知辅因子与CARD 11结合所需的，则在使用C9 orf 9缺陷型细胞的免疫共沉淀测定的蛋白质印迹中可检测到较少的辅因子。最后，C9 orf 9在 将阐明OCI-Ly 3 DLBCL细胞系。如果C9 orf 9是OCI-Ly 3存活所需的，则与用非靶发夹感染的OCI-Ly 3细胞相比，shRNA感染的OCI-Ly 3细胞将显示快速细胞死亡。所提出的研究试图证实C9 orf 9是ABC DLBCL中CARD 11-to-NF-κB通路的关键部分，并且是DLBCL治疗的新型治疗药物靶标。