Identifying Regulators of Oncogenic CARD11 in Diffuse Large B Cell Lymphoma

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）样亚型以其依赖于构成性NF- B活性而闻名。从ABC DLBCL患者样本中鉴定出了支架蛋白CARD11的突变形式。高通量蛋白-蛋白相互作用筛选鉴定出C9orf9蛋白是致癌CARD11活性的潜在调节剂。拟开展的研究旨在表征C9orf9在TCR-to-NF- B通路中的作用机制，包括表征Jurkat细胞中C9orf9被敲低时的表型，确认C9orf9在TCR-to-NF- B通路中的位置，以及识别C9orf9的关键区域或残基。进一步阐明C9orf9在DLBCL OCI-Ly3中的作用及机制。为了实现这些目标，将使用短发夹rna来敲除Jurkat中的C9orf9，并使用NF- B荧光素酶报告基因来比较C9orf9敲除Jurkat和对照Jurkat中的tcr -NF- B信号。发夹抗性C9orf9 cdna将用于挽救C9orf9敲低系中的任何表型，并证明TCR-to-NF- B通路特异性。据推测，C9orf9作用于PKC /的下游和IKK复合物的上游。这个位置将通过刺激C9orf9 Jurkat敲低系和使用CD3/ CD28抗体的控制系来确认。样品将在一段时间内收集，Western blots检测磷酸PKC和IKK复合物的ib底物。如果C9orf9的假设位置是正确的，那么在C9orf9敲低系和对照Jurkat系之间的磷酸化- PKC水平将是一致的。相反，如果C9orf9像预测的那样作用于IKK复合体之上，那么在C9orf9敲低的细胞系中，ib将不能被降解，并且将以比对照Jurkat细胞更高的水平被检测到。C9orf9的功能区域将通过在HEK293T细胞中测试一系列缺失和突变构建体来确定其激活NF- B荧光素酶报告蛋白的能力，其中缺乏功能区的构建体将不会激活报告蛋白。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，这种相互作用将通过Jurkat细胞中BRET信号的增加来测量。将通过免疫沉淀和Western blot在C9orf9 Jurkat敲除系和对照Jurkat系中测量CARD11及其辅助因子的刺激依赖性关联。如果已知的辅助因子与CARD11结合需要C9orf9，则使用C9orf9缺陷细胞的免疫印迹共免疫沉淀试验中检测到的辅助因子较少。最后，C9orf9在

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.