Characterization of a novel suppressor of oncogenic CARD11 signaling.

致癌 CARD11 信号传导新型抑制剂的表征。

• 批准号: 10642823
• 负责人: Nicole Marie Carter
• 金额: $ 2.28万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10642823
• 关键词: Acute T Cell Leukemia; American; Antigen Receptors; B Cell Proliferation; B-Cell Development; B-Cell Lymphomas; B-Lymphocytes; Binding; Biological Assay; Cause of Death; Cell Line; Cell Survival; Cells; Central Nervous System Lymphoma; Cessation of life; Co-Immunoprecipitations; Data; Development; Developmental Delay Disorders; Disease; Dose; Down-Regulation; Etiology; Event; FRAP1 gene; Flow Cytometry; Functional disorder; Growth; Immune signaling; Immunity; Incidence; Individual; Knock-out; Knowledge; Length; Link; LoxP-flanked allele; Luciferases; Lymphocyte; Lymphoma; MAPK8 gene; Malignant Neoplasms; Mantle Cell Lymphoma; Mass Spectrum Analysis; Mediating; Molecular; Monitor; Mus; Mutate; Mutation; Non-Hodgkin' s Lymphoma; Oncogenic; Output; Pathway interactions; Patients; Peripheral; Physiological; Proliferating; Proteins; Rag1 Mouse; Receptor Signaling; Regulation; Regulatory T-Lymphocyte; Reporter; Role; Sampling; Scaffolding Protein; Signal Transduction; Structure of germinal center of lymph node; T-Cell Lymphoma; T-Cell Receptor; T-Lymphocyte; Testing; The Cancer Genome Atlas; Transcriptional Activation; United States; Work; activated B cell like; adoptive B cell transfer; cancer therapy; checkpoint therapy; chondrodysplasia; cofactor; conditional knockout; deletion analysis; experimental study; gain of function; gain of function mutation; human disease; improved; inhibitor; insight; large cell Diffuse non-Hodgkin' s lymphoma; leukemia/lymphoma; lymphocyte proliferation; mouse model; mutant; novel; overexpression; pharmacologic; precision medicine; synergism; transcription factor; treatment strategy; tumor

Project Summary/Abstract Non-Hodgkin lymphomas are a major cause of death in the United States, yet the molecular causes are not thoroughly understood. In many lymphomas including Diffuse Large B Cell Lymphoma (DLBCL), the scaffold protein CARD11 is mutated, and this can drive aberrant activation of the transcription factor NF-κB, as well as JNK and mTOR. In healthy lymphocytes, CARD11 connects antigen receptor engagement to NF-κB, JNK, and mTOR activation, but in lymphoma, this signaling can become dysregulated and lead to aberrant lymphocyte proliferation, activation, and survival. Downregulation of CARD11 signaling can therefore be useful in treating lymphoma, but there is currently not a complete mechanistic understanding of signal transduction through CARD11 in healthy and diseased lymphocytes. Recent mass spectrometry screens have identified QRICH1 as a novel negative regulator of CARD11 signaling to NF-κB. QRICH1 is a highly conserved protein without a known function. Mutations in QRICH1 have been identified in individuals with developmental delay disorders and chondrodysplasia, but QRICH1 has not been thoroughly studied on the molecular level. Some DLBCL cases have QRICH1 copy number losses, but QRICH1 has never before been implicated in immune signaling or linked to CARD11. In preliminary studies, QRICH1 physically interacts with CARD11 and can inhibit NF-κB activation by wild-type and oncogenic CARD11. The proposed work aims to 1) determine the mechanism by which QRICH1 regulates CARD11 signaling, and 2) characterize the role of QRICH1 in B cell proliferation and survival. In order to elucidate the mechanism of CARD11 inhibition by QRICH1, a deletion analysis will be performed to determine the region(s) of QRICH1 that are necessary and/or sufficient for inhibition, and the interactions between QRICH1, CARD11, and known CARD11 cofactors will be characterized. The signaling step(s) that QRICH1 acts on will be determined by comparing CARD11 signaling events in wild-type and QRICH1-deficient Jurkat T cells. The effect of QRICH1 on CARD11 signaling to JNK and mTOR will be resolved by comparing JNK and mTOR activation markers in wild-type and QRICH1-deficient Jurkat T cells. To characterize the role of QRICH1 in B cell proliferation and survival, QRICH1 will be knocked out in DLBCL-derived cell lines, and the effect on growth/proliferation will be quantified using a flow-cytometry based assay. QRICH1 knockout primary B cells with and without a CARD11 gain-of-function mutation will then be injected into Rag1-/- mice, and injected B cell proliferation and survival will be quantified by flow-cytometry. Finally, a QRICH1fl/fl mouse has been generated and will be crossed to Mb1-Cre for conditional knockout of QRICH1 in B cells. B cell expansion and activation will be quantified by flow-cytometry, and QRICH1fl/fl Mb1-Cre mice with and without a CARD11 GOF mutation will be monitored for B cell lymphoma. This work will advance knowledge of CARD11 signaling regulation, which can be used to develop new treatments to downregulate CARD11’s signaling output in cancer. It will also improve the molecular understanding of DLBCL, which can improve precision medicine strategies for patients.

项目摘要/摘要 非霍奇金淋巴瘤是美国的主要死亡原因，但分子原因是 没有彻底理解。在包括弥漫性大B细胞淋巴瘤（DLBCL）的许多淋巴瘤中，支架 蛋白质card11被突变，这可能会驱动转录因子NF-κB的异常激活以及 JNK和MTOR。在健康的淋巴细胞中，Card11将抗原受体的参与度连接到NF-κB，JNK和 MTOR激活，但在淋巴瘤中，该信号传导可能失调并导致异常淋巴细胞 增殖，激活和生存。因此，Card11信号的下调可用于治疗 淋巴瘤，但目前尚无对信号转导的完全理解 在健康和疾病的淋巴细胞中的card11。最近的质谱屏幕已将QRICH1确定为 Card11信号的新型负调节剂对NF-κB。 Qrich1是一种高度保守的蛋白质，没有已知的 功能。在患有发育延迟疾病的个体中已经确定了QRICH1中的突变，并且 软骨增生，但Qrich1尚未在分子水平上进行彻底研究。一些DLBCL案例 有QRICH1拷贝数损失，但是QRICH1从未与免疫信号或链接有关 到card11。在初步研究中，QRICH1与Card11物理相互作用，可以抑制NF-κB激活 由野生型和致癌卡11。拟议的工作旨在1）确定Qrich1的机制 调节Card11信号传导，2）表征Qrich1在B细胞增殖和存活中的作用。为了 为了阐明QRICH1抑制Card11的机制，将进行删除分析以确定 QRICH1的区域是必要和/或足以抑制的，以及Qrich1之间的相互作用 Card11和已知的Card11辅助因子将被表征。 QRICH1在遗嘱上作用的信号传导步骤 通过比较野生型和Qrich1缺陷型Jurkat T细胞中的Card11信号传导事件来确定。这 QRICH1对JNK和MTOR的Card11信号的影响将通过比较JNK和MTOR解决 野生型和QRICH1缺乏的Jurkat T细胞中的激活标记。表征Qrich1在B细胞中的作用 增殖和存活，QRICH1将在DLBCL衍生的细胞系中淘汰，对 将使用基于流程度仪的测定法对生长/增殖进行定​​量。 Qrich1敲除原代B细胞与 然后，如果没有Card11的功能获得突变，将注入RAG1 - / - 小鼠，并注入B细胞 增殖和存活率将通过流程仪进行量化。最后，已经生成了QRICH1FL/FL鼠标 并将越过MB1-CRE，以使B细胞中的Qrich1有条件地敲除。 B细胞扩展和激活 将通过流程度仪和带有或没有Card11 GoF突变的QRICH1FL/FL MB1-CRE量化 将监测B细胞淋巴瘤。这项工作将提高对Card11信号法规的了解， 可用于开发新的治疗方法，以下调Card11在癌症中的信号输出。它也会改善 对DLBCL的分子理解，可以改善患者的精确医学策略。

项目成果

CARD11 signaling in regulatory T cell development and function.

• DOI: 10.1016/j.jbior.2022.100890
• 发表时间: 2022-05
• 期刊: Advances in biological regulation
• 作者: Carter, Nicole M.;Pomerantz, Joel L.
• 通讯作者: Pomerantz, Joel L.