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: 10064777
• 负责人: Nicole Marie Carter
• 金额: $ 4.55万
• 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/10064777
• 关键词: Acute T Cell Leukemia; Adoptive Transfer; American; Antigen Receptors; B Cell Proliferation; B-Cell Activation; 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; Lead; 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; Pharmacology; Physiological; 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; base; cancer therapy; checkpoint therapy; chondrodysplasia; cofactor; conditional knockout; deletion analysis; experimental study; gain of function; gain of function mutation; human disease; improved; inhibitor/antagonist; insight; large cell Diffuse non-Hodgkin' s lymphoma; leukemia/lymphoma; lymphocyte proliferation; mouse model; mutant; novel; overexpression; precision medicine; 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.

项目概要/摘要 非霍奇金淋巴瘤是美国的一个主要原因，但其分子原因是 没有彻底理解。在许多淋巴瘤中，包括弥漫性大 B 细胞淋巴瘤 (DLBCL)，支架 蛋白质 CARD11 发生突变，这会导致转录因子 NF-κB 的异常激活，以及 JNK 和 mTOR。在健康淋巴细胞中，CARD11 将抗原受体与 NF-κB、JNK 和 mTOR 激活，但在淋巴瘤中，这种信号传导可能失调并导致淋巴细胞异常 增殖、激活和存活。因此，CARD11 信号传导的下调可用于治疗 淋巴瘤，但目前还没有对信号转导的完整机制了解 健康和患病淋巴细胞中的 CARD11。最近的质谱筛选已将 QRICH1 鉴定为 CARD11 信号传导至 NF-κB 的新型负调节因子。 QRICH1 是一种高度保守的蛋白质，未知 功能。已在患有发育迟缓障碍的个体中发现了 QRICH1 突变 软骨发育不良，但 QRICH1 尚未在分子水平上得到彻底研究。部分DLBCL病例 有 QRICH1 拷贝数丢失，但 QRICH1 以前从未涉及免疫信号传导或链接 至卡 11。初步研究中，QRICH1 与 CARD11 发生物理相互作用，可以抑制 NF-κB 激活 由野生型和致癌 CARD11 引起。拟议工作的目的是 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，将进行缺失分析以确定 QRICH1 的抑制所必需和/或充分的区域，以及 QRICH1 之间的相互作用， CARD11 和已知的 CARD11 辅因子将被表征。 QRICH1 作用的信令步骤将 通过比较野生型和 QRICH1 缺陷型 Jurkat T 细胞中的 CARD11 信号转导事件来确定。这 QRICH1 对 JNK 和 mTOR 的 CARD11 信号传导的影响将通过比较 JNK 和 mTOR 来解决 野生型和 QRICH1 缺陷型 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衍生的细胞系中被敲除，并且对增殖和存活的影响 将使用基于流式细胞术的测定来量化生长/增殖。 QRICH1 敲除原代 B 细胞 然后将没有 CARD11 功能获得突变的小鼠注射到 Rag1-/- 小鼠中，并注射 B 细胞 增殖和存活将通过流式细胞术进行量化。最后，一只QRICH1fl/fl小鼠就生成了 并将与 Mb1-Cre 杂交，以在 B 细胞中条件性敲除 QRICH1。 B细胞扩增和激活 将通过流式细胞术进行定量，并且带有和不带有 CARD11 GOF 突变的 QRICH1fl/fl Mb1-Cre 小鼠 将监测 B 细胞淋巴瘤。这项工作将增进对 CARD11 信号传导调控的了解， 可用于开发新疗法来下调 CARD11 在癌症中的信号输出。也会改善 对 DLBCL 的分子理解，可以改善患者的精准医疗策略。