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: 10442742
• 负责人: Nicole Marie Carter
• 金额: $ 4.68万
• 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/10442742
• 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; 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.

项目总结/摘要 在美国，非霍奇金淋巴瘤是死亡的主要原因，但分子原因是 没有被彻底理解。在包括弥漫性大B细胞淋巴瘤（DLBCL）在内的许多淋巴瘤中， 蛋白质CARD 11突变，这可以驱动转录因子NF-κB的异常激活，以及 JNK和mTOR。在健康淋巴细胞中，CARD 11将抗原受体结合与NF-κB、JNK和 mTOR激活，但在淋巴瘤中，这种信号传导可能变得失调，并导致异常淋巴细胞 增殖、活化和存活。因此，CARD 11信号传导的下调可用于治疗 淋巴瘤，但目前还没有一个完整的机制，了解信号转导，通过 健康和患病淋巴细胞中的CARD 11。最近的质谱筛选已经将QRICH 1鉴定为 一种新的CARD 11信号转导至NF-κB的负调控因子。QRICH 1是一种高度保守的蛋白质， 功能QRICH 1的突变已在发育迟缓障碍的个体中被鉴定， 软骨发育不良，但QRICH 1尚未在分子水平上进行彻底研究。一些DLBCL病例 有QRICH 1拷贝数损失，但QRICH 1以前从未参与免疫信号传导或相关 到CARD 11。在初步研究中，QRICH 1与CARD 11物理相互作用，并可抑制NF-κB活化 野生型和致癌CARD 11。拟议的工作旨在1）确定QRICH 1 调节CARD 11信号传导，和2）表征QRICH 1在B细胞增殖和存活中的作用。为了 为了阐明QRICH 1抑制CARD 11的机制，将进行缺失分析以确定 抑制所必需和/或足够的QRICH 1区域，以及QRICH 1， CARD 11和已知的CARD 11辅因子将被表征。QRICH 1作用的信令步骤将 通过比较野生型和QRICH 1缺陷型Jurkat T细胞中的CARD 11信号传导事件来确定。的 QRICH 1对CARD 11信号传导至JNK和mTOR的影响将通过比较JNK和mTOR来解决 在野生型和QRICH 1缺陷型Jurkat T细胞中的活化标志物。为了表征QRICH 1在B细胞中的作用， 在DLBCL衍生的细胞系中，QRICH 1将被敲除，并且对细胞的增殖和存活的影响将被抑制。 将使用基于流式细胞术的测定来定量生长/增殖。QRICH 1敲除原代B细胞， 然后将没有CARD 11功能获得性突变的小鼠注射到Rag 1-/-小鼠中，并注射B细胞 增殖和存活将通过流式细胞术定量。最后，已经生成QRICH 1fl/fl小鼠 并将其与Mb 1-Cre杂交以在B细胞中条件性敲除QRICH 1。B细胞扩增和活化 将通过流式细胞术进行定量，并且具有和不具有CARD 11 GOF突变的QRICH 1fl/fl Mb 1-Cre小鼠 将监测是否有B细胞淋巴瘤。这项工作将推进CARD 11信号调节的知识， 可用于开发新的治疗方法，以下调CARD 11在癌症中的信号输出。还将完善 DLBCL的分子理解，这可以改善患者的精准医疗策略。