Characterization of a novel suppressor of oncogenic CARD11 signaling.

项目摘要/摘要 在美国，非霍奇金淋巴瘤是主要的死亡原因，但其分子原因是 不完全理解的。在包括弥漫性大B细胞淋巴瘤(DLBCL)在内的许多淋巴瘤中，支架 蛋白CARD11发生突变，这可能会导致转录因子NF-κB的异常激活，以及 JNK和mTOR。在健康淋巴细胞中，CARD11将抗原受体参与与核因子-κB、JNK和 MTOR激活，但在淋巴瘤中，这一信号可能变得失调并导致淋巴细胞异常 增殖、激活和存活。因此，下调CARD11信号转导可用于治疗 淋巴瘤，但目前还没有一个完整的机制了解信号转导通过 CARD11在健康和疾病淋巴细胞中的表达。最近的质谱仪屏幕已经确认QRICH1是 一种新的针对核因子κB的CARD11信号负调控因子QRICH1是一种高度保守的蛋白质，没有已知的 功能。QRICH1基因突变已在发育迟缓障碍患者和 软骨发育不良，但QRICH1在分子水平上还没有得到深入的研究。部分DLBCL病例 有QRICH1拷贝数丢失，但QRICH1以前从未与免疫信号有关或关联 至CARD11。在初步研究中，QRICH1与CARD11在物理上相互作用，并能抑制NF-κB的激活 野生型和致癌性CARD11。拟议的工作旨在1)确定QRICH1 2)研究QRICH1在B细胞增殖和存活中的作用。按顺序 为了阐明QRICH1抑制CARD11的机制，我们将进行缺失分析以确定 QRICH1对抑制是必要和/或充分的区域(S)，以及QRICH1， CARD11，以及已知的CARD11辅助因子将被描述。QRICH1作用于Will的信令步骤(S) 通过比较野生型和QRICH1缺陷Jurkat T细胞中的CARD11信号事件来确定。这个 QRICH1对JNK和mTOR的CARD11信号的影响将通过比较JNK和mTOR来解决 野生型和QRICH1缺陷Jurkat T细胞中的激活标记。QRICH1在B细胞中的作用 增殖和存活，QRICH1将在DLBCL来源的细胞系中被敲除，并对其影响 生长/增殖将使用基于流式细胞术的分析进行量化。QRICH1基因敲除的初级B细胞 在没有CARD11的情况下，功能获得突变将被注射到Rag1/-小鼠体内，并注射B细胞 增殖和存活率将通过流式细胞仪进行量化。最后，产生了一个QRICH1fl/fl小鼠 并将其与Mb1-Cre杂交，在B细胞中条件敲除QRICH1。B细胞的扩增和激活 将通过流式细胞仪进行量化，以及具有和不具有CARD11 GOF突变的QRICH1fl/fl Mb1-Cre小鼠 将被监测为B细胞淋巴瘤。这项工作将促进对CARD11信号调节的了解， 可用于开发新的治疗方法，下调CARD11‘S信号在癌症中的输出。它也将得到改善 对DLBCL的分子认识，可以提高患者的精准用药策略。

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.