Somatic mutation(s) and cellular changes in CCM pathogenesis

摘要-项目1 基于我们计划项目的前一个周期中的发现，项目1将深入探讨 CCM发病的细胞和分子事件。小鼠CCM病变的谱系追踪 我们表明，病变发生的最初事件是剩余的野生型拷贝的躯体丢失 CCM基因，然后克隆扩增携带相同体细胞突变的内皮细胞，以及 似乎是将更多的内皮细胞重新招募到不断增长的病变中--这些细胞不包含 体细胞突变。其次，基于卡恩实验室对激活的PI3激酶在 在小鼠CCM模型中，我们发现激活PIK3CA突变的比例超过50% 人类CCM损伤。这些合作发现表明，我们还没有完全理解 有助于CCM病变发生、生长和成熟的分子和细胞事件。在第一个 目的，我们将对大量人类CCM进行深度测序，以确定是否存在任何其他 癌基因有助于病变的发展。在第二个目标中，我们将使用单细胞基因组 DNA测序以确定单个内皮细胞是否同时需要体细胞CCM突变 和PIK3CA突变(和/或在AIM 1中发现的其他基因的突变)，或者相反，这些 基因在病变的不同内皮细胞隔间发生突变。在目标3中，由于不断增长 有证据表明，体细胞突变细胞毒化了非突变细胞，使它们重新进入生长阶段 CCM，我们将使用单细胞RNA测序来确定体细胞突变图谱如何 皮损细胞影响细胞的基因表达谱。除了增加基本面 了解CCM的发病机制，本工作具有治疗意义。PI3K-AKT信令 PATH是现有药物的靶点，其他药物正在开发中。因此，作为该项目的一部分，我们将 继续我们对高效疗法的追求，通过测试本发明或任何一种 三个项目，使用我们在过去的资金周期中开发的更具临床相关性的CCM小鼠模型。 随着对CCM病变细胞的分子和突变特征有了更全面的了解，我们 希望为CCM患者的药物再利用寻找新的靶点。

ABSTRACT - PROJECT 1 Based on discoveries made in the previous cycle of our program project, Project 1 will probe deeply into the cellular and molecular events of CCM pathogenesis. Using lineage tracing of murine CCM lesions we showed that the initial event of lesion genesis is somatic loss of the remaining wild-type copy of the Ccm gene, followed by a clonal expansion of endothelial cells harboring the same somatic mutation, and what appears to be recruitment of additional ECs into the growing lesion – cells that do not harbor the somatic mutation. Secondly, based on discoveries in the Kahn lab for a role for activated PI3 kinase in lesion development in mouse CCM models, we identified activating PIK3CA mutations in more than 50% of human CCM lesions. These collaborative discoveries suggest we have not yet fully understood the molecular and cellular events that contribute to CCM lesion genesis, growth and maturation. In the first Aim, we will deeply sequence bulk human CCMs to determine whether somatic mutation of any other oncogenes contribute to the development of lesions. In a second Aim we will use single-cell genomic DNA sequencing to determine whether individual endothelial cells require both the somatic CCM mutation and the PIK3CA mutation (and/or mutation of other genes discovered in Aim 1), or instead whether these genes are mutated in separate endothelial cell compartments of the lesion. In Aim 3, due to growing evidence that the somatically mutated cells poison the non-mutant cells to recruit them into the growing CCM, we will employ single-cell RNA sequencing to determine how the somatic mutation profile of the lesional cell influences the cell’s gene expression profile. In addition to increasing fundamental understanding of CCM pathogenesis, this work has therapeutic implications. The PI3K-AKT signaling pathway is a target of existing drugs with others under development. Thus as part of this project we will continue our quest for a highly effective therapy by testing compounds nominated in this or any of the three projects, using our more clinically-relevant CCM mouse models developed in the past funding cycle. With a more complete knowledge of the molecular and mutational signature of CCM lesional cells, we hope to identify new targets for drug repurposing for CCM patients.