(PQ1) Genomic characterization of mesenchymal stromal cells in Monoclonal Gammopathy of Undermined Significance (MGUS)

 描述(由申请人提供)：未确定意义的单克隆性伽马病(MGUS)是最常见的癌前疾病之一，影响50岁以上人口的约3.5%。这项拨款申请旨在测试挑衅性问题PQ1。对于来自癌前领域的肿瘤，该领域的细胞的哪些特性可以被用来设计抑制未来肿瘤发展的策略。最近的研究表明，肿瘤不仅仅是岛状的增殖癌细胞。相反，它们是由多种不同类型的细胞组成的复杂组织，这些细胞类型参与彼此之间的异型相互作用。在这里，我们假设与早期癌前MGUS细胞相邻的正常骨髓间充质基质细胞(MSCs)是肿瘤发生和克隆进化的积极参与者，而不是被动的旁观者；因此，这些细胞有助于多发性骨髓瘤(MM)的发展。我们将在三个具体目标上检验这一假设。在特定的目标1中，我们将阐明在MGUS癌前阶段管理MSCs变化的基因组/转录事件。我们将对MGUS细胞附近的MSCs在单个细胞水平上发生的基因组、表观基因组和转录水平的变化进行综合表征。对特定靶点的进一步验证将在蛋白质水平上进行，以使用细胞飞行时间质谱仪成像来确定这些改变的骨髓间充质干细胞在骨髓壁龛的空间定位。在特定的目标2中，我们将确定在小鼠模型中，在早期癌前阶段和疾病进展期间，MSCs中基因组损伤的顺序获取。在这里，我们将定义在癌前浆细胞的第一个遗传事件期间在MSCs中发生的变化，并确定在MGUS早期克隆浆细胞与存在于远端骨髓部位的克隆浆细胞之间发生的变化。我们将研究MSCs的改变是在早期的遗传冲击之前(允许的微环境)还是在MGUS发育之后获得的(获得性改变)。我们还将确定在这些小鼠中，随着干细胞的老化，MSCs中发生的基因组和基因表达的变化。在特定的目标3中，我们将在AIMS 1和2中进行的研究的基础上，使用基于CRISPR的高优先级基因敲除来验证特定的靶点，以在功能上询问它们对MSCs的特定作用以及它们在体外和体内模型中对肿瘤进展的调控。此外，我们将利用CRISPR-Cas9小鼠与OSX-cre小鼠杂交来开发MSC特异性基因组改变，以确定这些靶基因在调节小鼠MM模型中肿瘤启动和MGUS进展中的作用。这些重点研究将有助于确定MSCs在MGUS发展和向MM克隆进展的早期阶段所起的作用。通过识别在MGUS早期癌前阶段调节克隆进化的新靶点，我们可能能够开发出防止或延缓从MGUS向显性MM进展的治疗剂。事实上，通过在前体阶段根除疾病，MM可能成为一种可预防的疾病。

 DESCRIPTION (provided by applicant): Monoclonal gammopathy of undetermined significance (MGUS) is one of the most common pre-malignant disorders and affects approximately 3.5% of the population over 50 years of age. This grant application aims to test the provocative question PQ1. For tumors that arise from a pre-malignant field, what properties of cells in this field can be used to design strategies to inhibit the development of future tumors Recent studies showed that tumors are more than insular masses of proliferating cancer cells. Instead, they are complex tissues composed of multiple distinct cell types that participate in heterotypic interactions with one another. Here, we hypothesize that normal bone marrow mesenchymal stromal cells (MSCs) adjacent to the early premalignant MGUS cells are active participants in tumorigenesis and clonal evolution rather than passive bystanders; as such, these cells contribute to the development of multiple myeloma (MM). We will test this hypothesis in 3 specific Aims. In Specific Aim 1, we will elucidate genomic/transcriptomic events that govern alterations in MSCs at the premalignant stage of MGUS. We will perform integrative characterization of genomic, epigenomic and transcriptomic changes that occur in MSCs present near MGUS cells in bulk and at the single cell level. Further validation of specific target will be performed at the protein level to identify the spatial localization of these altered MSCs i the bone marrow niche using CyTOF mass spectrometry imaging. In Specific Aim 2, we will identify the sequential acquisition of genomic lesions in MSCs in the early premalignant stage and during disease progression in a murine model. Here, we will define the changes that occur in MSCs during the first genetic event in the premalignant plasma cells and identify changes that occur in the proximity of clonal plasma cells at the early MGUS stages vs. those that are present in distant bone marrow sites. We will examine whether MSCs alterations precede the early genetic hit (permissive microenvironment) or are acquired after MGUS development (acquired alterations). We will also define genomic and gene expression changes that occur in MSCs with stem cell aging in these mice. In Specific Aim 3, we will validate specific targets using CRISPR-based gene knockout of highly prioritized genes based on the studies performed in Aims 1 and 2 to functionally interrogate their specific role on MSCs and their regulation of tumor progression using in vitro and in vivo models. Furthermore, we will develop MSC-specific genomic alterations using CRISPR-cas9 mice crossed with Osx-cre mice to define the role of these target genes in regulating tumor initiation and MGUS progression in murine MM models. These focused research studies will help define the contributing role of MSCs in the early stages of MGUS development and clonal progression to MM. By identifying novel targets that regulate clonal evolution at the early premalignant stage of MGUS, we may be able to develop therapeutic agents that prevent or delay progression from MGUS to overt MM. Indeed, by eradicating the disease at the precursor stages, MM may become a preventable disease.