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

 描述（由申请方提供）：意义不明的单克隆丙种球蛋白病（MGUS）是最常见的癌前病变之一，影响约3.5%的50岁以上人群。这个补助金申请旨在测试挑衅性的问题PQ 1。对于从癌前区域产生的肿瘤，该区域中细胞的哪些特性可用于设计抑制未来肿瘤发展的策略最近的研究表明，肿瘤不仅仅是增殖癌细胞的孤立块。相反，它们是由多种不同的细胞类型组成的复杂组织，这些细胞类型参与彼此的异型相互作用。在这里，我们假设，正常骨髓间充质基质细胞（MSC）相邻的早期癌前MGUS细胞是积极的参与者在肿瘤发生和克隆进化，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中检验这一假设。在具体目标1中，我们将阐明基因组/转录组事件，在MGUS的癌前阶段的MSC的变化。我们将进行综合表征的基因组，表观基因组和转录组的变化，发生在骨髓间充质干细胞存在附近的MGUS细胞散装和在单细胞水平。将在蛋白质水平上对特异性靶标进行进一步验证，以使用CyTOF质谱成像鉴定这些改变的MSC在骨髓小生境中的空间定位。在特定目标2中，我们将在小鼠模型中鉴定早期癌前阶段和疾病进展期间MSC中基因组病变的顺序获得。在这里，我们将定义在恶性前浆细胞中的第一个遗传事件期间在MSC中发生的变化，并确定在早期MGUS阶段克隆浆细胞附近发生的变化与那些存在于远端骨髓部位的变化。我们将研究MSC的改变是否先于早期遗传命中（允许的微环境）或在MGUS发育后获得（获得性改变）。我们还将定义这些小鼠中随着干细胞老化而发生在MSC中的基因组和基因表达变化。在具体目标3中，我们将根据目标1和2中进行的研究，使用基于CRISPR的基因敲除高度优先化的基因来验证特定靶标，以功能性地询问它们对MSC的特定作用及其使用体外和体内模型调节肿瘤进展。此外，我们将使用与Osx-cre小鼠杂交的CRISPR-cas9小鼠开发MSC特异性基因组改变，以确定这些靶基因在调节小鼠MM模型中肿瘤起始和MGUS进展中的作用。这些集中的研究将有助于确定骨髓间充质干细胞在MGUS发展的早期阶段和MM的克隆进展的贡献作用。通过确定在MGUS的早期癌前阶段调节克隆进化的新靶点，我们可能能够开发预防或延迟从MGUS进展到明显MM的治疗药物。

 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.