Multiple Myeloma (MM) is a disease of plasma cells with specific localization in the bone marrow. Recent studies in several malignancies including MM have shown intraclonal architectural heterogeneity at diagnosis and at different stages of disease progression over time. The presence of clonal tides in MM represent a novel paradigm in myeloma evolutionary biology which will revolutionize the current modeling of MM tumorigenesis and progression and are likely to have profound therapeutic implications. However, the role of the supporting bone marrow niche, specifically mesenchymal stromal cells (MSCs) in the clonal evolution of MM and other malignancies has not been previously elucidated. Although many factors regulating tumor progression are tumor cell autonomous, they are insufficient to induce progression and metastasis, and a permissive microenvironment is required for frank malignancy to emerge. In this grant, we focus on MSCs as critical regulators of clonal evolution in MM that allows for more rapid dissemination and drug resistance during disease progression. Our overarching hypothesis is that MSCs are integral regulators of clonal evolution in MM inducing both tumor dissemination and drug resistance during progression. We will examine this in 3 Specific Aims. Specific Aim 1 will elucidate sequential molecular events that occur in MSCs during MM progression and explore mechanisms of cooperativity of these events with tumor clonal evolution. MM patient samples at different stages of disease progression (MGUS to MM) will be used to determine molecular changes that occur in MSCs that correlate with, or drive tumor clonal diversification. Specific Aim 2 will determine the role of MSCs in clonal evolution in MM that leads to disease progression. The hypothesis of this aim is that MSCs confer selective advantage of specific clones for tumor progression in MM. We will use in vivo tracking of clones distinguished by fluorophores, where clonal subsets can be molecularly interrogated sequentially to track the biography of cells that emerge as winner or loser MM clones in response to loss-of or gain-of-function studies of specific genes deregulated in MSCs. Specific Aim 3 will investigate the role of the MSCs in the regulation of drug resistance in MM. Our hypothesis is that specific molecular changes that occur in MSCs after high-dose chemotherapy allow outgrowth of aggressive drug-resistant subclones of MM. In this aim, we will use in vitro and in vivo model systems to determine molecular changes that occur in MSCs after high dose chemotherapy used in stem cell transplant in MM and investigate how this in turn plays a role in clonal evolution and drug resistance in MM. This grant is focused on using innovative and diverse methods to understand the role of MSCs in clonal evolution in MM. We combine patient samples with mouse models to examine, in high-throughput unbiased methods, the role of MSCs in inducing tumor growth, clonal heterogeneity and drug resistance.