Exploration of the role of neural-type receptors and the ECM proteins SPARC and Tenascin C in the bone marrow HSC-niche unit

骨髓增生异常综合征(MDS)是一种克隆性血液学疾病，以无效造血、外周血细胞减少和骨髓细胞发育不良为特征，进展为急性髓系白血病(AML)的风险增加。MDS不仅是一种造血细胞室的疾病，而且骨髓生态位的改变也影响着髓系肿瘤的发展。基于这一假设，在之前的联合工作中，我们已经建立了一个基于患者来源的CD34+造血干细胞(HSCs)和骨髓来源的基质细胞(MSCs)共同移植的人MDS的强大的异种移植模型。我们的工作揭示了MDS HSCs诱导的微环境的有益重塑，以优先支持其自身的生长。MDS来源的MSCs显著高表达的关键因素之一是分泌酸性和富含半胱氨酸的细胞外基质蛋白(SPARC)。SPARC被认为是介导肿瘤细胞与周围间质相互作用和多种实体瘤转移的中心因子。在恶性血液病中，我们可以证明骨髓间质中SPARC的缺失可以保护造血细胞免受化疗毒性的影响。在目前的提案中，我们的目标是使用我们独特的MDS异种移植模型来阐明SPARC的分子机制，以便可能确定针对髓系肿瘤中异常间质间隔的新的治疗策略。为了实现这一点，我们将以前的小鼠SPARC基因敲除株与NSG模型杂交，以产生NSG SPARC基因敲除小鼠。原代MDS来源和健康的骨髓样本将被异种移植到该模型中，以研究骨髓微环境中SPARC的缺失或存在的生物学效应。作为植入率以外的读数，MDS异种移植的克隆性骨髓成分将通过对原始患者样本和MDS异种移植的定量突变和免疫表型分析来分析。为了分析造血室和骨髓基质之间的指导性串扰，我们将在体内接触MDS HSCs后，对原始分选的基质细胞组分进行全转录RNA测序，包括内皮细胞、成骨细胞和间充质干细胞等亚组分。除此之外，我们将利用我们的模型作为临床前平台，测试是否可以通过添加或抑制外源性SPARC来操纵骨髓利基，对MDS生物学、克隆性成分或改善功能血液成分产生积极影响。在其他肿瘤实体如Bortezomib和NAB-Paclitaxel的背景下，SPARC生物学中涉及的药理物质的应用将被评估为针对MDS骨髓基质的潜在疗法。

Myelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with increased risk of evolution to acute myeloid leukemia (AML). MDS is not only a disease of the hematopoietic cell compartment but also alterations in the bone marrow niche influence the development of myeloid neoplasms. Based on this hypothesis, in previous joint collaborative work we have established a robust xenograft model of human MDS based on the co-transplantation of patient derived CD34+ hematopoietic stem cells (HSCs) and bone marrow (BM) derived stromal cells (MSCs). Our work revealed an instructive remodeling of the microenvironment induced by MDS HSCs to preferentially support their own growth. One of the key factors significantly overexpressed in MDS derived MSCs was the extracellular matrix protein secreted protein acidic and rich in cysteine (SPARC). SPARC has been implicated as a central factor in mediating tumor cell interaction with surrounding stroma and metastasis of a variety of solid tumors. In hematologic malignancies, we could show that loss of SPARC in the bone marrow stroma compartment protects hematopoietic cells from chemotherapy toxicity. In the current proposal we aim to use our unique MDS xenograft model to elucidate the molecular mechanisms of SPARC in order to possibly identify new therapeutic strategies that are targeted against the aberrant stroma compartment in myeloid neoplasms. To accomplish this we have crossed previous murine SPARC knockout strains with the NSG model in order to generate NSG SPARC knockout mice. Primary MDS derived and healthy bone marrow samples will be xenotransplanted into this model to study the biologic effect of absence or presence SPARC in the bone marrow niche. As a readout beside engraftment rates, the clonal bone marrow composition of the MDS xenografts will be analyzed via quantitative mutation and immunophenotypic analyses of primary patient samples and MDS xenografts. To analyze the instructive crosstalk between the hematopoietic compartment and the bone marrow stroma, we will perform whole transcriptome RNA sequencing in primary sorted stroma cell fractions, including subfractions such as endothelial cells, osteoblasts and mesenchymal stem cells after exposure to the MDS HSCs in vivo. Beyond this, we will use our model as a pre-clinical platform to test whether by manipulation of the bone marrow niche with either addition of exogenous SPARC or its inhibition, positive effects on MDS biology, clonal composition or improvement of functional blood components can be achieved. The application of pharmacologic substances that have been implicated in the biology of SPARC in the context of other tumor entities such as Bortezomib and nab-Paclitaxel will be evaluated as potential therapies targeting the bone marrow stroma of MDS.