Hypersensitivity of Multiple Myeloma Bone Disease to Vitamin D

描述(申请人提供)：溶骨性骨病是导致无法治愈的多发性骨髓瘤(MM)的发病率和死亡率的主要因素。活性1，25-二羟基维生素D(1，25-(OH)2D)刺激肿瘤细胞和骨细胞，导致骨髓瘤骨病(MBD)，尽管大多数MM患者维生素D不足。维生素D信号在MBD中的这种持续作用是由于肿瘤细胞和骨细胞中特定基因启动子对异常低浓度(10-11-10-10M)的1，25-(OH)2D的超敏。大量的初步数据支持1，25-(OH)2D在MBD中的一个新的作用机制：肿瘤细胞和骨细胞都通过转录激活因子TAF12的过度表达而对1，25-(OH)2D超敏。在正常细胞中，TAF12的表达水平很低，是一种广泛表达的常规转录因子复合体，但当它过度表达时，它会与维生素D受体(VDR)反应的启动子结合，并作为一种新的VDR共激活因子发挥作用。TAF12在5/8的原发MM中高表达，而TAF12的获得和功能丧失分别增加和降低了特异性转录对1，25-(OH)2D的敏感性。额外的维生素D过敏可能是由于ATF7(在MM中也过度表达)与TAF12结合，以及MM细胞中VDR共抑制物SMRT(NCoR2)的频繁丢失。目的1检测骨髓瘤和骨髓基质细胞对低浓度1，25-(OH)2D的增殖、黏附、细胞因子的产生和对破骨细胞形成的支持作用，以探讨骨髓瘤和骨髓基质细胞对低浓度1，25(OH)2D的生物学反应。我们将测试ATF7是否在原代MM细胞中持续过表达，以及它是否与TAF12协同发生。TAF12表达增加的机制也将被研究，并可能是由骨髓微环境中IL6和TNF增加引起的。目的研究肿瘤和骨基质细胞维生素D超敏的分子机制，包括TAF12和TAF12：ATF7复合体在两个维生素D反应启动子上的组装，用染色质免疫沉淀法检测TAF12和TAF12：ATF7对1，25-(OH)2D反应的VDR蛋白半衰期的影响，以及辅阻遏子SMRT逆转1，25-(OH)2D超敏反应的能力。在第二年，AIM 3将测试TAF12作为骨髓瘤的治疗靶点。将带有TAF12功能增强或丧失的MM细胞种植在免疫受损的溶骨性MBD小鼠模型中，以确定其对肿瘤负荷和骨破坏的影响。我们的目标是证明TAF12作为MBD的重要调节因子的实用性，因为共激活剂：VDR结合作用可以用小分子进行抑制。这些新型药物可以选择性和有效地靶向骨髓瘤骨病中肿瘤细胞和骨细胞对1，25-(OH)2D的病理反应，而不会出现维生素D拮抗剂的全身毒性。

DESCRIPTION (provided by applicant): Osteolytic bone disease is a major contributor to the morbidity and mortality caused by incurable multiple myeloma (MM). Active 1,25-dihydroxyvitamin D (1,25-(OH)2D) stimulates both tumor & bone cells, contributing to myeloma bone disease (MBD), despite vitamin D insufficiency in the majority of MM patients. This continuing function of vitamin D signaling in MBD is due to hypersensitivity of specific gene promoters in both tumor cells & bone cells to unusually low concentrations (10-11-10-10M) of 1,25-(OH)2D. Extensive preliminary data support a novel mechanism of action of 1,25-(OH)2D in MBD: Both tumor cells and bone cells are hypersensitized to 1,25-(OH)2D by overexpression of the transcriptional activating factor TAF12. At very low levels of expression in normal cells, TAF12 functions as a widely expressed, conventional transcription factor complex component, but when overexpressed it binds at vitamin D receptor (VDR)- responsive promoters and functions as a novel VDR co-activator. TAF12 was strongly overexpressed in 5/8 primary MM samples, while TAF12 gain and loss of function respectively increased and decreased sensitivity of specific transcription to 1,25-(OH)2D. Additional vitamin D hypersensitivity may be due to binding of ATF7 (also overexpressed in MM) to TAF12 & frequent loss of the VDR co-repressor SMRT (NCoR2) from MM cells. AIM 1 will investigate the biological responses of myeloma & marrow stromal cells to low concentrations of 1,25-(OH)2D by assaying MM cell proliferation, MM-stromal cell adhesion, cytokine production & support of osteoclast formation. We will test whether ATF7 is consistently overexpressed in primary MM cells and if it occurs in concert with TAF12. The mechanism by which TAF12 expression is increased will also be investigated and may be caused by increased IL6 & TNF¿ in the marrow microenvironment. AIM 2 will characterize the molecular mechanisms of vitamin D hypersensitivity in tumor & bone stromal cells, including assembly of TAF12 & TAF12:ATF7 complexes on two vitamin D-responsive promoters, assayed by chromatin immunoprecipitation The effects of TAF12 & TAF12:ATF7 on VDR protein half-life in response to 1,25-(OH)2D will be determined in MM and bone stromal cells, as will the ability of the co-repressor SMRT to reverse hypersensitivity to 1,25-(OH)2D. In Year 2, AIM 3 will test TAF12 as a therapeutic target in myeloma. MM cells with TAF12 gain or loss of function will be grown in an immunocompromised mouse model of osteolytic MBD to determine the effects on tumor burden and bone destruction. The goal is to demonstrate the utility of TAF12 as an important regulator of MBD, since co-activator:VDR binding interactions are druggable with small molecules. Such novel agents could selectively and effectively target the pathological responses to 1,25-(OH)2D of tumor cells and bone cells in myeloma bone disease, without the systemic toxicities caused by vitamin D antagonists.