Hypersensitivity of Multiple Myeloma Bone Disease to Vitamin D

描述（由申请方提供）：溶骨性骨病是导致不可治愈的多发性骨髓瘤（MM）发病率和死亡率的主要因素。活性1，25-二羟维生素D（1，25-（OH）2D）刺激肿瘤和骨细胞，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中的这种持续功能是由于肿瘤细胞和骨细胞中的特定基因启动子对异常低浓度（10-11-10- 10 M）的1，25-（OH）2D的超敏反应。广泛的初步数据支持1，25-（OH）2D在MBD中的新作用机制：肿瘤细胞和骨细胞通过转录激活因子TAF 12的过表达对1，25-（OH）2D超敏。在正常细胞中表达水平非常低时，TAF 12作为一种广泛表达的常规转录因子复合物组分发挥作用，但当过表达时，它结合维生素D受体（VDR）应答启动子并作为一种新的VDR共激活因子发挥作用。TAF 12在5/8例原发性MM样品中强烈过表达，而TAF 12功能的获得和丧失分别增加和降低特异性转录对1，25-（OH）2D的敏感性。额外的维生素D超敏反应可能是由于ATF 7（也在MM中过表达）与TAF 12的结合以及MM细胞中VDR辅阻遏物SMRT（NCoR 2）的频繁丢失。目的1通过检测骨髓瘤细胞增殖、骨髓基质细胞粘附、细胞因子产生和破骨细胞形成的支持作用，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ATF 7是否在原代MM细胞中持续过表达，以及它是否与TAF 12一致。TAF 12表达增加的机制也将被研究，并且可能是由骨髓微环境中增加的IL 6和TNF引起的。目的2将描述肿瘤和骨基质细胞中维生素D超敏反应的分子机制，包括通过染色质免疫沉淀法测定TAF 12和TAF 12：ATF 7复合物在两个维生素D响应启动子上的组装。将在MM和骨基质细胞中测定TAF 12和TAF 12：ATF 7对VDR蛋白半衰期响应1，25-（OH）2D的影响，以及辅阻遏物SMRT逆转对1，25-（OH）2D超敏反应的能力。在第二年，AIM 3将测试TAF 12作为骨髓瘤的治疗靶点。TAF 12功能增加或丧失的MM细胞将在溶骨性MBD的免疫受损小鼠模型中生长，以确定对肿瘤负荷和骨破坏的影响。目标是证明TAF 12作为MBD的重要调节剂的效用，因为辅激活剂：VDR结合相互作用是小分子可药物化的。这些新型药物可以选择性地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，而没有维生素D拮抗剂引起的全身毒性。

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.