GFI-1 and Osteoblast Suppression in Multiple Myeloma

多发性骨髓瘤中的 GFI-1 和成骨细胞抑制

基本信息

批准号：
8117780
负责人：
DEBORAH Lynn GALSON
金额：
$ 27.76万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-26 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8117780
关键词：
Ascorbic Acid BMP2 gene Binding Binding Sites Bone Diseases Bone Pain Cell Differentiation process Cells Cessation of life Clinical Detection Differentiation Antigens Disease remission Embryo Environment Enzymes Epigenetic Process Fracture Ganciclovir Gene Expression Genes Growth Healed Histones In Vitro Interleukin-6 Interleukin-7 Lesion Lytic Lytic Metastatic Lesion Maintenance Malignant Neoplasms Mediating Mesenchymal Multiple Myeloma Mus Osteoblasts Osteoclasts Osteogenesis Pathological fracture Patients Plasmids Play Production Recruitment Activity Reporting Repression Risk Role Skeleton Small Interfering RNA Stromal Cells TNF gene TNFSF11 gene Testing Thymidine Kinase Transcription Repressor/Corepressor Transfection Tumor Necrosis Factor Suppression Tumor Necrosis Factor-alpha Up-Regulation base bone cell growth chromatin remodeling deletion analysis economic impact healing in vivo in vivo Model killings lipid biosynthesis mouse model osteoblast differentiation prevent promoter public health relevance transcription factor

项目摘要

DESCRIPTION (provided by applicant): Myeloma (MM) is the most frequent cancer to involve the skeleton. Up to 90% of patients develop bone lesions that can result in severe bone pain and frequent pathologic fractures. Unfortunately, these bone lesions rarely heal even when patients are in long-term remission because of the permanent MM-induced suppression of osteoblast precursor (OBP) differentiation into functional bone-forming osteoblasts. MM induces an intrinsic and persistent change in the OBP differentiation potential, the basis of which is unknown. Preliminary studies with a murine in vivo model of MM-induced OB suppression using 5TGM1 MM cells (which we modified to express GFP for detection and thymidine kinase, which allows them to be selectively killed by ganciclovir) demonstrate a persistent inhibition of OB differentiation even in the absence of MM cells. The OBP from these mice maintained low levels of the critical OB transcription factor, Runx2 even when induced to differentiate and had elevated expression of the transcriptional repressor Gfi-1, which can mediate chromatin remodeling. Importantly, Gfi-1 levels in OBP from 7/7 MM patients were elevated compared to 3 normals. 5TGM1 MM cells inhibited OB differentiation in vitro by producing TNF-1 and IL-7, which increased Gfi-1 in a mouse OBP line (MC4). Further, mouse Runx2 promoter analysis identified a 1003 bp region (-992/+111) that is responsible for suppression of Runx2 expression by MM cells and contains 29 putative Gfi-1 binding sites. This region is also repressed by TNF-1 and by co-transfection with a Gfi-1 expression plasmid. Importantly, knockdown of Gfi-1 expression, using a specific siRNA, significantly restored expression of Runx2 as well as the expression of several OB markers in both MC4 cells pretreated with MM cells and in MSC from 2 MM patients. These results suggest the hypothesis that MM cells secrete soluble factors (TNF-1 and IL-7) that increase Gfi-1 expression in OBPs. Gfi-1 then suppresses Runx2 production and thereby inhibits osteoblastogenesis. Further, since Gfi-1 can recruit to genes histone-modifying enzymes that create epigenetic changes, this results in long-term suppression of Runx2 that is maintained in the absence of MM cells. However, the role of Gfi-1 plays in OBP differentiation and in MM in particular is currently unknown and is the focus of this proposal. The following specific aims will be pursued to test this hypothesis: (1) Determine if Gfi-1 up-regulation in MSC is necessary for MM or TNF-1/IL-7 suppression of OB differentiation. (2) Determine if elevated Gfi-1 is sufficient for OB suppression and/or for increased IL-6 and RANKL production by MSC and does it act by direct binding to the Runx2 gene. (3) Determine if MM cells induce epigenetic changes in the Runx2 gene in MSC via Gfi-1, and if they are responsible for long-term OB suppression by assessing if altering the epigenetic status of the Runx2 gene relieves the differentiation block. (4) Determine if Gfi-1 deficiency in MSC in vivo prevents MM-induced suppression of OB differentiation. PUBLIC HEALTH RELEVANCE: Multiple Myeloma (MM) is the most frequent cancer to involve the skeleton, with up to 90% of patients developing bone lesions that can result in severe bone pain and frequent pathologic fractures. Unfortunately, these lytic bone lesions rarely heal even when patients are in long-term complete remission because of the permanent MM-induced block of osteoblast precursor differentiation into functional bone-forming osteoblasts. This application proposes to investigate what intrinsic changes to the osteoblast precursor are triggered by MM cells and are responsible for the selective differentiation block.

描述（由申请人提供）：骨髓瘤（MM）是涉及骨骼的最常见癌症。多达90％的患者患有骨骼病变，可导致严重的骨痛和频繁的病理性骨折。不幸的是，这些骨骼病变即使患者长期缓解，由于永久性MM诱导的成骨细胞前体（OBP）分化为功能性骨形成骨成骨细胞，因此这些骨骼病变也很少愈合。 MM引起OBP分化潜力的内在和持续变化，其基础尚不清楚。使用5TGM1 MM细胞的MM诱导的OB抑制的鼠体内模型的初步研究（我们修改为表达GFP以进行检测和胸苷激酶，这使得它们可以被Ganciclovir选择性地杀死），这表明在没有MM细胞的情况下，持续的OB分化持续抑制。这些小鼠的OBP保持临界OB转录因子的较低水平，即使诱导分化并具有升高转录抑制剂GFI-1的表达，这可以介导染色质重塑。重要的是，与3个正常患者相比，来自7/7 mM患者的OBP中的GFI-1水平升高。 5TGM1 MM细胞通过产生TNF-1和IL-7在体外抑制OB分化，从而增加了小鼠OBP系中的GFI-1（MC4）。此外，小鼠RUNX2启动子分析确定了1003 bp区域（-992/+111），该区域负责MM细胞抑制Runx2表达，并包含29个假定的GFI-1结合位点。 TNF-1也抑制了该区域，并与GFI-1表达质粒共转染。重要的是，使用特定的siRNA敲低GFI-1表达，可显着恢复RUNX2的表达，以及在用MM细胞预处理的两个MC4细胞中的几个OB标记和来自2 mM患者的MSC中的表达。这些结果表明，MM细胞分泌可溶性因子（TNF-1和IL-7），从而增加了OBP中的GFI-1表达。然后，GFI-1抑制RUNX2的产生，从而抑制成骨细胞生成。此外，由于GFI-1可以募集产生表观遗传变化的酶组蛋白修饰的基因，因此这会导致长期抑制Runx2，而Runx2在没有MM细胞的情况下保持了。但是，GFI-1在OBP分化中的作用尤其是目前尚不清楚，这是该提案的重点。将追求以下具体目的来检验此假设：（1）确定MSC中的GFI-1上调是否对于MM或TNF-1/IL-7抑制OB分化是必需的。（2）确定升高的GFI-1是否足以抑制OB和/或增加MSC的IL-6和RANKL产生，并通过直接结合Runx2基因来起作用。（3）确定MM细胞是否通过GFI-1诱导MSC中RUNX2基因的表观遗传变化，以及是否通过评估RUNX2基因的表观遗传状态来缓解差异障碍而负责长期OB抑制。（4）确定体内MSC中的GFI-1缺乏是否可以防止MM诱导的OB分化抑制。公共卫生相关性：多发性骨髓瘤（MM）是涉及骨骼的最常见癌症，多达90％的患者患有骨骼病变，可导致严重的骨痛和频繁的病理性骨折。不幸的是，这些裂解的骨骼病变即使患者长期完全缓解，由于永久性MM诱导的成骨细胞前体块分化为功能性骨形成骨成骨细胞，因此很少愈合。该应用程序建议研究MM细胞触发成骨细胞前体的内在变化，并负责选择性分化块。