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

• 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
• 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

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）是累及骨骼的最常见癌症。高达90%的患者会出现骨病变，导致严重的骨痛和频繁的病理性骨折。不幸的是，这些骨病变很少愈合，即使患者在长期缓解，因为永久MM诱导的成骨细胞前体（OBP）分化成功能性骨形成成骨细胞的抑制。MM诱导OBP分化潜能的内在和持续变化，其基础尚不清楚。使用5 TGM 1 MM细胞（我们对其进行了修饰以表达用于检测的GFP和胸苷激酶，这使得它们能够被更昔洛韦选择性地杀死）对MM诱导的OB抑制的鼠体内模型的初步研究表明，即使在没有MM细胞的情况下，OB分化也受到持续抑制。来自这些小鼠的OBP保持低水平的关键OB转录因子Runx 2，即使在诱导分化时也是如此，并且具有转录抑制因子Gfi-1的升高表达，其可以介导染色质重塑。重要的是，与3名正常人相比，7/7名MM患者的OBP中的Gfi-1水平升高。5 TGM 1 MM细胞通过产生TNF-1和IL-7抑制体外OB分化，这增加了小鼠OBP系（MC 4）中的Gfi-1。此外，小鼠Runx 2启动子分析鉴定了1003 bp区域（-992/+111），其负责MM细胞对Runx 2表达的抑制，并且含有29个推定的Gfi-1结合位点。该区域也被TNF-1和与Gfi-1表达质粒共转染抑制。重要的是，使用特异性siRNA敲低Gfi-1表达，显著恢复了用MM细胞预处理的MC 4细胞和来自2名MM患者的MSC中Runx 2的表达以及几种OB标志物的表达。这些结果表明MM细胞分泌可溶性因子（TNF-1和IL-7）增加OBP中Gfi-1表达的假设。然后Gfi-1抑制Runx 2的产生，从而抑制成骨细胞生成。此外，由于Gfi-1可以招募基因组蛋白修饰酶，产生表观遗传变化，这导致Runx 2的长期抑制，在MM细胞不存在的情况下维持。然而，Gfi-1在OBP分化中的作用，特别是在MM中的作用目前尚不清楚，并且是该提议的焦点。将追求以下具体目标来检验该假设：（1）确定MSC中的Gfi-1上调是否是MM或TNF-1/IL-7抑制OB分化所必需的。(2)确定升高的Gfi-1是否足以抑制OB和/或增加MSC产生的IL-6和RANKL，以及它是否通过直接结合Runx 2基因起作用。(3)确定MM细胞是否通过Gfi-1诱导MSC中Runx 2基因的表观遗传变化，以及通过评估改变Runx 2基因的表观遗传状态是否缓解分化阻滞来确定它们是否负责长期OB抑制。(4)确定体内MSC中Gfi-1缺陷是否阻止MM诱导的OB分化抑制。 公共卫生相关性：多发性骨髓瘤（MM）是最常见的累及骨骼的癌症，高达90%的患者发生骨病变，可导致严重的骨痛和频繁的病理性骨折。不幸的是，这些溶解性骨病变很少愈合，即使当患者处于长期完全缓解，因为永久MM诱导的成骨细胞前体分化成功能性骨形成成骨细胞的阻滞。本申请提出研究MM细胞引发的成骨细胞前体的内在变化，并负责选择性分化阻滞。