GFI-1 and Osteoblast Suppression in Multiple Myeloma

描述（申请人提供）：骨髓瘤（MM）是最常见的涉及骨骼的癌症。高达90%的患者发生骨损伤，可导致严重的骨痛和频繁的病理性骨折。不幸的是，即使患者处于长期缓解期，这些骨病变也很少愈合，因为mm诱导的成骨细胞前体（OBP）向功能性成骨细胞分化的永久性抑制。MM诱导OBP分化电位的内在和持续变化，其基础尚不清楚。在MM诱导OB抑制的小鼠体内模型中，使用5TGM1 MM细胞（我们对其进行了修饰，使其表达GFP用于检测和胸苷激酶，这使得它们可以被更昔洛韦选择性杀死）进行的初步研究表明，即使在没有MM细胞的情况下，OB分化也能持续抑制。这些小鼠的OBP即使被诱导分化，也保持了低水平的关键OB转录因子Runx2，并且转录抑制因子Gfi-1的表达升高，Gfi-1可以介导染色质重塑。重要的是，与3名正常人相比，7/7 MM患者的OBP中Gfi-1水平升高。5TGM1 MM细胞通过产生TNF-1和IL-7抑制OB的体外分化，从而增加小鼠OBP细胞系（MC4）的Gfi-1。此外，小鼠Runx2启动子分析发现了一个1003bp的区域（-992/+111），该区域负责抑制MM细胞的Runx2表达，包含29个假定的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，使用特定的siRNA敲低Gfi-1的表达，在MM细胞预处理的MC4细胞和2mm患者的MSC中，显著恢复Runx2的表达以及几种OB标记物的表达。这些结果提出了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Runx2的产生，从而抑制成骨细胞的形成。此外，由于Gfi-1可以招募产生表观遗传变化的组蛋白修饰酶，这导致Runx2的长期抑制，这种抑制在MM细胞缺失的情况下得以维持。然而，Gfi-1在OBP分化，特别是MM中的作用目前尚不清楚，这是本提案的重点。为了验证这一假设，我们将追求以下具体目标：(1)确定骨髓间质中Gfi-1的上调对于MM或TNF-1/IL-7抑制OB分化是否必要。(2)确定升高的Gfi-1是否足以抑制OB和/或增加MSC中IL-6和RANKL的产生，以及它是否通过直接结合Runx2基因起作用。(3)通过评估改变Runx2基因的表观遗传状态是否减轻分化阻滞，确定MM细胞是否通过Gfi-1诱导MSC中Runx2基因的表观遗传改变，以及它们是否负责长期OB抑制。(4)确定体内间充质干细胞中Gfi-1缺乏是否能阻止mm诱导的OB分化抑制。

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.