Molecular Pathogenesis of Multiple Myeloma

我们继续鉴定和描述多发性骨髓瘤（MM）和意义不明的单克隆丙种球蛋白病（MGUS）的致癌事件及其后果。过去一年的进展包括：MM肿瘤和细胞系突变激活NF-κ B（NF κ B）通路。我们最初发现，两个人MM细胞系（HMCL）组成性激活NF κ B途径的NF κ B诱导激酶（NIK）的过度表达，由于易位或基因扩增。IkB激酶β（IKK 2）抑制剂导致约50%的HMCL细胞分裂停止或死亡，包括过表达NIK的两种HMCL。该抑制剂对几种HMCL的作用用于产生NFkB活性的11个基因分子标记。使用该NFkB指数分析来自基因表达阵列的数据，我们发现50%的HMCL、82%的MM肿瘤、95%的MGUS肿瘤和所有正常骨髓浆细胞具有NFkB通路的实质性激活。聚焦于NIK和其他调节NFkB途径突变的基因，我们发现两种阳性NFkB调节剂NIK和CD 40的表达在一些MM或HMCL样本中升高，而四种阴性调节剂TRAF 3，CYLD，BIRC 2和BIRC 3在其他样本中缺失或失活。此外，在一些HMCL中，发现NFKB 1基因的过表达或NFKB 2基因的截短形式激活NFkB途径。在所有情况下，这些突变都在具有高NFkB指数的肿瘤或HMCL中发现。在未经治疗的MM肿瘤中，突变的发生率估计为15-20%，在HMCL中>35%，表明突变可能在肿瘤进展期间发生。HMCL中的大多数突变激活经典和替代NFkB途径，但我们的数据表明，经典途径激活可能是最关键的，因为IKK 2抑制剂选择性靶向经典途径。其他人报道，来自正常骨髓基质细胞的外源性信号激活NFkB通路对于正常小鼠浆细胞的存活至关重要。因此，我们的研究结果表明，NFkB通路的激活-无论是通过外在信号还是内在突变，可能降低肿瘤对骨髓微环境的依赖性，对大多数MGUS和MM肿瘤的生存和生长都很重要。MGUS和MM肿瘤对活化的NFkB通路的可能成瘾表明这些肿瘤可能对制药公司正在开发的各种NFkB通路抑制剂敏感。HMCL和增殖性MM肿瘤中RB通路的失调。细胞周期蛋白D基因的失调/过度表达是几乎所有MGUS和MM肿瘤中的统一事件。此外，我们还发现在大多数HMCL和MM肿瘤中p16 INK 4A表达缺失或极低，尽管p16基因在>90%的HMCL中甲基化，但在MGUS和MM肿瘤中仅20-30%甲基化;这表明缺乏p16表达不需要甲基化。尽管存在细胞周期蛋白D基因的失调和p16的低表达或不表达，但大多数MM肿瘤具有极低的增殖指数。我们已经确定，p18 INK 4C的表达，这似乎是一个关键的调节器的G1>S转换在正常的浆细胞，提供了一个重要的标志物的增殖。在约30%的HMCL和近10%的增殖性MM肿瘤中，p18的双等位基因缺失似乎是与增殖增加相关的关键事件。外源性p18在表达很少或不表达p18的HMCL中的表达由于G1>S转换的阻断而显著降低HMCL中的增殖。然而，奇怪的是，与正常浆细胞相比，约60%的高度增殖的HMCL和MM肿瘤具有显著增加的p18水平，这表明这些肿瘤细胞对由于增殖增加而发生的p18水平增加不敏感。事实上，p18 RNA表达的增加与患者存活率的降低有关。大约15%的HMCL和增殖性肿瘤表达高水平的p18，使RB 1失活，这可以解释它们对高水平p18的不敏感性。因此，我们得出结论，MM肿瘤进展中增殖的增加至少在某些情况下可以通过RB通路失调的连续步骤（p18或RB 1的失活）来解释。我们正在继续努力，以确定当p18水平高时导致增殖增加的其他机制。

We continue to identify and characterize oncogenic events and their consequences in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Progress during the past year includes the following: Activation of NF-kappaB (NFkB) pathway by mutations in MM tumors and cell lines. We initially found that two human MM cell lines (HMCL) have constitutively activated the NFkB pathway by over-expression of NFkB-inducing kinase (NIK) due to either a translocation or gene amplification. An inhibitor of IkB kinase beta (IKK2) caused cessation of cell division or death in approximately 50% of HMCL, including the two HMCL that over-express NIK. The effect of this inhibitor on several HMCL was used to generate an 11 gene molecular signature of NFkB activity. Using this NFkB index to analyze data from gene expression arrays, we found that 50% of HMCL, 82% of MM tumors, 95% of MGUS tumors, and all normal bone marrow plasma cells have substantial activation of the NFkB pathway. Focusing on NIK and other genes that regulate the NFkB pathway for mutations, we found that the expression of two positive NFkB regulators NIK and CD40 is elevated in some MM or HMCL samples, while four negative regulators TRAF3, CYLD, BIRC2, and BIRC3 are absent or inactivated in other samples. In addition, over-expression of the NFKB1 gene or truncated forms of the NFKB2 gene were found to activate the NFkB pathway in some HMCL. In all cases, these mutations were found in tumors or HMCL that have a high NFkB index. The prevalence of mutations is estimated to be 15-20% in untreated MM tumors, and >35% in HMCL, suggesting that the mutations are likely to occur during tumor progression. Most mutations in HMCL activate both the classical and alternative NFkB pathways, but our data suggest that the classical pathway activation may be most critical in view of results with the IKK2 inhibitor that selectively targets the classical pathway. Others had reported that activation of the NFkB pathway by extrinsic signals from normal bone marrow stromal cells is critical for survival of normal mouse plasma cells. Therefore, our results suggest that activation of the NFkB pathway - either by extrinsic signals or intrinsic mutations that presumably decrease the dependence of the tumor on the bone marrow microenvironment is important for the survival and growth of most MGUS and MM tumors. The possible addiction of MGUS and MM tumors to an activated NFkB pathway suggests that these tumors may be susceptible to a variety of NFkB pathway inhibitors that are being developed by pharmaceutical companies. Dysregulation of the RB pathway in HMCL and proliferative MM tumors. The dysregulation/over-expression of a CYCLIN D gene is a unifying event in virtually all MGUS and MM tumors. In addition, we have found that p16INK4A expression is absent or extremely low in most HMCL and MM tumors, even though the p16 gene is methylated in >90% of HMCL but only 20-30% of MGUS and MM tumors; this indicates that the lack of p16 expression does not require methylation. Despite the dysregulation of a CYCLIN D gene and low or absent expression of p16, most MM tumors have an extremely low proliferation index. We have determined that the expression p18INK4C, which seems to be a key regulator of the G1>S transition in normal plasma cells, provides an important marker of proliferation. In approximately 30% of HMCL and nearly 10% of proliferative MM tumors, the bi-allelic deletion of p18 appears to be a critical event that is associated with increased proliferation. Expression of exogenous p18 in HMCL that express little or no p18 markedly decreases proliferation in HMCL due to a block in the G1>S transition. Paradoxically, however, about 60% of both HMCL and MM tumors that are hightly proliferative have substantially increased levels of p18 compared to normal plasma cells, suggesting that these tumor cells are insensitive to the increased levels of p18 that occur as a consequence of increased proliferation. In fact increased expression of p18 RNA is associated with decreased patient survival. About 15% of HMCL and proliferative tumors that express high levels of p18 have inactivated RB1, which would explain their insensitivity to high p18 levels. Therefore, we conclude that an increase in proliferation with progression of MM tumors is explained in at least some cases by sequential steps (inactivation of p18 or RB1) that dysregulate the RB pathway. We are continuing efforts to identify additional mechanisms responsible for increased proliferation when there are high p18 levels