Regulation, integration and impact of NFkB-signaling within the oncogenic signaling network in multiple myeloma

在第一个资助期，我们发现NFκB系统在骨髓瘤细胞中远未被最大限度地激活，因此其活性可以通过刺激TNF受体或用SMAC模拟物治疗外源性增强。值得注意的是，这种对NFκB系统的外源性刺激对多种靶向致癌途径的药物没有明显的保护作用，甚至通过上调内源性凋亡诱导剂CD95L受体CD95，增强了其对CD95L的敏感性。我们还发现，使用IKK2特异性抑制剂TPCA1、nedd8激活酶抑制剂MLN4924或IKK1和IKK2敲低对经典和替代NFκB通路的单独和双重抑制在短期（12-48小时）检测中不会引起细胞毒性作用或产生相当轻微的细胞毒性作用。然而，基质细胞的骨髓瘤支持活性被nf - κ b信号传导抑制剂强烈降低。因此，NFκB系统对骨髓瘤细胞存活的作用似乎不像最初预期的那么重要，但除了单纯的凋亡保护外，它可能与骨髓瘤细胞有至关重要的关系，并且可能进一步需要维持骨髓瘤兼容的微环境。在此基础上，我们希望解决以下四个问题：首先，鉴于我们自己对两种TNF受体TNFR1和TNFR2对骨髓瘤细胞外源性凋亡的复杂调节作用的发现，以及TNF-TNF受体系统在MM中的相关性的临床证据，我们希望更详细地评估TNF和TNF受体靶向试剂在MM中的治疗潜力。为此，我们将与Z1项目合作，分析同源MM模型中的治疗方案，这些模型允许选择性外源刺激TNFR1或TNFR2，同时或不同时阻断内源性TNF。大多数nf κ b相关突变影响NIK和IKKs上游的分子。由于这些分子也在NFκB系统之外起作用，我们将其次探讨这种与NFκB无关的功能有助于MM表型的可能性。具体来说，我们将研究在骨髓瘤细胞中，NIK和/或IKK1与Wnt、Notch和STAT3信号之间是否存在串接。其三，在MM中发现的许多NFκB相关突变影响TRAF2-cIAP1/2复合物的成分或诱诱剂，这不仅抑制了替代的NFκB信号传导，还刺激了经典的NFκB信号传导，并且抑制了caspase-8激活的核酶体。因此，我们将在体外和体内评估MM细胞是否特别容易受到激活SMAC模拟BV6的核酶体的治疗效果。最后，为了以公正的方式确定NFκB依赖性和非依赖性IKK/ nik调节靶点，将与Z4N项目合作，使用针对NFκB信号传导不同阶段的各种抑制剂进行SILAC/质谱筛选。

In the first funding period we found that the NFκB system is far from being maximally activated in myeloma cells and its activity could consequently be boosted exogenously by stimulation of TNF receptors or treatment with SMAC mimetics. Noteworthy, such exogenous stimulation of the NFκB system showed no major protective effect against various drugs targeting oncogenic pathways and even enhanced sensitivity for the endogenous apoptosis inducer CD95L by upregulation of its receptor CD95. We also found that individual as well as dual inhibition of the classical and alternative NFκB pathways using the IKK2-specifc inhibitors TPCA1, the NEDD8-activating enzyme inhibitor MLN4924 or IKK1 and IKK2 knockdown elicited no or a rather mild cytotoxic effect in short term assays (12-48 h). The myeloma-supporting activity of stromal cells, however, was strongly reduced by inhibitors of NFκB signaling. Thus, the NFκB system appears less important for myeloma cell survival as initially anticipated but could be of crucial relevance for myeloma cells beyond sole apoptosis protection and might further be required to maintain a myeloma compatible microenvironment.Based on these results, we now want to address the following four issues: First, in view of our own findings on the complex regulatory role of the two TNF receptors TNFR1 and TNFR2 on extrinsic apoptosis of myeloma cells and clinical evidence arguing for relevance of the TNF-TNF receptor system in MM, we want to evaluate in more detail the therapeutic potential of TNF and TNF receptor targeting reagents in MM. For this, we will analyze in cooperation with project Z1 treatment regimes in syngeneic MM models that allow selective exogenous stimulation of TNFR1 or TNFR2 with and without concomitant blockade of endogenous TNF. Most NFκB-related mutations affect molecules that act upstream of NIK and the IKKs. Because these molecules also act outside the NFκB system, we will secondly address the possibility that such NFκB-independent functions contribute to the MM phenotype. Specifically, we will investigate whether in myeloma cells crosstalk between NIK and/or IKK1 with Wnt, Notch and STAT3 signaling exists. Thirdly, many of the NFκB-related mutations found in MM affect components or inducers of the TRAF2-cIAP1/2 complex, which not only inhibits alternative NFκB signaling but also stimulates classical NFκB signaling and moreover inhibits the caspase-8 activating ripoptosome. Therefore, we will evaluate in vitro and in vivo whether MM cells are particularly prone to the therapeutic effects of the ripoptosome activating SMAC mimetic BV6. Finally, to identify in an unbiased fashion NFκB-dependent and -independent IKK/NIK-regulated targets, SILAC/mass spectrometry screens will be performed in co-operation with project Z4N using various inhibitors targeting distinct stages of NFκB signaling.