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

在第一个资助期，我们发现核因子κB系统在骨髓瘤细胞中远未被最大限度地激活，因此它的活性可以通过刺激肿瘤坏死因子受体或用Smac类似物治疗而被外源性增强。值得注意的是，这种外源性刺激对多种针对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的受体κ来增强其敏感性。我们还发现，在短期内(12-48小时)，使用IKK2特异性抑制剂TPCA1、NEDD8激活酶抑制剂MLN4924或IKK1和IKK2基因敲除单独或双重抑制经典和替代的NFκB通路会引起NO或相当轻微的细胞毒效应。然而，基质细胞的支持骨髓瘤的活性被NFκB信号的抑制剂强烈地降低。基于这些结果，我们现在想解决以下四个问题：首先，鉴于我们自己对两个κ受体对骨髓瘤细胞外源性凋亡的复杂调控作用的发现，以及支持肿瘤坏死因子-肿瘤坏死因子受体系统在多发性骨髓瘤中的相关性的临床证据，我们希望更详细地评估肿瘤坏死因子及其受体靶向试剂在多发性骨髓瘤中的治疗潜力。我们将与Z1项目合作分析同基因多发性骨髓瘤模型中允许选择性外源性刺激TNFR1或TNFR2并伴随和不伴随内源性肿瘤坏死因子阻断的治疗方案。大多数与NFκB相关的突变都会影响作用于NIK和IKK上游的分子。由于这些分子也在NFκB系统之外发挥作用，我们将其次讨论这种NFκB独立功能对MM表型的贡献的可能性。具体地说，我们将研究在骨髓瘤细胞中是否存在带有Wnt、Notch和STAT3信号的Nik和/或IKK1之间的串扰。第三，在多发性骨髓瘤中发现的许多与κB相关的突变影响TRAF2-κ1/2复合体的成分或诱导物，不仅抑制交替的NFκB信号转导，还刺激经典的NF Caspase B信号转导，进而抑制caspase-8激活的核小体。因此，我们将在体外和体内评估MM细胞是否特别容易受到核小体激活Smac模拟BV6的治疗效果的影响。最后，为了以公正的方式识别依赖和独立的IKK/NIK调节的靶标，将与Z4N项目合作，使用针对不同阶段的NFκB信号转导的各种抑制剂进行SILAC/MS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.