In vivo models for the functional analysis of YB-1 in multiple myeloma

YB-1是一种DNA/RNA结合蛋白，参与DNA转录和mRNA翻译的调节。在乳腺癌和非小细胞肺癌中，该蛋白的核定位与患者的预后相关。该项目的基本假设是YB-1在多发性骨髓瘤(MM)的早期肿瘤发展以及维持恶性生长方面发挥关键作用。在研究这个问题时，我们建立了几种不同的小鼠模型和肿瘤细胞系，每一种模型都描述了浆细胞(PC)瘤的不同方面，从非恶性PC到去分化的PC肿瘤。通过从疾病谱的两端进行工作，我们的目标是确定这种痛苦和致命疾病的治疗靶点。为了研究YB-1在PC恶性转化中的作用，我们使用了F.YB-1敲入模型，其中YB-1转基因是在晚期B细胞启动子的作用下进行的。在最初的一年半内，这些动物还没有表现出任何PC肿瘤的发展。然而，与野生型相比，在骨髓和肠道绒毛中发现了过多的具有正常形态的PC。因此，YB-1单独并不是该病的致病原因；因此，其他癌基因也是恶性PC转化所必需的。基于这些新的发现，我们假设YB-1与MYC和/或其他YB-1非调控非调控信号通路(NOTCH1和CCND1/2)的合作是必要的。为了解决这些问题，我们将在来自F.YB-1动物的野生型和转基因脾细胞中过表达特定的蛋白，并分析重建后的肿瘤发展。我们已经证明YB-1在多发性骨髓瘤中过表达，并且在体外有助于肿瘤细胞的增殖、存活和耐药。最近，我们已经证明在髓外PC肿瘤(人和小鼠)中MYC的表达与YB-1的表达相关，并且YB-1在恶性PC中作为mRNA翻译的关键调节因子发挥作用。在人多发性骨髓瘤细胞系中，MYC蛋白的表达依赖于YB-1介导的mRNA翻译，而MYC则参与YB-1基因的转录。值得注意的是，这两种蛋白中的任何一种都可以诱导HMCL细胞凋亡，这表明这种转录/翻译电路对去分化侵袭性生长的MM细胞的生存至关重要。自那以后，这些数据已经扩大到包括对YB-1功能的活体分析。利用多发性骨髓瘤小鼠模型(使用MOPC315.BM.luc细胞系)，YB-1的部分敲除可以延缓体内肿瘤的生长，这表明YB-1在肿瘤维持中起着关键作用。这些实验得出的假设是，YB-1依赖的翻译对去分化、侵袭性生长的MM细胞在体外和体内的存活至关重要。为了验证这一假设，我们将使用基于锌指核酸酶的策略来产生YB-1缺失的MM细胞系，并使用条件性YB-1表达来确定该蛋白是否对侵袭性生长的去分化肿瘤细胞的生存至关重要。此外，我们将使用Click-SILAC技术识别YB-1依赖的翻译调控的mRNAs/蛋白质，这些mRNAs/蛋白质可能介导其促肿瘤作用。然后将对合适的候选者进行功能鉴定，以使用体内的小shRNA筛选来测试他们对肿瘤维持的贡献。我们相信，这些方法将有助于阐明YB-1在骨髓增生症中的作用，并且它们也有望识别关键的YB-1依赖的生存介质，这些介质可以作为目前尚不能用药的YB-1蛋白本身的治疗相关代理。

YB-1 is a DNA/RNA binding protein implicated in the regulation of DNA transcription and mRNA translation. Nuclear localization of the protein has been correlated to patient prognosis in breast carcinoma and non-small cell lung carcinoma. The underlying hypothesis of this project is that YB-1 plays a critical role in both early tumor development as well as maintenance of malignant growth in multiple myeloma (MM). In studying this question we have implemented several different mouse models and tumor cell lines, each of which characterizes different aspects of plasma cell (PC) neoplasia ranging from non-malignant PC to dedifferentiated PC tumors. By working from both ends of the disease spectrum we aim to identify therapeutic targets for this painful and deadly illness.To study the role of YB-1 in the malignant transformation of PCs we use the F.YB-1 knock-in model where the YB-1 transgene is under a late B cell promoter. Within the first 1½ years these animals did not yet show any PC tumor development. However, an excess of PCs with normal morphology was found in the bone marrow and gut villi relative to wild-type littermates. Hence, YB-1 alone is not responsible for the disease pathogenesis; therefore, other oncogenes are (additionally) required for malignant PC transformation. Based on these new findings we hypothesize that co-operation of YB-1 with MYC and/or other YB-1- independent deregulated signaling pathways (NOTCH1 and CCND1/2) is required. To address these questions we will overexpress specified proteins in wild-type and transgenic spleenocytes from F.YB-1 animals and analyze tumor development after reconstitution. We have shown that YB-1 is overexpressed in MM and that it contributes to tumor cell proliferation, survival and drug resistance in vitro. Most recently, we have demonstrated that MYC expression correlates with YB-1 expression in extramedullary PC tumors (human and mouse) and that YB-1 functions as a critical regulator of mRNA translation in malignant PCs. In human MM cell lines (HMCLs) MYC protein expression depends on YB-1-mediated mRNA translation while MYC is involved in YB-1 gene transcription. Notably, knockdown of either of these proteins induces apoptosis in HMCLs demonstrating that this transcriptional/- translational circuit is critical for the survival of dedifferentiated aggressively growing MM cells. These data have since been expanded to include in vivo analyses of YB-1 function. Using a murine model for MM (employing the MOPC315.BM.luc cell line), a partial knockdown of YB-1 delayed tumor growth in vivo, suggesting a critical role for YB-1 in tumor maintenance. These experiments led to the hypothesis that YB-1-dependent translation is critical for dedifferentiated aggressively growing MM cell survival both in vitro and in vivo. To test this hypothesis, we will use a zinc-finger nuclease-based strategy to produce a YB-1 null MM cell line and use conditional YB-1 expression to determine if the protein is critical for the survival of aggressively growing dedifferentiated tumor cells. Furthermore, we will identify YB-1-dependent translationally regulated mRNAs/proteins potentially mediating its tumorpromoting effects using Click-SILAC technology. Suitable candidates will then be functionally characterized to test their contribution to tumor maintenance using a small shRNA in vivo screen. We believe that together these approaches will help to clarify the role of YB-1 in myelomagenesis and that they also hold promise to identify critical YB-1-dependent mediators of survival that could serve as therapeutically relevant proxies for the as yet undruggable YB-1 protein itself.