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翻译的调控。该蛋白的核定位与乳腺癌和非小细胞肺癌患者的预后有关。本项目的基本假设是YB-1在多发性骨髓瘤（MM）的早期肿瘤发展和恶性生长维持中发挥关键作用。在研究这个问题时，我们采用了几种不同的小鼠模型和肿瘤细胞系，每一种都具有浆细胞（PC）肿瘤的不同方面的特征，从非恶性的PC到去分化的PC肿瘤。通过对疾病谱系两端的研究，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F.YB-1敲入模型，其中YB-1转基因位于一个晚期B细胞启动子下。在头一年半的时间里，这些动物还没有表现出任何PC肿瘤的发展。然而，在骨髓和肠绒毛中发现与野生型仔鼠相比，正常形态的pc数量较多。因此，单独的YB-1不负责疾病的发病机制；因此，恶性PC转化需要其他癌基因。基于这些新发现，我们假设YB-1与MYC和/或其他与YB-1无关的解除调控信号通路（NOTCH1和CCND1/2）的合作是必需的。为了解决这些问题，我们将在野生型和转基因F.YB-1动物脾细胞中过表达特定蛋白，并分析重建后的肿瘤发展情况。我们已经证明YB-1在MM中过表达，并有助于肿瘤细胞的增殖、存活和体外耐药。最近，我们已经证明髓外PC肿瘤（人和小鼠）中MYC表达与YB-1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MM细胞系（hmcs）中，MYC蛋白的表达依赖于YB-1介导的mRNA翻译，而MYC参与YB-1基因的转录。值得注意的是，敲低这两种蛋白中的任何一种都会诱导hmcs细胞凋亡，这表明这种转录/翻译回路对去分化的侵袭性生长MM细胞的存活至关重要。这些数据已经扩展到包括YB-1功能的体内分析。采用小鼠MM模型（采用MOPC315.BM）。在体内，部分敲低YB-1可延缓肿瘤生长，提示YB-1在肿瘤维持中起关键作用。这些实验得出了一个假设，即依赖yb -1的翻译对于体外和体内去分化的侵袭性生长的MM细胞的存活至关重要。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YB-1表达来确定该蛋白是否对积极生长的去分化肿瘤细胞的存活至关重要。此外，我们将使用Click-SILAC技术鉴定可能介导其促肿瘤作用的yb -1依赖性翻译调节mrna /蛋白。然后将对合适的候选者进行功能表征，以使用小shRNA在体内筛选来测试它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在骨髓瘤形成中的作用，并且它们也有望确定关键的YB-1依赖的生存介质，这些介质可以作为迄今无法治疗的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.