Functional characterization of the CRBN E3 ubiquitin ligase, the target of lenalidomide

免疫调节药物来那度胺（IMiD）在伴有del（5q）的多发性骨髓瘤和骨髓增生异常综合征（MDS）中高度活跃。2014年，我们发现来那度胺调节小脑（CRBN）的底物特异性，而小脑是CRBNCRL4 E3泛素连接酶的底物接头。来那度胺及其类似物沙利度胺和泊马度胺均可诱导泛素化和淋巴转录因子ikaros （IKZF1）和aiolos （IKZF3）的降解，从而导致多发性骨髓瘤细胞生长停滞并诱导T细胞释放白细胞介素-2。在Emmy noetheri - program的第一个资助期，我们可以阐明来那度胺在del(5q) MDS中的作用机制：来那度胺，但其他IMiDs不能通过CRBN-CRL4诱导酪蛋白激酶1a （CK1a）的降解。del(5q) MDS细胞中CK1a的单倍性不足为来那度胺诱导的CK1a降解导致TP53激活和细胞凋亡打开了治疗窗口。我们进一步发现，CRBN中的非保守氨基酸是小鼠对imid不敏感的原因。人类CRBN的表达克服了小鼠细胞的这种天然抗性。在下一个资助期，我们的目标是建立新的肿瘤小鼠模型，这将允许首次在体内评估IMiDs和联合治疗的直接和间接影响。我们进一步生成并验证了基于imid的靶向嵌合体（PROTAC）作为CRBN的首个化学抑制剂。我们将利用该化合物进一步研究CRBN在不同模型系统中的作用和IMiDs的影响。crbn结合蛋白FAM46C在多发性骨髓瘤中反复突变，在多发性骨髓瘤中被认为是影响来那度胺敏感性的肿瘤抑制因子。在正在进行的研究中，我们的目标是确定FAM46C突变在多发性骨髓瘤中的特异性作用，并分析FAM46C失活在体内的影响。在第三个目的中，IKZF1 RNA表达水平被确定为来那度胺治疗患者的预后标志物。治疗前和治疗进展中获得的原发性多发性骨髓瘤样本的外显子组和RNA测序显示，来那度胺耐药细胞中的基因突变和异常表达基因具有高度异质性。在第二个资助期，我们将通过定量蛋白质组学来补充我们的DNA和RNA分析，并将使用基于crispr - cas9的混合筛选对细胞系中的发现进行功能分析。这些项目的结果将加深我们对CRBN-CRL4 E3连接酶和IMiDs分子机制的理解，可能转化为更好的多发性骨髓瘤和MDS患者的治疗方法。鉴于改变泛素连接酶以靶向其他蛋白质（如基于imid的PROTACS）的药物的最新发展，我们的发现对癌症治疗和更广泛的医学治疗具有重要意义。

The immunomodulatory drug (IMiD) lenalidomide is highly active in multiple myeloma and myelodysplastic syndrome (MDS) with del(5q). In 2014, we found that lenalidomide modulates the substrate specificity of cereblon (CRBN) which is the substrate adaptor of the CRBNCRL4 E3 ubiquitin ligase. Lenalidomide and its analogs thalidomide and pomalidomide all induce ubiquitination and degradation of the lymphoid transcription factors ikaros (IKZF1) and aiolos (IKZF3) what results in growth arrest of mutlilple myeloma cells and induces interleukin-2 release in T cells. In the first funding period of the Emmy Noether-Program we could elucidate the mechanism of lenalidomide in del(5q) MDS: Lenalidomide, but not the other IMiDs induce degradation of casein kinase 1a (CK1a) via CRBN-CRL4. Haploinsufficiency of CK1a in del(5q) MDS cells opens a therapeutic window for lenalidomide-induced degradation of CK1a resulting in TP53 activation and apoptosis. We further found that non-conserved amino acids in CRBN are responsible for the IMiD-insensitivity of mice. Expression of human CRBN overcomes this natural resistance of mouse cells. In the next funding period we aim to establish new tumor mouse models that will allow for the first time to assess the direct and indirect effects of IMiDs and combination treatments in vivo. We further generated and validated an IMiD-based homodimeric proteolysis targeting chimera (PROTAC) as first chemical inhibitor of CRBN. We will use this compound to further study the role of CRBN and effects of IMiDs in different model systems.The CRBN-binding protein FAM46C, which is recurrently mutated in multiple myeloma, was characterized as a tumor-suppressor in multiple myeloma that is affecting lenalidomide sensitivity. In ongoing studies we aim to identify the multiple myeloma-specific role of FAM46C mutations and to analyze the effects of FAM46C inactivation in vivo.In the third aim IKZF1 RNA expression levels were identified as prognostic marker in lenalidomide-treated patients. Exome and RNA sequencing of primary multiple myeloma samples obtained pre-treatment and at progression revealed a high heterogeneity of gene mutations and aberrantly expressed genes in lenalidomide-resistant cells. In the second funding period we will complement our DNA and RNA analyses by quantitative proteomics and will functionally analyze the findings in cell lines using pooled CRISPR-Cas9-based screens.The results of these projects will deepen our understanding of CRBN-CRL4 E3 ligase and the molecular mechanism of IMiDs what may translate into better treatments for multiple myeloma and MDS patients. Given the recent developments of drugs that alter ubiquitin ligases to target other proteins like IMiD-based PROTACS, our findings have important implications for cancer therapy and medical therapeutics more broadly.