Investigating the Role of Cdc73 in T-cell Acute Lymphoblastic Leukemia (T-ALL)

项目摘要 大约60%的T细胞急性淋巴细胞白血病（T-ALL）患者具有激活的NOTCH 1突变，使得 NOTCH 1是这种癌症中最常见的癌基因。在早期的临床试验中，泛Notch抑制剂，如gamma- 由于Notch在组织中具有重要的功能，分泌酶抑制剂（GSI）引起过度毒性 体内平衡为了避免这种毒性，我的实验室和其他人的工作支持一种替代策略， Notch激活其增强子所需的上下文依赖性转录辅因子。从理论上讲， 抑制特异性Notch辅因子可能比抑制所有Notch功能具有更小的毒性。细胞分裂周期 73（Cdc 73）是聚合酶相关因子复合物（PAF 1c）的支架组分。就像有变种人的苍蝇 Notch，携带突变PAF 1c的果蝇显示有缺口的翅膀和Notch靶基因表达受损，这表明 PAF 1c与Notch通路有关。我的初步数据表明Cdc 73与Notch 1相互作用， 复合物在T细胞特异性Notch依赖性MYC增强子上的表达，并促进Notch诱导的白血病发生。 vivo. MYC是T-ALL中一个重要的Notch诱导的癌基因。PAF 1c的典型功能是激活mRNA 通过将组蛋白修饰酶和转录机器募集到一个小的子集来在启动子处进行合成 转录起始位点。其在增强子中的非典型功能知之甚少。我的建议审查了 Cdc 73在原代人T-ALL细胞增强子激活和增殖中的作用。我推测Cdc 73 促进T细胞中驱动Notch依赖性致癌基因表达程序的增强子的激活。 所有.因此，Cdc 73的缺失将通过使已知驱动白血病细胞增殖的增强子失活来抑制白血病细胞增殖。 T-ALL。我的影响是通过靶向CDC 73辅因子， 损伤致癌Notch转录复合物。我的第一个目标是确定Cdc 73在T-ALL中的作用。 通过测量Notch激活的原发性骨髓瘤细胞中Cdc 73撤除的抗白血病作用， 人患者样品和Notch激活的T-ALL的临床相关转基因小鼠模型中。我的第二 目的是确定Cdc 73增强Notch 1靶基因转录的机制， 通过蛋白质组学和分子生物学方法研究Cdc 73缺失对mRNA/eRNA转录和增强子激活的影响， 转录基因组方法。我希望能证明增强子结合的Cdc 73/PAF 1c激活了一个亚群， 增强子富集功能性eRNA，从而促进将来鉴定支持T-ALL的eRNA。我 我还希望将Cdc 73确定为Notch驱动的白血病细胞的一个新的脆弱性，它将Notch连接到白血病细胞。 转录机制和染色质修饰酶激活增强子。成功完成 该项目将证明Notch作为辅因子依赖性反式激活因子的果蝇模型的原理 可以应用于开发人类抗癌策略。我还将展示Paf 1C的第一个实例， 一个重要的表观遗传调节因子，已经被很好地建立，以激活mRNA转录，可能参与 在非典型位点的典型因子，以促进增强子RNA转录和增强子激活。

PROJECT SUMMARY About 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients have activating NOTCH1 mutations, making NOTCH1 the most prevalent oncogene in this cancer. In early clinical trials, pan-Notch inhibitors like gamma- secretase inhibitors (GSI) caused excessive toxicity because Notch has essential functions in tissue homeostasis. To circumvent this toxicity, work by my lab and others supports an alternative strategy of targeting context-dependent transcriptional cofactors that are required for Notch to activate its enhancers. In theory, inhibiting specific Notch cofactors might have less toxicity than inhibiting all Notch functions. Cell division cycle 73 (Cdc73) is a scaffold component of the polymerase-associated factor complex (PAF1c). Like flies with mutant Notch, flies with mutant PAF1c display notched wings and impaired expression of Notch target genes, suggesting that PAF1c is linked to the Notch pathway. My preliminary data suggests that Cdc73 interacts with the Notch1 complex at the T-cell specific Notch-dependent MYC enhancer and promotes Notch-induced leukemogenesis in vivo. MYC is a critical Notch-induced oncogene in T-ALL. The canonical function of PAF1c is to activate mRNA synthesis at promoters by recruiting histone modifier enzymes and transcriptional machinery to a small subset of transcription start sites. Its non-canonical function at enhancers is poorly understood. My proposal examines the role of Cdc73 in enhancer activation and proliferation of primary human T-ALL cells. I hypothesize that Cdc73 facilitates the activation of enhancers that drive Notch-dependent oncogenic gene expression programs in T- ALL. Therefore, deletion of Cdc73 will inhibit leukemic cell proliferation by inactivating enhancers known to drive T-ALL. My impact is to shift the T-ALL field away from pan-Notch inhibitors by targeting the CDC73 cofactor to impair oncogenic Notch transcriptional complexes. My first aim is to determine the role of Cdc73 in T-ALL proliferation in vivo by measuring the anti-leukemic effects of Cdc73 withdrawal in Notch-activated primary human patient samples and in a clinically relevant transgenic mouse model of Notch-activated T-ALL. My second aim is to determine the mechanism by which Cdc73 enhances Notch1 target gene transcription by quantifying the effects of Cdc73 deletion on mRNA/eRNA transcription and enhancer activation through proteomic and transcriptional genomic approaches. I expect to show that enhancer-bound Cdc73/PAF1c activates a subset of enhancers enriched for functional eRNAs, thus facilitating future identification of eRNAs that support T-ALL. I also expect to establish Cdc73 as a new vulnerability of Notch-driven leukemic cells that connects Notch to the transcriptional machinery and chromatin modifying enzymes that activate enhancers. Successful completion of this project will show proof-of-principle that the Drosophila model of Notch as a cofactor-dependent transactivator can be applied to develop human anti-cancer strategies. I will also show the first instance that Paf1C, an important epigenetic regulator that has been well established to activate mRNA transcription, might engage canonical factors at non-canonical sites to promote enhancer RNA transcription and enhancer activation.