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

项目总结 大约60%的T细胞急性淋巴细胞白血病(T-ALL)患者有激活的NOTCH1突变，使 NOTCH1是这种癌症中最常见的癌基因。在早期的临床试验中，PAN-Notch抑制剂，如伽马- 分泌酶抑制剂(GSI)引起过度毒性，因为Notch在组织中具有必要的功能 动态平衡。为了规避这种毒性，我的实验室和其他人的工作支持另一种定向策略 Notch激活其增强子所需的上下文依赖的转录辅因子。从理论上讲， 抑制特定的Notch辅助因子可能比抑制所有Notch功能的毒性小。细胞分裂周期 73(CDC73)是聚合酶相关因子复合体(PAF1c)的骨架成分。就像有变种人的苍蝇 Notch，携带突变PAF1c的果蝇显示出缺口翅膀和Notch靶基因表达受损，这表明 PAF1c与Notch通路相连。我的初步数据表明，CDC73与Notch1相互作用 T细胞特异性Notch依赖的MYC增强子的复合体和促进Notch诱导的白血病的发生 活着。MYC是T-ALL中一个关键的Notch诱导癌基因。PAF1c的典型功能是激活mRNA 通过将组蛋白修饰酶和转录机制招募到一个小的子集在启动子进行合成 转录起始点。人们对它在增强剂中的非正则功能知之甚少。我的建议是审查 CDC73在人原代T-ALL细胞增强子激活和增殖中的作用我假设CDC73 促进增强子的激活，驱动T-T细胞中依赖Notch的致癌基因表达程序 全。因此，CDC73的缺失将通过灭活已知的驱动增强剂来抑制白血病细胞的增殖。 全部都是。我的影响是通过靶向CDC73辅因子来将T-ALL领域从泛诺奇抑制剂转移到 破坏致癌的Notch转录复合体。我的第一个目标是确定CDC73在T-ALL中的作用 在Notch激活的原代细胞中检测CDC73停用对白血病的抑制作用 人类患者样本和临床相关的Notch激活的T-ALL转基因小鼠模型。我的第二个 目的是通过量化方法确定CDC73促进Notch1靶基因转录的机制 CDC73缺失通过蛋白质组学和蛋白质组学途径对mRNA/Erna转录和增强子激活的影响 转录基因组学方法。我希望展示增强子结合的CDC73/PAF1c激活一个子集 富含功能性eRNA的增强子，从而促进未来识别支持T-ALL的eRNA。我 也希望将CDC73确定为Notch驱动的白血病细胞的一个新的脆弱性，它将Notch与 转录机制和激活增强子的染色质修饰酶。成功完成 这个项目将证明作为辅因子依赖的反式激活因子的Notch的果蝇模型 可应用于开发人类抗癌策略。我还将在第一个实例中展示Paf1C和 重要的表观遗传调节因子，已经被很好地建立来激活mRNA转录，可能参与 促进增强子RNA转录和增强子激活的非正则位点的正则因子。

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.