Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development

摘要 T细胞急性淋巴细胞白血病(T-ALL)患者NOTCH1基因突变的发现带来了希望 用于治疗这种癌症的泛诺奇抑制剂。不幸的是，在临床试验中，这些药物毒性太大，因为 凹槽具有基本的正常功能。因此，挑战是选择性地靶向T-ALL细胞中的Notch。自.以来 缺口活性需要在其增强剂上的辅因子来创造有利的细胞类型特定的“染色质环境”，我们 设想以这些辅因子为靶点可能会避免潘-切迹抑制的不可耐受的影响。因此，我们的 长期目标是了解Notch辅助因子的T细胞生物学。例如，我们发现 PIAS样辅活化子Zmiz1是Notch1的直接辅因子，选择性地促进T细胞上的Notch活性 特定的Myc增强子。Zmiz1退出或中断Zmiz1-Notch1相互作用损害Myc依赖 前T细胞和白血病母细胞的增殖。重要的是，Zmiz1的无处不在的失活没有重大的 对非T细胞组织的影响，提示比Notch抑制作用更具T细胞特异性。对于这项建议，我们 观察到Zmiz1在前T细胞中的表达从稳态水平显著增加 胸腺再生。相应地，Zmiz1缺陷的Pre-T细胞缺陷在照射后放大4倍 与稳定状态相比。因此，Zmiz1被招募来紧急恢复细胞减少后的T细胞免疫。这个 Zmiz1途径也可能作为Zmiz1扩展的Pre-T的超生理学激活而具有治疗作用 细胞和原始早期胸腺祖细胞(ETP)的体外培养。随着Zmiz1抑制Notch-ETP被扩展 诱导T细胞分化信号，从而保护ETP免受过度分化。在这里，我们的 目的是了解Zmiz1的这些新的阶段特异性作用。我们的假设是激活Zmiz1 途径诱导阶段特异性转录程序，促进前T细胞增殖和ETP 维修。为了测试这一点，我们将确定Zmiz1是如何在胸腺再生过程中被诱导的，以及Zmiz1是如何 通过重塑染色质来促进转录因子的活性。我们还将确定Zmiz1如何 操纵协因子和靶基因以促进未分化的ETP增殖。最后，我们将提高 Zmiz1向超生理水平发出信号，以促进体内胸腺的恢复。T细胞延长引起的感染 各种癌症治疗后的不足是一个主要的临床问题。将平移凹槽激活作为一种策略 再生T血统是有问题的。生理上的缺口激活通过促进ETP耗尽 过度的T细胞承诺。相反，Zmiz1保留ETP细胞，促进增殖，并且本身 不能诱发白血病。因此，我们的项目意义重大，因为它将阐明一个直接的Notch1辅因子 导致白血病，并在促进早期T细胞增殖同时抑制T细胞增殖方面发挥重要的阶段特异性作用 差异化。我们将学习抗击白血病和促进胸腺再生的新策略。我们的项目 具有创新性，因为它首次研究了Notch辅因子调节胸腺种群的情况 以促进平衡Notch诱导的胸腺再生的方式进行动力学研究。

ABSTRACT The discovery of NOTCH1 mutations in T-cell Acute Lymphoblastic Leukemia (T-ALL) patients raised hopes for pan-Notch inhibitors to treat this cancer. Unfortunately, in clinical trials, these drugs were too toxic because Notch has essential normal functions. Thus, the challenge is to selectively target Notch in T-ALL cells. Since Notch activity requires cofactors at its enhancers to create favorable cell type-specific “chromatin contexts”, we envision that targeting these cofactors might avoid the intolerable effects of pan-Notch inhibition. Thus, our long-term goal is to understand the T-cell biology of Notch cofactors. For example, we discovered that the PIAS-like coactivator Zmiz1 is a direct cofactor of Notch1 that selectively promotes Notch activity at the T-cell specific Myc enhancer. Zmiz1 withdrawal or disrupting the Zmiz1-Notch1 interaction impaired Myc-dependent proliferation of pre-T cells and leukemic blasts. Importantly, ubiquitous inactivation of Zmiz1 did not have major effects on non-T cell tissues, suggesting more T-cell specific effects than Notch inhibition. For this proposal, we observed that the expression of Zmiz1 in pre-T cells dramatically increases from steady state levels during thymic regeneration. Accordingly, the Zmiz1-deficient pre-T cell defect is magnified 4-fold after irradiation compared to steady state. Thus, Zmiz1 is recruited to urgently restore T-cell immunity after cytoreduction. The Zmiz1 pathway might also have therapeutic utility as supraphysiological activation of Zmiz1 expanded pre-T cells and primitive early thymic progenitors (ETPs) in vitro. ETPs were expanded as Zmiz1 restrains Notch- induced T-cell differentiation signals, thereby protecting ETPs from excessive differentiation. Here, our objective is to understand these novel stage-specific roles of Zmiz1. Our hypothesis is that activating the Zmiz1 pathway induces stage-specific transcriptional programs that promote pre-T cell proliferation and ETP maintenance. To test this, we will determine how Zmiz1 is induced during thymic regeneration and how Zmiz1 facilitates transcription factor activities by remodeling chromatin. We will also determine how Zmiz1 manipulates cofactors and target genes to promote undifferentiated ETP proliferation. Finally, we will raise Zmiz1 signals to supraphysiological levels to enhance thymic recovery in vivo. Infection due to prolonged T-cell deficiency after various cancer therapies is a major clinical problem. Pan-Notch activation as a strategy to regenerate the T-lineage is problematic. Supraphysiological Notch activation depletes ETPs by promoting excessive T-cell commitment. In contrast, Zmiz1 preserves ETP cells, promotes proliferation, and by itself cannot induce leukemia. Thus, our project is significant because it will elucidate a direct Notch1 cofactor that drives leukemia and plays important stage-specific roles in enhancing early T-cell proliferation while restraining differentiation. We will learn new strategies to combat leukemia and promote thymic regeneration. Our project is innovative because it investigates the first instance that a Notch cofactor regulates thymic population dynamics in a manner that would promote balanced Notch-induced thymic regeneration.