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) 患者中 NOTCH1 突变的发现给人们带来了希望 寻找泛Notch抑制剂来治疗这种癌症。不幸的是，在临床试验中，这些药物毒性太大，因为 Notch 具有基本的正常功能。因此，挑战在于选择性地靶向 T-ALL 细胞中的 Notch。自从 Notch 活性需要其增强子上的辅因子来创建有利的细胞类型特异性“染色质环境”，我们 设想针对这些辅助因子可能会避免泛Notch抑制带来的无法忍受的影响。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 样辅激活因子 Zmiz1 是 Notch1 的直接辅因子，可选择性促进 T 细胞的 Notch 活性 特异性 Myc 增强子。 Zmiz1 撤回或破坏 Zmiz1-Notch1 相互作用受损 Myc 依赖性 前 T 细胞和白血病母细胞的增殖。重要的是，Zmiz1 的普遍失活并没有造成重大影响。 对非 T 细胞组织的影响，表明 T 细胞特异性作用比 Notch 抑制更多。对于这个提案，我们 观察到 Zmiz1 在前 T 细胞中的表达从稳态水平急剧增加 胸腺再生。因此，Zmiz1缺陷的前T细胞缺陷在辐射后放大了4倍 与稳态相比。因此，Zmiz1 被招募来在细胞减灭后紧急恢复 T 细胞免疫。的 Zmiz1 通路也可能具有治疗效用，因为 Zmiz1 的超生理激活在 T 前扩展 细胞和原始早期胸腺祖细胞（ETP）在体外。由于 Zmiz1 抑制 Notch，ETP 得到扩展- 诱导T细胞分化信号，从而保护ETP免于过度分化。在这里，我们的 目标是了解 Zmiz1 的这些新颖的特定于阶段的角色。我们的假设是激活 Zmiz1 途径诱导促进前 T 细胞增殖和 ETP 的阶段特异性转录程序 维护。为了测试这一点，我们将确定 Zmiz1 在胸腺再生过程中如何被诱导以及 Zmiz1 如何 通过重塑染色质促进转录因子活性。我们还将确定 Zmiz1 如何 操纵辅助因子和靶基因以促进未分化的 ETP 增殖。最后，我们将提出 Zmiz1 向超生理水平发出信号，以增强体内胸腺恢复。 T细胞延长引起的感染 各种癌症治疗后的缺乏是一个主要的临床问题。泛缺口激活作为一种策略 再生 T 谱系是有问题的。超生理Notch激活通过促进来耗尽ETP T 细胞过度投入。相比之下，Zmiz1 可以保护 ETP 细胞，促进增殖，并且通过其自身 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， 驱动白血病，并在增强早期 T 细胞增殖同时抑制 差异化。我们将学习对抗白血病和促进胸腺再生的新策略。我们的项目 具有创新性，因为它研究了 Notch 辅助因子调节胸腺群体的第一个实例 以促进平衡的Notch诱导的胸腺再生的方式进行动力学。

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.