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

摘要 在T细胞急性淋巴细胞白血病（T-ALL）患者中发现NOTCH 1突变带来了希望 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， 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。以来 Notch活性需要辅因子在其增强子上产生有利的细胞类型特异性“染色质环境”，我们 设想靶向这些辅因子可能避免pan-Notch抑制的不可忍受的作用。所以我们 长期目标是了解Notch辅因子的T细胞生物学。例如，我们发现， PIAS样辅激活因子Zmiz 1是Notch 1的直接辅因子，其选择性地促进T细胞上的Notch活性。 特异性Myc增强子。Zmiz 1撤除或破坏Zmiz 1-Notch 1相互作用损害Myc依赖性 前T细胞和白血病母细胞的增殖。重要的是，Zmiz 1的普遍失活并不具有主要的 对非T细胞组织的作用，表明比Notch抑制更具有T细胞特异性作用。对于这一建议，我们 观察到Zmiz 1在前T细胞中的表达从稳态水平急剧增加， 胸腺再生因此，Zmiz 1缺陷型前T细胞缺陷在照射后放大4倍 与稳态相比。因此，Zmiz 1被募集以在细胞减少后紧急恢复T细胞免疫。的 Zmiz 1通路也可能具有治疗效用，因为Zmiz 1的超生理激活扩展了pre-T 细胞和原始早期胸腺祖细胞（ETP）。当Zmiz 1抑制Notch时，ETP扩增- 诱导的T细胞分化信号，从而保护ETP免于过度分化。这里我们的 目的是了解Zmiz 1的这些新的阶段特异性作用。我们的假设是激活Zmiz 1 途径诱导促进前T细胞增殖和ETP的阶段特异性转录程序 上维护为了验证这一点，我们将确定在胸腺再生过程中Zmiz 1是如何被诱导的，以及Zmiz 1是如何被诱导的。 通过重塑染色质促进转录因子的活性。我们还将确定Zmiz 1 操纵辅因子和靶基因以促进未分化的ETP增殖。最后，我们将提高 Zmiz 1信号超生理水平，以提高胸腺恢复体内。由于T细胞延长而感染 各种癌症治疗后的缺乏是一个主要的临床问题。Pan-Notch激活作为一种策略， 再生T细胞系是有问题的超生理学Notch激活通过促进ETP消耗 过度的T细胞定型相比之下，Zmiz 1保护ETP细胞，促进增殖， 不会诱发白血病。因此，我们的项目是重要的，因为它将阐明一个直接的Notch 1辅因子， 驱动白血病，并在增强早期T细胞增殖，同时抑制 分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 是创新的，因为它调查了Notch辅因子调节胸腺群体的第一例 动力学的方式，将促进平衡的Notch诱导的胸腺再生。

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.