Direct coregulation of Notch1 by Zmiz1 in T-cell leukemia

 描述（由申请人提供）：在大多数人类T-ALL病例中发现NOTCH 1作为驱动突变，这为这种癌症的靶向治疗带来了希望。NOTCH也被认为是多种实体癌和血液癌中的重要致癌基因。然而，完全阻断Notch途径是不切实际的，因为无法忍受的靶向副作用。也有人担心这会导致癌症。因此，临床试验被迫使用较低剂量的Notch抑制剂。然而，这些剂量效果不佳，因为协作网络放大了微弱的Notch信号并驱动了抗性。我们的长期目标是识别和了解与Notch合作的网络。我们的初步数据表明，皮亚斯共激活剂Zmiz 1是Notch 1的一种新的直接和选择性调节剂。ZMIZ 1和活化的NOTCH 1在原代人T-ALL标本的重要子集中共表达。Zmiz 1抑制减缓增殖并克服对Notch抑制剂的抗性。在这里，我们展示了Zmiz 1和Notch 1在Notch 1调控位点的一个子集上的共结合; Zmiz 1对Notch 1靶基因的一个子集的调控;以及一个关键的结构调控。 Zmiz 1与Notch 1直接相连的结构域。与Notch缺陷类似，Zmiz 1缺陷导致所有T细胞亚群的细胞自主损失。与Notch缺陷相反，Zmiz 1缺陷不会引起骨髓增生性疾病或严重的GI毒性。重要的是，删除 Zmiz 1在已建立的T-ALL中导致肿瘤消退和延长生存期。本申请的目的是阐明Zmiz 1增强Notch 1激活的T-ALL的机制。我们的中心假设是，Zmiz 1通过直接与Notch 1相互作用，与其他因素一起驱动T-ALL，从而调节Notch 1信号的致癌子集。在我们的第一个目标中，我们将确定Zmiz 1和Notch 1选择性共结合和共调节Notch靶基因的致癌亚组的机制。我们将绘制氨基酸水平上的相互作用。我们将确定在基因调控中控制Zmiz 1选择性的因素。在我们的第二 我们将使用我们新产生的Zmiz 1基因敲除小鼠来确定Zmiz 1对白血病起始细胞的影响。我们将了解Zmiz 1抑制是否可以有效和安全地与可耐受剂量的Notch抑制剂相结合。我们的项目意义重大，因为它将阐明一种新的直接和选择性Notch调节因子，该调节因子是T-ALL维持所需的，并有助于GSI抗性，但对肠道稳态和骨髓肿瘤抑制不利。因此，它可以导致Notch定向的癌症治疗，而没有泛Notch抑制的不可接受的后果。我们的初步分析表明，Zmiz 1在多种癌症中表达并具有功能活性。因此，我们的工作可能具有广泛的影响。我们的项目是创新的，因为据我们所知，这是第一个转录调节因子直接参与核心Notch复合物以异质性调节Notch靶基因的实例。我们采用了新的基因敲除小鼠和技术，提高了T-ALL领域人类样本验证的标准，以确保我们的项目具有最大的临床转化潜力。

 DESCRIPTION (provided by applicant): The discovery of NOTCH1 as a driver mutation in the majority of human T-ALL cases has raised hopes for targeted therapy in this cancer. NOTCH has also been implicated as an important oncogene in a diverse range of solid and hematologic cancers. However, complete blockade of the Notch pathway is impractical because of intolerable on-target side effects. There is also concern that this would cause cancer. Thus, clinical trials are forced to use lower doses of Notch inhibitors. However, these doses are poorly effective because collaborative networks amplify weak Notch signals and drive resistance. Our long-term goal is to identify and understand the networks that collaborate with Notch. Our preliminary data implicate the PIAS coactivator Zmiz1 as a new direct and selective regulator of Notch1. ZMIZ1 and activated NOTCH1 were co-expressed in a significant subset of primary human T-ALL specimens. Zmiz1 inhibition slowed proliferation and overcame resistance to Notch inhibitors. Here we show co-binding of Zmiz1 and Notch1 at a subset of Notch1 regulatory sites; regulation by Zmiz1 of a subset of Notch1 target genes; and a critical structural domain that attaches Zmiz1 directly to Notch1. Similar to Notch deficiency, Zmiz1 deficiency caused a cell autonomous loss of all T-cell subsets. In contrast to Notch deficiency, Zmiz1 deficiency did not cause myeloproliferative disease or severe GI toxicity. Importantly, deletion of Zmiz1 in established T-ALL led to tumor regression and prolonged survival. Our objective in this application is to elucidate the mechanism by which Zmiz1 enhances Notch1-activated T- ALL. Our central hypothesis is that Zmiz1 regulates an oncogenic subset of Notch1 signals by directly interacting with Notch1 in concert with other factors to drive T-ALL. In our first aim, we will determine the mechanisms by which Zmiz1 and Notch1 selectively co-bind and coregulate an oncogenic subset of Notch target genes. We will map the interactions at the amino acid level. We will identify the factors controlling the selectivity of Zmiz1 in gene regulation. In our second aim, we will use our newly generated Zmiz1 knockout mice to determine the effects of Zmiz1 on leukemia initiating cells. We will learn whether Zmiz1 inhibition could be effectively and safely combined with tolerable doses of Notch inhibitors. Our project is significant because it will elucidate a new direct and selective Notch regulator that is required for T-ALL maintenance and contributes to GSI resistance, but is dispensable for intestinal homeostasis and myeloid tumor suppression. Thus, it may lead to Notch-directed cancer therapy without the unacceptable consequences of pan-Notch inhibition. Our preliminary analysis suggests that Zmiz1 is expressed and functionally active in diverse cancers. Thus, our work may have broad implications. Our project is innovative because it is the first instance, to our knowledge, that a transcriptional regulator directly engages the core Notch complex to heterogeneously regulate Notch target genes. We employ new knockout mice and technologies that raise the standards of human sample validation in the T-ALL field to ensure that our project has the highest potential for clinical translation.