No-nonsense approach to treat myeloid malignancies with ASXL1 nonsense mutations

描述（由申请人提供）：通读化合物（RTC）能够促进提前终止密码子（PTC）的核糖体通读，导致PTC被氨基酸取代并产生全长蛋白质产物。RTC包括氨基糖苷类（如庆大霉素）和非氨基糖苷类（如阿他卢仑和RTC 13）化合物，它们对PTC具有特异性并且不干扰正常的终止密码子。这些RTC已被证明在培养物和动物模型中对CFTR和肌营养不良蛋白基因中的无义突变具有通读活性。有趣的是，在无义突变型肌营养不良症和囊性纤维化患者中进行的临床试验表明，RTC诱导了这两种疾病中一些功能性的、以前缺失的蛋白质的表达，并具有一些治疗益处。 额外性梳样1（ASXL 1）在多种形式的骨髓恶性肿瘤中以高频率突变，包括MDS、MPN、CMML和AML。新生ASXL 1突变导致Bohring-Opitz综合征。ASXL 1中的突变是无义/移码，通过截短关键的PHD结构域导致功能丧失。ASXL 1突变与预后不良相关。大约1/3的具有ASXL 1突变的骨髓恶性肿瘤病例是无义的，这是基于PTC的通读的有吸引力的靶标。在本申请中，我们将探索以下假设：RTC能够逆转ASXL 1-PTC并诱导造血干/祖细胞（HSC/HSPC）中足量的全长功能性ASXL 1表达，从而减弱ASXL 1缺陷/单倍不足介导的异常HSC/HPC功能和血液学表型。提出了三个具体目标来检验这一假设。 目标1：确定RTC（庆大霉素、阿他卢仑和RTC 13）逆转Asxl 1-PTC并在Asxl 1-PTCTg;MxCre小鼠的各种造血细胞群中产生足量全长功能性Asxl 1表达的能力。目标二：通过检查RTC治疗是否能够预防或治愈Asxl 1f/f;MxCre; Asxl 1-PTCTg小鼠中Asxl 1缺陷介导的骨髓恶性肿瘤发病机制（诱导型Asxl 1失活伴Asxl 1-PTC转基因表达），评估RTC的治疗潜力。目的3：使用来自具有ASXL 1无义突变的骨髓恶性肿瘤患者的原代细胞验证RTCs的治疗潜力。我们将确定RTC在来自具有ASXL 1无义突变的骨髓恶性肿瘤患者的各种造血细胞群体中促进不同ASXL 1-PTC（TGA、TAA、TAG）的核糖体通读的能力。我们将检查RTC治疗是否能够纠正/减弱NSG小鼠中具有ASXL 1无义突变的骨髓恶性肿瘤HSC/HPC的异常细胞功能。 这些研究的成功将使这种新的治疗策略不仅适用于ASXL 1-PTC疾病，而且适用于许多由于无义突变介导的翻译早期终止而导致的血液恶性肿瘤。

DESCRIPTION (provided by applicant): Read-through compounds (RTCs) are capable of promoting ribosomal read-through of premature termination codons (PTCs), resulting in substitution of the PTC with an amino acid and generation of full-length protein product. The RTCs include aminoglycosides (such as gentamicin) and non-aminoglycoside (such as ataluren and RTC13) compounds, which are specific for PTCs and do not interfere with normal stop codons. These RTCs have been shown to have read-through activity on nonsense mutations in the CFTR and the dystrophin genes in both cultures and animal models. Intriguingly, clinical trials in patients with nonsense mutation muscular dystrophy and cystic fibrosis showed that RTCs induced some expression of functional, previously missing, proteins in both diseases and had some therapeutic benefits. Additional sex comb-like 1 (ASXL1) is mutated at high frequencies in multiple forms of myeloid malignancies, including MDS, MPN, CMML, and AML. De novo ASXL1 mutations cause Bohring-Opitz syndrome. Mutations in ASXL1 are nonsense/frameshift, leading to loss-of-function by truncating the critical PHD domain. Mutations in ASXL1 are associated with poor prognosis. Approximately 1/3 of the myeloid malignancy cases with ASXL1 mutations are nonsense, which are appealing targets for PTC-based read- through. In this application, we will explore the hypothesis that RTCs are capable of reversing ASXL1-PTCs and induce adequate amount of full-length, functional ASXL1 expression in hematopoietic stem/progenitor cells (HSCs/HSPCs), therefore attenuating the ASXL1-deficiency/haploinsufficiency mediated abnormal HSC/HPC function and hematological phenotype. Three specific aims are proposed to test this hypothesis. Aim 1: To determine the ability of RTCs (gentamicin, ataluren and RTC13) to reverse Asxl1-PTCs and yield adequate amount of full-length, functional Asxl1 expression in various hematopoietic cell populations from Asxl1-PTCTg;MxCre mice. Aim 2: To assess the therapeutic potential of RTCs by examining if RTC treatment is capable of preventing or curing the Asxl1-deficiency mediated pathogenesis of myeloid malignancies in Asxl1f/f;MxCre;Asxl1-PTCTg mice (inducible Asxl1-inactivation with simultaneous Asxl1-PTC transgene expression). Aim 3: To validate the therapeutic potential of RTCs using primary cells from myeloid malignancy patients with ASXL1 nonsense mutations. We will determine the ability of RTCs in promoting ribosomal read- through of different ASXL1-PTCs (TGA, TAA, TAG) in various hematopoietic cell populations from myeloid malignancy patients with ASXL1 nonsense mutations. We will examine if RTC treatment is capable of correcting/attenuating the abnormal cellular function of myeloid malignancy HSC/HPCs with ASXL1 nonsense mutations in NSG mice. Success of these studies will advance this novel treatment strategy into not only ASXL1-PTC diseases, but also many hematological malignancies due to nonsense mutation-mediated early termination of translation.