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）能够促进过早终止密码子（PTC）的核糖体通读，导致PTC被氨基酸取代并产生全长蛋白产物。rtc包括氨基糖苷类（如庆大霉素）和非氨基糖苷类（如ataluren和RTC13）化合物，它们是ptc特有的，不会干扰正常的停止密码子。在培养和动物模型中，这些rtc已被证明对CFTR和肌营养不良蛋白基因的无义突变具有解读活性。有趣的是，对无意义突变肌营养不良和囊性纤维化患者的临床试验表明，rtc在这两种疾病中诱导了一些功能性、先前缺失的蛋白质的表达，并具有一些治疗益处。额外的性梳状样1 （ASXL1）在多种髓系恶性肿瘤中发生高频率突变，包括MDS、MPN、CMML和AML。新生ASXL1突变导致bohling - opitz综合征。ASXL1中的突变是无意义的/移码的，通过截断关键的PHD结构域导致功能丧失。ASXL1基因突变与不良预后相关。大约1/3的ASXL1突变的髓系恶性肿瘤病例是无意义的，这是基于ptc的通读的有吸引力的目标。在本应用中，我们将探索rtc能够逆转ASXL1- ptc的假设，并在造血干细胞/祖细胞（HSC /HSPCs）中诱导足量的全长、功能性ASXL1表达，从而减弱ASXL1缺乏/单倍不足介导的异常HSC/HPC功能和血液学表型。为了验证这一假设，提出了三个具体目标。目的1：确定rtc（庆大霉素、ataluren和RTC13）逆转Asxl1- ptcs的能力，并在Asxl1- ptctg的各种造血细胞群中产生足够数量的全长、功能性Asxl1表达；MxCre老鼠。目的2：通过检查RTC治疗是否能够预防或治愈asxl1缺陷介导的髓系恶性肿瘤的发病机制，评估RTC的治疗潜力；Asxl1-PTCTg小鼠（诱导asxl1失活并同时表达Asxl1-PTC转基因）。目的3：利用ASXL1无义突变髓系恶性肿瘤患者的原代细胞验证rtc的治疗潜力。我们将确定rtc促进不同ASXL1- ptc （TGA， TAA， TAG）在来自ASXL1无义突变髓系恶性肿瘤患者的各种造血细胞群中的核糖体读透的能力。我们将研究RTC治疗是否能够纠正/减弱NSG小鼠中ASXL1无意义突变的髓系恶性HSC/HPCs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ASXL1-PTC疾病，还适用于许多由于无义突变介导的早期翻译终止而导致的血液系统恶性肿瘤。

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.