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.
Read-through compounds (RTCs) are capable of promoting ribosomal read-through of premature termination codons (PTCs), resulting in generation of full-length protein product. ASXL1-PTCs are frequent in myeloid malignancies and Bohring-Opitz syndrome. The major goal of this application is to establish the therapeutic potential of RTCs in ASXL1-PTC diseases using the combination of our newly developed innovative ASXL1- PTC mouse model and primary patient specimens with ASXL1-PTCs.