Role of Asxl1 in normal hematopoiesis and pathogenesis of myeloid malignancies

Asxl1在正常造血和骨髓恶性肿瘤发病机制中的作用

基本信息

批准号：
10543761
负责人：
Mingjiang Xu
金额：
$ 23.3万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-02 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10543761
关键词：
Acetylation Affinity BRD2 gene Behavior Binding Biological Biological Assay Biological Process Blood Bromodomain C-terminal Cells ChIP-seq Chromatin Complex DNA biosynthesis Data Development Doxycycline Drosophila genus Dysmyelopoietic Syndromes Event Exhibits Family Family member Frequencies Gene Expression Genes Genetic Transcription Goals Hematopoiesis Hematopoietic Hematopoietic stem cells Homologous Gene Human Impairment Knowledge Lysine Malignant Neoplasms Mediating Molecular Mus Mutation Myeloproliferative disease Oncogenic PHD Finger Pathogenesis Pathologic Patients Phenotype Physiological Polycomb Prognosis Protein Truncation Proteins Proteomics Regulation Regulator Genes Reporting Role Sampling Therapeutic Xenograft procedure anticancer activity cohesin epigenetic regulation gene repression genome-wide in vivo inhibitor member mouse model new therapeutic target novel prevent protein complex sex success targeted treatment therapeutic target therapeutically effective transgene expression translational impact transplant model tumorigenesis ubiquitin isopeptidase

项目摘要

Additional sex comb-like (ASXL) genes are human homologues of Drosophila-Asx gene which encode important regulators of gene expression. ASXL1 mutations occur at high frequencies in multiple forms of myeloid malignancy patients with poor prognosis. ASXL1 mutations are mostly nonsense/frameshift, causing truncation of the protein lacking the C-terminal PHD finger, which is detectable in patient samples with ASXL1 mutations. We have recently shown that transgenic expression of an ASXL1 truncation in mice (Asxl1Y588XTg) results in increased HSC/HPC pools and pathogenesis of myeloid malignancies (Blood 2017). BAP1 is activated by ASXL1 to deubiquitinate H2AK119 during polycomb protein-mediated gene repression. BAP1 mutations also occur in patients with MDS. Despite the significant impact of ASXL1 truncation mutations on the pathogenesis of myeloid malignancies, the underlying mechanisms remain largely unknown, hindering the development of effective targeted therapeutics. Our proteomics studies discovered that ASXL1aa1-587 exhibits an increased binding affinity to BAP1 compared to ASXL1FL and gains an interaction with BRD4, a member of the BET family. BRD4 is involved in multiple biologic processes, including transcription, DNA replication, epigenetic regulation, and tumorigenesis. We hypothesize that truncated ASXL1 dysregulates HSC/HPCs and causes myeloid malignancies through altering the function of the BAP1 deubiquitinase complex and gaining interaction with BRD4. Challenging this critical question has great translational impact and is the major goal of this application. In Aim 1, we will use our recently generated Tet-on/off Asxl1Y588XTg mice to assess in prove- of-concept whether silencing transgene expression by withdrawing doxycycline can eradicate the ASXL1aa1-587- mediated abnormal hematopoietic phenotype and myeloid malignancies. We will apply bPPI-seq, a novel sensitive assay to confirm/identify the true ASXL1aa1-587 interactors at physiological conditions in vivo. In Aim 2, we will determine the role of BRD4-ASXL1aa1-587 interaction in truncated ASXL1-mediated HSC/HPC dysregulation and myeloid malignancy development. We will examine whether BRD4 inhibitor (EP11313) treatment is capable of preventing and/or rescuing the abnormal hematopoietic phenotype and myeloid malignancies in Asxl1Y588XTg mice. In Aim 3, we will determine the role of BAP1 in truncated ASXL1-mediated abnormal HSC/HPC behavior and pathogenesis of myeloid malignancies in vivo. We will decipher how ASXL1aa1-587 alters the function of BAP1 in HSC/HPCs in vivo. The effects of ASXL1aa1-587 on genome-wide BAP1 and H2AK119Ub occupancy in LK cells will be determined by ChIP-seq and correlated with the gene expression data. These studies are timely and fundamentally important for advancing our knowledge on ASXL1 truncation mutation-mediated HSC/HPC dysregulation and myeloid malignancy development. The success of this project will likely identify novel therapeutic targets in the gained-interactors for ASXL1 truncation, BRD4 and BAP1, for the treatment of myeloid malignancies with ASXL1 truncation mutations.

额外的性梳状 (ASXL) 基因是果蝇 Asx 基因的人类同源物，编码基因表达的重要调节因子。 ASXL1 突变以多种形式高频率发生骨髓恶性肿瘤患者预后较差。 ASXL1 突变大多是无义/移码，导致缺少 C 端 PHD 指的蛋白质被截断，该蛋白在 ASXL1 患者样本中可检测到突变。我们最近证明 ASXL1 截短体在小鼠中的转基因表达 (Asxl1Y588XTg) 导致 HSC/HPC 池增加和骨髓恶性肿瘤的发病机制 (Blood 2017)。 BAP1 是在多梳蛋白介导的基因抑制过程中，ASXL1 激活使 H2AK119 去泛素化。 BAP1 突变也发生在MDS患者中。尽管 ASXL1 截短突变对骨髓恶性肿瘤的发病机制，其潜在机制仍然很大程度上未知，阻碍了开发有效的靶向治疗方法。我们的蛋白质组学研究发现 ASXL1aa1-587 表现出与 ASXL1FL 相比，对 BAP1 的结合亲和力增加，并与 BRD4 发生相互作用，BRD4 是 BET 家族。 BRD4 参与多种生物过程，包括转录、DNA 复制、表观遗传调控和肿瘤发生。我们假设截短的 ASXL1 会导致 HSC/HPC 失调，通过改变 BAP1 去泛素酶复合物的功能并获得髓系恶性肿瘤与 BRD4 相互作用。挑战这个关键问题具有巨大的转化影响，也是我们的主要目标这个应用程序。在目标 1 中，我们将使用最近生成的 Tet-on/off Asxl1Y588XTg 小鼠来评估证明- 概念上通过撤回多西环素来沉默转基因表达是否可以根除 ASXL1aa1-587- 介导异常造血表型和骨髓恶性肿瘤。我们将应用bPPI-seq，一种新颖的灵敏的测定，以在体内生理条件下确认/鉴定真正的 ASXL1aa1-587 相互作用因子。在目标 2 中，我们将确定 BRD4-ASXL1aa1-587 相互作用在截短的 ASXL1 介导的 HSC/HPC 中的作用失调和骨髓恶性肿瘤的发展。我们将检查BRD4抑制剂（EP11313）是否治疗能够预防和/或挽救异常的造血表型和骨髓细胞 Asxl1Y588XTg 小鼠的恶性肿瘤。在目标 3 中，我们将确定 BAP1 在截短的 ASXL1 介导的中的作用异常 HSC/HPC 行为和体内骨髓恶性肿瘤的发病机制。我们将如何破译 ASXL1aa1-587 改变体内 HSC/HPC 中 BAP1 的功能。 ASXL1aa1-587 对全基因组的影响 LK 细胞中 BAP1 和 H2AK119Ub 的占用率将通过 ChIP-seq 确定并与基因相关表达数据。这些研究对于增进我们的知识是及时且具有根本性重要的 ASXL1 截短突变介导的 HSC/HPC 失调和骨髓恶性肿瘤的发展。这该项目的成功可能会在获得的 ASXL1 相互作用物中确定新的治疗靶点截短、BRD4 和 BAP1，用于治疗具有 ASXL1 截短突变的骨髓恶性肿瘤。