Functional characterization of CUX1 regulators for the treatment of myelodysplastic syndromes

项目总结/摘要 骨髓增生异常综合征（MDS）是一组异质性疾病的特点是血细胞减少 以及由于无效造血作用导致的一种或多种骨髓细胞系的发育不良。患者表现为 贫血的体征和症状，伴随感染和出血，导致严重的发病率。损失 7号染色体（-7）或7号长臂[del（7 q）]在高危MDS患者中常见。治疗 选择有限，高危患者的生存期不到一年。切割样同源框1（CUX 1），a 在7 q22的通常缺失片段中编码的转录因子，在单倍不足时表达， -7/del（7 q）骨髓疾病中的转录物和蛋白质水平。CUX 1的敲除引起自发的 具有人MDS的许多特征的造血障碍，包括红细胞发育不良和致命性贫血， 这表明这一单一基因的缺失足以导致疾病。值得注意的是，CUX 1再表达逆转了 这为当前的提议提供了前提，即CUX 1的恢复是可行的。 MDS患者的治疗策略。然而，我们对这一问题的理解存在重大差距。 CUX 1水平调节的机制。初步研究表明，糖原合成酶激酶- 3（GSK 3）抑制增加细胞CUX 1蛋白水平。核心假设是， 诱导CUX 1水平将恢复造血干细胞稳态并逆转受损的分化 在CUX 1缺陷型骨髓疾病模型中。将通过追求两个具体目标来检验中心假设： 1)定义调节CUX 1水平的细胞和分子机制，以及2）驱动正常 通过CUX 1的治疗性恢复，-7/del（7 q）骨髓增生异常的红系分化。根据第一项 目的，将使用位点- 永生化人血细胞系中的定向突变分析。此外，全基因组 CRISPR/Cas9功能丧失筛选将用于鉴定CUX 1的新型调节剂。对于第二个目标， 将使用CD 34+人-7/del（7 q）MDS细胞和CUX 1缺陷小鼠模型确定疗效 GSK 3亚型选择性抑制剂在恢复CUX 1蛋白和逆转红系分化缺陷中的作用。 这项拟议中的研究有望揭示CUX 1恢复其水平的药物和调节途径 作为逆转-7/del（7 q）MDS患者贫血和发育不良的一种方法。

PROJECT SUMMARY/ABSTRACT The myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by cytopenias and dysplasia in one or more of the myeloid cell lines due to ineffective hematopoiesis. Patients present with signs and symptoms of anemia, accompanied by infections and bleeding leading to serious morbidity. Loss of chromosome 7 (-7) or the long-arm of 7 [del(7q)] is commonly seen in high-risk MDS patients. Treatment options are limited and high-risk patients have less than one-year survival. Cut-like homeobox 1 (CUX1), a transcription factor encoded in the commonly deleted segment of 7q22, is expressed at haploinsufficient transcript and protein levels in -7/del(7q) myeloid diseases. Knockdown of CUX1 causes a spontaneous hematopoietic disorder with many features of human MDS, including erythroid dysplasia, and fatal anemia, indicating that loss of this single gene is sufficient to cause disease. Remarkably, CUX1 re-expression reverses MDS in mice, which provides the premise for the current proposal, that restoration of CUX1 is a viable therapeutic strategy for MDS patients. However, there exists a major gap in our understanding of the mechanisms by which CUX1 levels are regulated. Preliminary studies indicate that Glycogen synthase kinase- 3 (GSK3) inhibition increases cellular CUX1 protein levels. The central hypothesis is that pharmacological induction of CUX1 levels will restore hematopoietic stem cell homeostasis and reverse impaired differentiation in CUX1 deficient myeloid disease models. The central hypothesis will be tested by pursuing two specific aims: 1) define the cellular and molecular mechanisms by which CUX1 levels are regulated, and 2) drive normal erythroid differentiation of -7/del(7q) myelodysplasia through therapeutic restoration of CUX1. Under the first aim, GSK3 mediated post-translational regulatory mechanism of CUX1 levels will be determined using site- directed mutational analysis in immortalized human blood cell lines. Additionally, a genome-wide CRISPR/Cas9 loss of function screen will be utilized to identify novel regulators of CUX1. For the second aim, CD34+ human -7/del(7q) MDS cells and CUX1-deficient mouse models will be used to determine the efficacy of GSK3 isoform selective inhibitors in restoring CUX1 protein and reversing erythroid differentiation defects. The proposed research is expected to reveal druggable and regulatory pathways of CUX1 to restore its levels as a way of reversing anemia and dysplasia in -7/del(7q) MDS patients.