Functional characterization of CUX1 regulators for the treatment of myelodysplastic syndromes

项目摘要/摘要 骨髓增生异常综合征(MDS)是一组以细胞减少为特征的异质性疾病 以及一个或多个髓系细胞系由于无效的造血而发育不良。出席的患者有 贫血的体征和症状，并伴有感染和出血，导致严重的发病率。损失 7号染色体(-7)或7号染色体的长臂[del(7q)]常见于高危MDS患者。治疗 选择有限，高风险患者的存活期不到一年。类切割同源异型盒1(CUX1)，a 编码在7q22共同缺失片段中的转录因子在单倍体不足时表达 -7/del(7q)髓系疾病的转录本和蛋白水平。CUX1基因敲除导致自发性 具有人类MDS许多特征的造血障碍，包括红系发育不良和致死性贫血， 这表明，这种单一基因的缺失足以导致疾病。值得注意的是，CUX1重新表达逆转 在小鼠中的MDS，这为目前的提议提供了前提，即恢复CUX1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很大差距 调节CUX1水平的机制。初步研究表明，糖原合成酶激酶- 3(GSK3)抑制可增加细胞内CUX1蛋白水平。中心假设是药理学上的 诱导CUX1水平将恢复造血干细胞的动态平衡并逆转受损的分化 在CUX1缺陷的髓系疾病模型中。核心假设将通过追求两个具体目标来检验： 1)确定调节CUX1水平的细胞和分子机制；2)驱动正常 通过CUX1的治疗修复-7/del(7q)骨髓发育不良的红系分化。在第一个下 目的研究GSK3介导的CUX1水平的翻译后调控机制。 永生化人类血细胞系的定向突变分析。此外，全基因组 CRISPR/Cas9功能丧失筛查将被用来识别CUX1的新调节子。对于第二个目的， CD34人-7/del(7q)MDS细胞和CUX1缺陷小鼠模型将用于确定疗效 GSK3亚型选择性抑制剂在修复CUX1蛋白和逆转红系分化缺陷中的作用 这项拟议的研究有望揭示CUX1的可用药和调节途径，以恢复其水平 作为逆转-7/del(7q)MDS患者贫血和异型增生的一种方法。

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.