KDM5 histone lysine demethylases as potential novel myeloid tumor suppressors

摘要：KDM5组蛋白赖氨酸去甲基酶是潜在的新型髓系肿瘤抑制因子。 异柠檬酸脱氢酶(IDH1和IDH2)突变存在于超过20%的新近正常的病例中 核型AML和10%-20%的继发性AML患者由白血病转化所致 骨髓增生异常综合征(MDS)或骨髓增生性肿瘤(MPN)。突变体IDH通过以下方式转化细胞 产生R-2-羟基戊二酸(R-2HG)，这是一种能抑制许多细胞活性的肿瘤代谢物 酶，包括TET2，一种调节DNA甲基化状态的髓系肿瘤抑制因子。它不是 已知R-2HG在AML中除TET2外是否还有其他致病靶点。然而，IDH突变体的表型 与TET2突变的髓系疾病有很大的不同。这一观察结果构成了我们的 提出R-2HG对其他途径的抑制作用有助于突变IDH 转型。 我们进行了无偏正选择CRISPR-Cas9筛查以确定新的肿瘤抑制因子 AML，我们发现两个组蛋白赖氨酸去甲基酶KDM5A和KDM5C是潜在的致病靶点 IDH突变型AML中R-2HG的表达。我们的中心假设是KDM5A和KDM5C调节造血干细胞 细胞功能，以及R-2HG对KDM5A和KDM5C活性破坏导致突变的IDH 转型。我们建议的研究通过提出三个关键问题来解决这一假设。首先，什么是 KDM5缺失促进TF-1细胞非细胞因子依赖性增殖的机制 建立因子依赖型人急性髓系白血病细胞系？日本血吸虫的基因拯救实验及基因组图谱 组蛋白赖氨酸甲基化和转录将与KDM5的遗传操作结合使用 酶，阐明KDM5亚型在AML中的独特和共有的功能。第二，有什么证据 在IDH突变体AML中，KDM5酶被R-2HG抑制。我们将对组蛋白进行分析 原始IDH突变体和IDH野生型AML患者样本的甲基化状态，并将表征 表达野生型和突变型IDH的等基因细胞系的表观遗传和转录状态以确定是否 R-2HG对KDM5酶的抑制有助于突变株IDH介导的转化。最后， KDM5A基因缺失对小鼠正常和克隆性造血的影响 失去典型的髓系肿瘤抑制因子？我们将使用有条件的KDM5A基因敲除小鼠来 对单独缺乏KDM5A和联合缺乏KDM5A的小鼠进行详细的造血分析 使用DNMT3A、NF1或TET2。 这些问题的答案将使我们更好地理解KDM5去甲基酶在 正常和恶性造血，尤其是IDH突变型AML。这项工作是概念性的 创新之处在于它将建立一种有助于急性髓系白血病的新的表观遗传机制，并在 它有可能导致治疗白血病患者的新的治疗方法。

ABSTRACT: KDM5 histone lysine demethylases as potential novel myeloid tumor suppressors. Mutations in Isocitrate Dehydrogenase (IDH1 and IDH2) are present in over 20% of cases of de novo normal karyotype AML and in 10-20% of cases of secondary AML that result from leukemic transformation of myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN). Mutant IDH transforms cells by producing R-2-hydroxyglutarate (R-2HG), an oncometabolite that can inhibit the activity of a number of cellular enzymes, including TET2, a myeloid tumor suppressor that regulates the methylation state of DNA. It is not known if R-2HG has other pathogenic targets besides TET2 in AML. However, the phenotypes of IDH mutant and TET2 mutant myeloid diseases are quite different. This observation forms the basis of the premise of our proposal, which is that inhibition of other pathways by R-2HG contributes to mutant IDH-mediated transformation. We performed an unbiased positive-selection CRISPR-Cas9 screen to identify novel tumor suppressors in AML, and we identified two histone lysine demethylases, KDM5A and KDM5C, as potential pathogenic targets of R-2HG in IDH mutant AML. Our central hypothesis is that KDM5A and KDM5C regulate hematopoietic stem cell function, and that disruption of KDM5A and KDM5C activity by R-2HG contributes to mutant IDH-mediated transformation. Our proposed studies address this hypothesis by asking three key questions. First, what is the mechanism by which KDM5 loss promotes cytokine-independent proliferation of TF-1 cells, an established factor-dependent human AML cell line? cDNA rescue experiments and genomic profiling of histone lysine methylation and transcription will be used, in conjunction with genetic manipulation of KDM5 enzymes, to elucidate the unique and shared functions of KDM5 isoforms in AML. Second, what evidence is there that KDM5 enzymes are inhibited by R-2HG in IDH mutant AML? We will profile the histone methylation state of primary IDH mutant and IDH wild-type AML patient samples, and will characterize the epigenetic and transcriptional states of isogenic cell lines that express wild-type and mutant IDH to determine if inhibition of KDM5 enzymes by R-2HG contributes to the mutant IDH-mediated transformation. And finally, how does loss of Kdm5a affect normal murine hematopoiesis and clonal hematopoiesis induced by loss of canonical myeloid tumor suppressors? We will employ conditional Kdm5a knock-out mice to perform detailed analyses of hematopoiesis in mice that lack Kdm5a alone and that lack Kdm5a in combination with Dnmt3a, Nf1 or Tet2. The answers to these questions will give us a greater understanding of the role of KDM5 demethylases in normal and malignant hematopoiesis, and in IDH mutant AML in particular. This work is conceptually innovative in that it will establish a novel epigenetic mechanism that contributes to AML, and is significant in that it has the potential to lead to novel therapeutic approaches to treat patients with leukemia.