KDM5 histone lysine demethylases as potential novel myeloid tumor suppressors

摘要：KDM 5组蛋白赖氨酸脱甲基酶作为潜在的新型髓系肿瘤抑制剂。 异柠檬酸脱氢酶（IDH 1和IDH 2）的突变存在于超过20%的新生正常病例中。 核型AML和10-20%的继发性AML病例中， 骨髓增生异常综合征（MDS）或骨髓增生性肿瘤（MPN）。突变IDH通过以下方式转化细胞： 产生R-2-羟基戊二酸（R-2 HG），这是一种可以抑制许多细胞活性的癌代谢物， 酶，包括TET 2，一种调节DNA甲基化状态的骨髓肿瘤抑制因子。不 已知R-2 HG在AML中是否具有除TET 2之外的其他致病靶点。然而，IDH突变体的表型 和TET 2突变型髓系疾病是完全不同的。这一观察构成了我们的前提的基础 建议，这是抑制其他途径的R-2 HG有助于突变IDH介导的 转型 我们进行了一项无偏的阳性选择CRISPR-Cas9筛选，以鉴定新的肿瘤抑制因子， 我们鉴定了两种组蛋白赖氨酸脱甲基酶KDM 5A和KDM 5C作为潜在的致病靶点， IDH突变型AML中R-2 HG的表达。我们的中心假设是KDM 5A和KDM 5C调节造血干细胞 细胞功能，以及R-2 HG对KDM 5A和KDM 5C活性的破坏有助于突变IDH介导的细胞凋亡。 转型我们提出的研究通过提出三个关键问题来解决这一假设。第一，什么是 KDM 5缺失促进TF-1细胞非依赖于嘌呤的增殖的机制， 建立因子依赖性人AML细胞系？cDNA拯救实验和基因组分析 组蛋白赖氨酸甲基化和转录将与KDM 5的遗传操作结合使用 酶，以阐明AML中KDM 5同种型的独特和共享功能。第二，证据是什么？ 在IDH突变AML中，R-2 HG抑制KDM 5酶。我们将分析组蛋白 本发明的目的是检测原发性IDH突变体和IDH野生型AML患者样品的甲基化状态，并将表征原发性IDH突变体和IDH野生型AML患者样品的甲基化状态。 表达野生型和突变IDH的等基因细胞系的表观遗传和转录状态，以确定 R-2 HG对KDM 5酶的抑制有助于突变体IDH介导的转化。最后， Kdm 5a缺失如何影响正常小鼠造血和由Kdm 5a诱导的克隆造血 典型髓系肿瘤抑制因子的缺失我们将采用条件性Kdm 5a敲除小鼠， 在单独缺乏Kdm 5a和联合缺乏Kdm 5a的小鼠中进行造血的详细分析 Dnmt 3a、Nf 1或Tet 2。 这些问题的答案将使我们更好地了解KDM 5去甲基化酶在 正常和恶性造血，特别是IDH突变型AML。这项工作在概念上 创新之处在于它将建立一种有助于AML的新的表观遗传机制， 它有可能导致新的治疗方法来治疗白血病患者。

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.