Investigating the role of KAT6A in MLL-rearranged acute myeloid leukemia

研究 KAT6A 在 MLL 重排急性髓系白血病中的作用

• 批准号: 10344289
• 负责人: Mario Andres Blanco
• 金额: $ 37.3万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10344289
• 关键词: Acute Myelocytic Leukemia; Binding; Biochemical; Biological Assay; CRISPR screen; Cell Cycle; Cell Differentiation process; Cell Proliferation; Cell Survival; Cells; ChIP-seq; Chimeric Proteins; Chromatin; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Differentiation Therapy; Disease; Disease Progression; Disease remission; ENL Protein; Epigenetic Process; FLT3 gene; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Hematologic Neoplasms; Hematopoiesis; Human; Impairment; In Vitro; Individual; KDM1A gene; Knock-out; MLL-AF9; MLL-rearranged leukemia; Mass Spectrum Analysis; Methodology; Methods; Methylcellulose; Modeling; Molecular; Mus; Myelogenous; Oncogenes; Patients; Pharmaceutical Preparations; Play; Proliferating; Reader; Role; Sampling; Scheme; Tertiary Protein Structure; Testing; Therapeutic; Toxic effect; Work; Xenograft Model; acute myeloid leukemia cell; burden of illness; chemotherapy; combinatorial; design; epigenome; epigenomics; histone acetyltransferase; histone-binding proteins; in vivo; inhibitor; mouse model; mutant; novel; programs; promoter; recruit; response; self-renewal; small molecule; stemness; success; targeted treatment; therapeutic evaluation; therapeutic target; transcription factor; transcriptome; transcriptome sequencing; transplant model; treatment effect

Acute myeloid leukemia (AML) is a disease of blocked differentiation in which blasts fail to mature and proliferate continuously. Differentiation therapy, which aims to reactivate latent maturation programs and induce cell cycle exit, is curative in the promyelocytic (APL) AML subtype, but not in other AML subtypes. Epigenetic factors help sustain the differentiation block, and inhibitors of regulators such as the LSD1, BRD4, and DOT1L has recently been shown to re-activate myeloid differentiation programs in selected AML models. However, these inhibitors generally do not achieve terminal differentiation and disease remission. Accordingly, there is significant need to identify more regulators of the AML differentiation block and to test whether their inhibitors can induce terminal differentiation when used individually or in combination regiments. To identify novel regulators of AML differentiation, we recently performed a chromatin-focused CRISPR sgRNA screen using gain-of- differentiation as a readout. This screen identified the H3K9 histone acetyltransferase KAT6A as a key driver of AML differentiation arrest, and mechanistic work showed that KAT6A and the H3K9ac histone binding protein ENL closely cooperate to active promoters of AML oncogenes. We confirmed that both genetic (CRISPR) and small molecule inhibition of KAT6A markedly induces differentiation and reduces proliferation most commonly in MLL-rearranged (MLL-r) AMLs, and also in selected MLL-wild type (MLL-WT) AMLs. Further, KAT6A inhibitors synergize with inhibitors of either LSD1 or DOT1L to induce near-terminal differentiation and fully halt proliferation in vitro. This proposal has three goals: First, we will determine the mechanisms by which KAT6A and ENL are recruited to chromatin and activate transcription. We will identify the protein domains in KAT6A and the MOZ complex it resides in that are responsible for its binding to chromatin at MLL-AF9 targets and non- MLL-AF9 targets. We will also identify any transcription factors interacting with KAT6A and ENL and test their effect on recruitment of the KAT6A-ENL module to chromatin. Our second goal is to test the therapeutic potential of targeting KAT6A, individually or in combination with LSD1 or DOT1L inhibitors, in genetically-defined AML mouse models. We will employ an Mll-Af9 model and a Dnm3a/Flt3-ITD model and test the effect of inhibitor treatment schemes on disease progression and overall survival. We will also test the effect of inhibitor treatments on normal hematopoiesis. Our third goal will be to test the response of clinical AML patient samples to inhibition of KAT6A, individually or in combination with LSD1 or DOT1L inhibitors. We will perform drug response assays in MLL-r and MLL-WT samples in vitro using OP9 feeder layer culturing methodology, and will also perform PDX transplant models and test the response to KAT6A and LSD1 or DOT1L inhibitors in vivo.

急性髓系白血病（AML）是一种分化受阻的疾病，其中原始细胞不能成熟 并不断增殖。分化治疗，旨在重新激活潜在的成熟 程序并诱导细胞周期退出，在早幼粒细胞（APL）AML亚型中是治愈性的，但在 其他AML亚型。表观遗传因素有助于维持分化阻滞， 调节剂如LSD 1、BRD 4和DOT 1 L最近被证明可以重新激活骨髓细胞， 在选定的AML模型中的分化程序。然而，这些抑制剂通常不能实现 终末分化和疾病缓解。因此，非常需要确定更多的 AML分化的调节剂阻断并测试其抑制剂是否可以诱导终末分化 当单独使用或组合使用时，可以区分。确定AML的新型调节剂 分化，我们最近进行了一个染色质聚焦CRISPR sgRNA屏幕使用增益- 差异化作为一种表现。该筛选将H3 K9组蛋白乙酰转移酶KAT 6A鉴定为H3 K9组蛋白乙酰转移酶。 AML分化阻滞的关键驱动因素，机制研究表明，KAT 6A和H3 K9 ac 组蛋白结合蛋白ENL与AML癌基因的活性启动子密切合作。我们证实 KAT 6A的遗传（CRISPR）和小分子抑制都显著诱导分化， 并减少增殖最常见于MLL重排（MLL-r）AML，也在选定的 MLL-野生型（MLL-WT）AML。此外，KAT 6A抑制剂与LSD 1或LSD 2的抑制剂协同作用。 DOT 1 L在体外诱导近终末分化并完全停止增殖。这一建议 三个目标：首先，我们将确定KAT 6A和ENL被招募的机制， 染色质和激活转录。我们将鉴定KAT 6A和MOZ中的蛋白质结构域， 它所处的复合物负责其在MLL-AF 9靶点和非MLL-AF 9靶点处与染色质结合。 MLL-AF 9目标。我们还将鉴定与KAT 6A和ENL相互作用的任何转录因子， 测试它们对KAT 6A-ENL模块向染色质募集的作用。第二个目标是测试 靶向KAT 6A的治疗潜力，单独或与LSD 1或DOT 1 L组合 抑制剂，在基因定义的AML小鼠模型中。我们将采用Mll-Af 9模型和 Dnm 3a/Flt 3-ITD模型，并测试抑制剂治疗方案对疾病进展的影响， 总体生存率。我们还将测试抑制剂治疗对正常造血的影响。我们的第三 目标是测试临床AML患者样本对KAT 6A抑制的反应，单独或 与LSD 1或DOT 1 L抑制剂组合。我们将在MLL-r中进行药物反应试验， MLL-WT样本采用OP 9饲养层培养方法进行体外培养，还将进行PDX 移植模型，并在体内测试对KAT 6A和LSD 1或DOT 1 L抑制剂的反应。