Biology and novel therapy of AML expressing somatic or germline mutant RUNX1

表达体细胞或种系突变体 RUNX1 的 AML 的生物学和新疗法

• 批准号: 10308449
• 负责人: KAPIL BHALLA
• 金额: $ 48.7万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10308449
• 关键词: Age; Apoptosis; BCL2 gene; Back; Binding; Biology; Bromodomain; C-terminal; CRISPR screen; Cell Line; Cell model; Cells; Chromatin; Complex; Core-Binding Factor; Coupled; DNA Binding; DNA Polymerase II; Dependence; Dysmyelopoietic Syndromes; Enhancers; Exhibits; Exons; Familial Platelet Disorder; Genes; Genetic Transcription; Germ Lines; Germ-Line Mutation; Growth; Hematopoiesis; Hematopoietic stem cells; Homologous Transplantation; In Vitro; Introns; Length; Libraries; Malignant - descriptor; Mediating; Messenger RNA; Mus; Mutation; Myeloproliferative disease; Network-based; Oncogenes; Outcome; Patient risk; Patients; Prognosis; Protac; Protein Isoforms; Proteins; RNA; RUNX1 gene; Reader; Recurrent disease; Repression; Residual state; Resistance; Somatic Mutation; Time; Transcriptional Activation Domain; antagonist; base; chemotherapy; chromatin protein; epigenome; graft vs host disease; homoharringtonine; improved; in vivo; inhibitor; knock-down; loss of function mutation; mutant; new therapeutic target; novel; novel therapeutics; patient derived xenograft model; pre-clinical; preclinical efficacy; recruit; small hairpin RNA; stem cells; targeted treatment; transcription factor; transcriptome

Project Summary: RUNX1 is the DNA-binding subunit of the core binding factor (CBF) complex and a master-regulator transcription factor, which is involved in normal and malignant hematopoiesis. Somatic, heterozygous RUNX1 mutations commonly occur in Myelodysplastic Syndrome (MDS) (10%), as well as in secondary (s) or de novo AML (~10%). Germline mutations in RUNX1 cause the highly penetrant (~40%) autosomal dominant, Familial Platelet Disorder (FPD), which can evolve into myeloid malignancy (FPD-MM). Majority of mutant (mt) RUNX1 behave mostly as loss of function mutations, conferring relative therapy-resistance and poorer survival in patients with AML. Consequently, there is a strong unmet need to develop novel therapies for AML expressing somatic or germline mtRUNX1. Our preliminary studies have demonstrated for the first time that shRNA- mediated knockdown of RUNX1 (mutant and wild-type) or disruption of its binding to CBFβ induces greater lethality in AML progenitor cells (HPCs) expressing mtRUNX1 compared to wild-type (wt) RUNX1. We also found that the +24kb enhancer (eR1) within the intragenic super-enhancer (SE) of RUNX1 regulates its transcription in AML cells. The chromatin reader BET (Bromodomain Extra-terminal) protein (BETP) BRD4 promotes transcription of RUNX1 and its targets. BRD4 degradation or eviction from chromatin, or gene-editing of the +24kb RUNX1 eR1, induces lethality in AML cells. By determining and utilizing the mRNA signature from RUNX1-depleted (by shRNA) AML cells, we queried, through LINCS1000-CMap (Connectivity Mapping) analysis, for expression mimickers (EMs). We identified novel EMs that repress RUNX1 and its targets and induce significantly more apoptosis of AML cells expressing mtRUNX1 versus wtRUNX1. Therefore, the hypothesis motivating our studies is that knocking down of levels of RUNX1 and its targets will induce lethality not only in AML blasts expressing somatic mtRUNX1 but also in FPD/MM HPCs expressing germline mtRUNX1. The specific aims of studies proposed are: AIM 1: To determine impact on active enhancers, transcriptome and pre-clinical in vitro and in vivo efficacy of BETP antagonist along with its co-repression of RUNX1, BCL2 and CDK6, alone or in combination with BCL2 or CDK6 inhibitor, in AML blasts and patient- derived xenograft (PDX) models expressing somatic mutant RUNX1. Additionally, we will evaluate pre-clinical efficacy of co-targeting CRISPR-Cas9 screen-discovered top ‘druggable’ dependencies along with BETP antagonist against AML blasts expressing somatic mtRUNX1. AIM 2: To elucidate pre-clinical in vitro and in vivo efficacy of the EMs homoharringtonine (omacetaxine) or fedratinib alone and in combination with BETP antagonists against patient-derived AML blasts and PDX models expressing somatic mtRUNX1. AIM 3: To determine pre-clinical in vitro and in vivo efficacy of selected EMs that repress RUNX1 and its targets against patient-derived HPCs from FPD-MM expressing germline mtRUNX1 and other somatic co-mutations versus HPCs from RUNX1-FPD expressing only germline mtRUNX1.

项目总结： RUNX1是核心结合因子(CBF)复合体的DNA结合亚基，是一个主调节因子 转录因子，参与正常和恶性造血。体细胞杂合子RUNX1 突变通常发生在骨髓增生异常综合征(MDS)(10%)，以及继发性(S)或新发 急性髓系白血病(~10%)。RUNX1的胚系突变导致高渗透性(~40%)常染色体显性家族性 血小板紊乱(FPD)，可演变为髓系恶性肿瘤(FPD-MM)。大多数突变型(Mt)RUNX1 主要表现为功能突变的丧失，导致相对治疗耐药和较差的生存 急性髓系白血病患者。因此，有一个强烈的未得到满足的需求，即开发AML表达的新疗法 体细胞或生殖系mtRUNX1。我们的初步研究首次证明了shRNA- 介导的RUNX1(突变型和野生型)的敲除或其与CBFβ结合的破坏引起更大的 表达mtRUNX1的AML祖细胞(HPC)与野生型(Wt)RUNX1的致死性比较。我们也 发现RUNX1基因内超增强子(SE)中的+24kb增强子(ER1)调节其 AML细胞中的转录。染色质阅读器BET(溴域额外末端)蛋白(BETP)BRD4 促进RUNX1及其靶点的转录。BRD4降解或从染色质中驱逐，或基因编辑 +24kb的RUNX1ER1基因可诱导急性髓系白血病细胞致死。通过确定和利用来自 RUNX1-耗尽(由shRNA)AML细胞，我们通过LINCS1000-Cmap(连接映射)进行查询 分析，用于表达模拟因子(EM)。我们发现了抑制RUNX1及其靶点的新型EM 诱导表达mtRUNX1的AML细胞的凋亡率明显高于表达wtRUNX1的AML细胞。因此， 激励我们研究的假设是，下调RUNX1及其靶点的水平将导致致命性 不仅在表达体细胞mtRUNX1的AML细胞中，而且在表达生殖系的FPD/MM HPC中也是如此 MtRUNX1.建议的研究的具体目标是：目标1：确定对活性促进剂的影响， BETP拮抗剂的转录组和临床前体内外效应及其共抑制作用 RUNX1、BCL2和CDK6单独或与BCL2或CDK6抑制剂联合应用于AML原始细胞和患者- 表达体细胞突变RUNX1的衍生异种移植(PDX)模型此外，我们将评估临床前 联合靶向CRISPR-Cas9筛查发现的顶级可药物依赖性与BETP的疗效 拮抗表达体细胞mtRUNX1的AML细胞。目的2：阐明临床前在体外和在 EMS高三尖杉酯碱(奥乙酰克辛)或非卓替尼单独及与BETP联合应用的体内疗效 拮抗患者来源的AML细胞和表达体细胞mtRUNX1的PDX模型。目标3：实现 体外和体内筛选抑制RUNX1及其靶点的EM的临床前疗效 表达种系mtRUNX1和其他体细胞共突变的FPD-MM患者来源的HPC与 来自RUNX1-FPD的HPC仅表达胚系mtRUNX1。