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

项目摘要： RUNX 1是核心结合因子（CBF）复合物的DNA结合亚基，也是一个主调节因子 转录因子，其参与正常和恶性造血。体细胞，杂合子RUNX 1 突变通常发生在骨髓增生异常综合征（MDS）（10%），以及继发性或新发 AML（~10%）。RUNX 1的生殖系突变导致高度外显（~40%）的常染色体显性遗传，家族性 血小板疾病（FPD），可演变为骨髓恶性肿瘤（FPD-MM）。大多数突变体（mt）RUNX 1 主要表现为功能突变的丧失，赋予相对的治疗抗性和较差的生存率， AML患者。因此，对于开发用于表达AML的新疗法存在强烈的未满足的需求。 体细胞或生殖系mtRUNX 1。我们的初步研究首次证明了shRNA- 介导的RUNX 1（突变体和野生型）敲低或其与CBFβ结合的破坏诱导了更大的 与野生型（wt）RUNX 1相比，表达mtRUNX 1的AML祖细胞（HPC）的致死率。我们也 发现RUNX 1基因内超级增强子（SE）中的+24kb增强子（eR 1）调节其 在AML细胞中的转录。染色质阅读器BET（Bromodomain Extra-terminal）蛋白（BETP）BRD 4 促进RUNX 1及其靶点的转录。BRD 4降解或从染色质中驱逐，或基因编辑 +24kbRUNX1 eR 1的表达可诱导急性髓细胞白血病细胞的致死性。通过确定和利用mRNA签名， 我们通过LINCS 1000-CMap（Connectivity Mapping）查询了RUNX 1缺失（通过shRNA）的AML细胞 分析，用于表达模拟物（EM）。我们鉴定了抑制RUNX 1及其靶点的新型EM， 诱导表达mtRUNX 1的AML细胞比表达wtRUNX 1的AML细胞显著更多的凋亡。因此 激发我们研究的假设是RUNX 1及其靶点的水平敲低将诱导致死性 不仅在表达体细胞mtRUNX 1的AML母细胞中，而且在表达生殖系的FPD/MM HPC中， mtRUNX1.提出的研究的具体目的是：目的1：为了确定对活性增强剂的影响， BETP拮抗剂沿着其共抑制的转录组和临床前体外和体内功效 RUNX 1、BCL 2和CDK 6单独或与BCL 2或CDK 6抑制剂联合在AML原始细胞和患者中的作用 表达体细胞突变体RUNX 1的衍生异种移植（PDX）模型。此外，我们将评估临床前 共靶向CRISPR-Cas9筛选发现的与BETP一起沿着的顶级“可药物化”依赖性的功效 针对表达体细胞mtRUNX 1的AML母细胞的拮抗剂。目的2：阐明临床前体外和体内 EMs高三尖杉酯碱（omacetaxine）或非德拉替尼单独使用以及与BETP联合使用的体内疗效 针对患者来源的AML母细胞和表达体细胞mtRUNX 1的PDX模型的拮抗剂。目标3： 确定抑制RUNX 1及其靶点的选定EM的临床前体外和体内功效， 来自表达生殖系mtRUNX 1和其他体细胞共突变的FPD-MM的患者源性HPC与 来自RUNX 1-FPD的HPC仅表达种系mtRUNX 1。

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.