Project 3 - Genomics of Secondary AML Progression.

项目3：急性髓系白血病继发性进展的基因组学。该项目的长期目标是定义 骨髓增生异常综合征进展过程中亚克隆扩大的机制 (MDS)到辅助AML(SAML)。约30%的MDS患者，最常见的成人髓系 恶性肿瘤在美国，进展为迅速致命的继发性急性髓细胞白血病。我们已经证明了从MDS开始的进展 TO SAML的特征是发现克隆突变的持久性，以及一个或多个亚克隆的扩展。 来自我们小组和其他人的初步数据表明，信号基因(例如，NRAS)的获得性突变 或髓系转录因子(例如，RUNX1)可能有助于高达50%的亚克隆和原始细胞扩增 MDS患者。目前尚不清楚是什么推动了其余50%患者的亚克隆扩张。我们 假设替代遗传(即，非编码或结构变异)或表观遗传改变也可能 为进步作出贡献。我们将确定在此项目中驱动亚克隆扩展的获得性更改。在……里面 具体目标1，我们将定义在进化过程中上升的亚克隆的基因组和转录本 从MDS到SAML。我们将对三个样本(皮肤、MDS和 SAML骨髓)，以全面定义样本的克隆结构，包括上升的亚克隆 在进程中。同时，我们将对MDS/SAML配对进行单细胞RNA测序(scRNA-seq) 用于标识进化为导致SAML的子克隆的表达式签名的样本。通过对样本进行排序 无论有没有已知的亚克隆驱动基因突变，我们将测试是否所有正在崛起的亚克隆都有 控制正常髓系成熟的基因或途径表达失调。这些研究将是 从项目1、2和4中收集的从头开始和TP53突变的急性髓细胞白血病样本中收集的数据进一步了解情况。 总而言之，这些研究应该确定亚克隆扩张的驱动因素，这些驱动因素可能是潜在的靶向 防止SAML进展。在特定的目标2中，我们将从功能上验证有助于 从MDS到SAML过程中的亚克隆扩展。与创建克隆人协同工作的基因 驱动亚克隆扩张的突变并不总是已知的。我们假设普通的MDS启动 表观遗传修饰基因和剪接体基因突变可能使造血干/祖细胞“启动” (HSPC)，并通过协同突变使其更容易进展；这反过来表明 突变的获得顺序可能在SAML的发病机制中起重要作用。我们将测试亚克隆的重要性 通过将它们导入原代小鼠HSPC，这些小鼠HSPC使用病毒进行原代克隆突变 过度表达，或CRISPR/Cas9技术，用于功能丧失和监测自我更新、增殖和 受体小鼠的克隆性扩增。我们预测，只有特定的突变组合--在 正确的顺序--将合作诱导克隆扩张。我们将测试靶向药物的疗效 使用临床前小鼠和患者来源的异种移植模型建立克隆和/或亚克隆。

Project 3: Genomics of secondary AML progression. The long-term goal of this project is to define the mechanisms underlying subclone expansion during progression from myelodysplastic syndromes (MDS) to secondary AML (sAML). Approximately 30% of patients with MDS, the most common adult myeloid malignancy in the US, progress to a rapidly fatal secondary AML. We have shown that progression from MDS to sAML is characterized by persistence of founding clone mutations, and expansion of one or more subclones. Preliminary data from our group and others suggest that an acquired mutation in a signaling gene (e.g., NRAS) or a myeloid transcription factor (e.g., RUNX1) may contribute to subclone and blast expansion in up to 50% of MDS patients. What drives subclone expansion in the remaining 50% of patients is not yet clear. We hypothesize that alternative genetic (i.e., non-coding or structural variants) or epigenetic alterations may also contribute to progression. We will identify acquired alterations that drive subclone expansion in this project. In Specific Aim 1, we will define the genomes and transcriptomes of rising subclones during progression from MDS to sAML. We will perform enhanced whole genome sequencing on sample trios (skin, MDS, and sAML bone marrow) to comprehensively define the clonal architecture of samples, including rising subclones during progression. In parallel, we will perform single-cell RNA-sequencing (scRNA-seq) on MDS/sAML paired samples to identify the expression signatures of subclones that evolve to cause sAML. By sequencing samples with and without known subclonal driver gene mutations, we will test whether all rising subclones have dysregulated expression of genes or pathways that control normal myeloid maturation. These studies will be further informed by the data collected on de novo and TP53 mutated AML samples in projects 1, 2, and 4. Collectively, these studies should define drivers of subclonal expansion that could potentially be targeted to prevent sAML progression. In Specific Aim 2, we will functionally validate mutations that contribute to subclone expansion during progression from MDS to sAML. The genes that cooperate with founding clone mutations to drive subclone expansion are not always known. We hypothesize that common MDS-initiating mutations in epigenetic modifier and spliceosome genes may “prime” a hematopoietic stem/progenitor cell (HSPC), and make it more susceptible to progression by cooperating mutations; this in turn suggests that the order of mutation acquisition may be important for sAML pathogenesis. We will test the importance of subclone mutations by introducing them into primary mouse HSPCs that harbor a founding clone mutation using viral over-expression, or CRISPR/Cas9 technology for loss-of-function and monitor self-renewal, proliferation, and clonal expansion in recipient mice. We predict that only specific combinations of mutations--acquired in the correct order--will cooperate to induce clonal expansion. We will test the efficacy of drugs targeting the founding clone and/or the subclone using preclinical mouse and patient-derived xenograft models.