Project 3 - Genomics of Secondary AML Progression.

项目3：继发性AML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增的机制 (MDS)继发性AML（sAML）大约30%的MDS患者是最常见的成人髓系白血病， 在美国，恶性肿瘤进展为快速致死性继发性AML。我们已经证明MDS的进展 转化为sAML的特征在于建立克隆突变的持续性和一个或多个亚克隆的扩增。 来自我们小组和其他人的初步数据表明，信号基因中的获得性突变（例如，NRAS） 或骨髓转录因子（例如，RUNX 1）可能有助于亚克隆和原始细胞扩增高达50%的 MDS患者。是什么驱动了其余50%患者的亚克隆扩增尚不清楚。我们 假设替代基因（即，非编码或结构变体）或表观遗传改变也可 有助于进步。在这个项目中，我们将确定驱动亚克隆扩展的获得性改变。在 具体目标1，我们将确定基因组和转录组的发展过程中上升的亚克隆 从MDS到sAML。我们将对样本三重（皮肤、MDS和 sAML骨髓），以全面定义样品的克隆结构，包括上升的亚克隆 在进展过程中。同时，我们将对MDS/sAML配对进行单细胞RNA测序（scRNA-seq）， 样本，以确定亚克隆的表达特征，演变为导致sAML。通过测序样本 有和没有已知的亚克隆驱动基因突变，我们将测试是否所有上升的亚克隆都有 控制正常骨髓成熟的基因或途径的表达失调。这些研究报告将 通过在项目1、2和4中收集的从头和TP 53突变AML样本的数据进一步了解。 总的来说，这些研究应该定义亚克隆扩增的驱动因素，这些驱动因素可能是针对 预防sAML进展。在具体目标2中，我们将在功能上验证有助于 从MDS进展为sAML期间的亚克隆扩增。与建立克隆相配合的基因 驱动亚克隆扩增的突变并不总是已知的。我们假设常见的MDS引发 表观遗传修饰基因和剪接体基因的突变可以“引发”造血干/祖细胞 （HSPC），并通过合作突变使其更容易进展;这反过来又表明， 突变获得的顺序对于sAML发病机制可能是重要的。我们将测试亚克隆的重要性 通过使用病毒载体将它们引入到具有基础克隆突变的原代小鼠HSPC中， 过表达或CRISPR/Cas9技术用于功能丧失，并监测自我更新、增殖， 受体小鼠的克隆扩增。我们预测，只有特定的突变组合--在 正确的顺序-将合作诱导克隆扩增。我们将测试药物的有效性， 使用临床前小鼠和患者来源的异种移植物模型建立克隆和/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.