Experimental and preclinical modeling of NUP98-rearranged acute leukemia

项目摘要 最初的FusOnC2联盟奖的首要目标是实验和临床前建模 NUP98重排急性白血病的研究有助于了解NUP98 FO驱动的机制基础 白血病的发生，并开发和翻译针对这种形式的白血病的新的、更有效的治疗方法 白血病。该联盟在项目1中的工作旨在开发和描述更忠实的实验 NUP98重排白血病的模型，并利用这些模型来提高我们对生物学的理解 以及针对这一急性白血病高危亚群的潜在治疗靶点。在此应用程序中，我们建议 整合细胞条形码和多组单细胞测序在小鼠和人类模型中的应用 NUP98-重排白血病剖析NUP98 FO、细胞起源和转录失调是如何相交的 推动白血病发生和白血病细胞表型。这项拟议的研究是对之的补充，但不是 与我们目前资助的项目重叠。此外，它还将提供与Dr。 沙林·奈克和马克·道森以及他们的研究小组成员，他们以前曾使用过 血统追踪以研究造血发育和恶性肿瘤。为了实现这一免费赠送的目标 关于补充申请，我们提出了以下具体目标： 目的1：鉴定人巨噬细胞瘤的基因表达谱、染色质可及性和免疫表型。 小鼠HSPC对NUP98 FO驱动白血病的耐受性。在这里，我们将获得单细胞图谱和谱系 跟踪(Splintr)来自我们合作者的条形码文库，并优化它们在小鼠造血中的使用 干细胞和祖细胞(HSPC)。然后，我们将利用NUP98：：KDM5A和KDM5A的穿透鼠标模型 NUP98：：LNP1融合癌蛋白(FOS)，已在Mullight an和Klco实验室开发， 并将Splintr条形码与空矢量、NUP98：：KDM5A或NUP98：：LNP1 FO组合。FO+条码+单元格 将在体外条件下以及移植和体内白血病发生后进行分析。 样品将在引入之前和之后进行多组单细胞图谱(DOGMA-SEQ) 以确定NUP98驱动的基因表达、染色质可及性和免疫表型的变化 重新编排。此外，白血病小鼠骨髓和脾样本的单细胞测序 将使我们能够评估与白血病发生有关的基因调控状态和干细胞/祖细胞亚型 在活体内。 目的2：研究NUP98低聚果糖诱导的细胞分化和基因调控模式 适用于多血统人类造血。为此，我们将Splintr条形码应用于人体模型 NUP98重排。Splintr条形码将被引入脐带血CD34+HSPC，这将 随后被转导以表达空载体NUP98：：KDM5A或NUP98：：LNP1。单元格将被暴露 以体外条件适合多系造血，并改变基因表达、染色质 可及性和免疫表型将通过DOGMA-SEQ确定，以确定FO表达的影响 关于基因调控和分化。此外，我们将进行类似的研究，然后进行移植 将FO+条形码+CD34+细胞注入免疫缺陷小鼠。这些研究将对FO诱导的 并将它们与我们在体外、在小鼠模型中观察到的结果进行比较(目标1)， 在NUP98-重新排列的患者样本中。

Project Summary The overarching goals of the original FusOnC2 consortium award titled Experimental and Preclinical Modeling of NUP98-rearranged Acute Leukemia are to understand the mechanistic basis of NUP98 FO-driven leukemogenesis and to develop and translate new, more effective therapeutic approaches for this form of leukemia. Work in Project 1 of this consortium seeks to develop and characterize more faithful experimental models of NUP98-rearranged leukemia and leverage these models to improve our understanding of the biology and potential therapeutic targeting of this high-risk subset of acute leukemias. In this application, we propose the use of integrated cell barcoding and multi-omic single-cell sequencing in mouse and human models of NUP98-rearranged leukemia to dissect how NUP98 FO, cell-of-origin, and transcriptional dysregulation intersect to drive leukemogenesis and leukemia cell phenotype. The proposed research is complimentary to but not overlapping with our currently funded projects. Additionally, it will provide interactive collaborations with Drs. Shalin Naik and Mark Dawson, as well as with members of their research groups, who have previously used lineage tracing to study hematopoietic development and malignancy. To achieve the goals of this complimentary supplemental application, we propose the following specific aims: Aim 1: To identify the gene expression profile, chromatin accessibility landscape, and immunophenotype of mouse HSPCs permissive to NUP98 FO-driven leukemia. Here, we will obtain single-cell profiling and lineage tracing (SPLINTR) barcoding libraries from our collaborators, and optimize their use in mouse hematopoietic stem and progenitor cells (HSPCs). We will then leverage penetrant mouse models of NUP98::KDM5A and NUP98::LNP1 fusion oncoproteins (FOs), which have been developed in the Mullighan and Klco laboratories, and combine SPLINTR barcodes with empty vector, NUP98::KDM5A, or NUP98::LNP1 FO. FO+barcode+ cells will be profiled under in vitro conditions as well as after transplantation and leukemia development in vivo. Samples will be subjected to multi-omic single-cell profiling (DOGMA-seq) before and after the introduction of FO to identify changes in gene expression, chromatin accessibility, and immunophenotype driven by NUP98 rearrangement. Furthermore, single-cell sequencing of bone marrow and spleen samples from leukemic mice will allow us to assess the gene regulatory state and stem cell/progenitor subtypes permissive to leukemogenesis in vivo. Aim 2: To characterize the differentiation and gene regulation patterns induced by NUP98 FOs in conditions suitable for multilineage human hematopoiesis. In this aim, we will apply SPLINTR barcoding to human models of NUP98 rearrangement. SPLINTR barcodes will be introduced in cord blood CD34+ HSPCs, which will subsequently be transduced to express empty vector, NUP98::KDM5A, or NUP98::LNP1. Cells will be exposed to in vitro conditions suitable for multilineage hematopoiesis, and changes in gene expression, chromatin accessibility, and immunophenotype will be determined via DOGMA-seq to identify the effect of FO expression on gene regulation and differentiation. Furthermore, we will perform similar studies followed by transplantation of FO+barcode+ CD34+ cells into immunodeficient mice. These studies will provide insights on the FO-induced changes that occur in vivo and compare them with the results we observe in in vitro, in mouse models (Aim 1), and in NUP98-rearranged patient samples.