Experimental and preclinical modeling of NUP98-rearranged acute leukemia

NUP98重排急性白血病的实验和临床前模型

• 批准号: 10829603
• 负责人: Charles G. Mullighan
• 金额: $ 16.15万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10829603
• 关键词: Acceleration; Acute leukemia; Award; Bar Codes; Biology; Bone Marrow; CD34 gene; Cells; Chromatin; Collaborations; Communities; Development; Disease; Experimental Models; Exposure to; Funding; Fusion Oncogene Proteins; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic; Genetic Transcription; Goals; Hematologic Neoplasms; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Immunodeficient Mouse; Immunophenotyping; In Vitro; Laboratories; Leukemic Cell; Libraries; Malignant Neoplasms; Modeling; Mus; NUP98 gene; Patients; Pattern; Phenotype; Pre-Clinical Model; Regulator Genes; Reporting; Research; Sampling; Spleen; Therapeutic; Translating; Transplantation; Umbilical Cord Blood; Work; experimental study; high risk; human model; improved; in vivo; insight; leukemia; leukemogenesis; member; mouse model; multiple omics; non-genetic; permissiveness; progenitor; single cell sequencing; stem cells; therapeutic target; therapeutically effective; transcriptomics; vector

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.

项目摘要 原始FUSONC2联盟奖的总体目标标题为实验和临床前建模 NUP98重新培养的急性白血病的理解是了解NUP98 FO驱动的机械基础 白血病生成并开发和翻译这种形式的新的，更有效的治疗方法 白血病。该财团的项目1中的工作旨在发展和表征更忠实的实验 NUP98重新培养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 急性白血病的这一高风险子集的潜在治疗靶向。在此应用程序中，我们建议 在小鼠和人类模型中使用集成的细胞条形码和多摩尼克单细胞测序 NUP98重新培养的白血病，以剖析NUP98 FO，Or-Of-of-of-of-of-of-of-of-of-of-ofergin和转录失调相交 驱动白血病和白血病细胞表型。拟议的研究符合但不是 与我们当前资助的项目重叠。此外，它将与DRS提供互动合作。 Shalin Naik和Mark Dawson以及其研究小组的成员，他们以前使用过 谱系追踪研究造血发育和恶性肿瘤。为了实现这个免费的目标 补充应用，我们提出以下具体目的： 目标1：确定基因表达谱，染色质访问性景观和免疫表型 小鼠HSPC允许NUP98 FO驱动的白血病。在这里，我们将获得单细胞分析和谱系 跟踪（Splintr）的条形码图书馆，并优化其在鼠标造血中的用途 茎和祖细胞（HSPC）。然后，我们将利用NUP98 :: KDM5A的渗透鼠标模型和 NUP98 :: LNP1融合量蛋白（FOS），已在Mullighan和Klco实验室中开发， 并将Splintr条形码与空矢量，NUP98 :: KDM5A或NUP98 :: LNP1 FO相结合。 FO+条形码+单元 将在体外的体外条件下以及体内的白血病发育后进行分类。 在引入之前和之后 FO确定由NUP98驱动的基因表达，染色质可及性和免疫表型的变化 重排。此外，从白血病小鼠的骨髓和脾样品的单细胞测序 将允许我们评估允许白血病生成的基因调节状态和干细胞/祖细胞亚型 体内。 目标2：表征NUP98 FOS在条件下引起的分化和基因调节模式 适用于多素的人造血。在此目标中，我们将对人类模型应用Splintr条形码 NUP98重排。 Splintr条形码将在脐带血CD34+ HSPC中引入 随后将其转导为表达空矢量，NUP98 :: KDM5A或NUP98 :: LNP1。细胞将暴露 体外条件适用于多肾化造血和基因表达的变化，染色质的变化 可访问性和免疫表型将通过Dogma-seq确定以确定FO表达的效果 关于基因调节和分化。此外，我们将进行类似的研究，然后进行移植 FO+条形码+ CD34+细胞的of降入免疫缺陷小鼠。这些研究将提供有关FO诱导的见解 体内发生的变化，并将其与我们在体外观察到的结果相比，在小鼠模型中（AIM 1）， 并在NUP98重新培养的患者样品中。