Mechanistic and Therapeutic Studies using a Xenotransplanted RUNX1-Haploinsufficient Murine Model

使用异种移植 RUNX1-单倍体不足小鼠模型进行机制和治疗研究

• 批准号: 10721954
• 负责人: Mortimer Poncz
• 金额: $ 17.8万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10721954
• 关键词: Acute Myelocytic Leukemia; Address; Adult; Age; Bar Codes; Biological Models; Biology; Blood Platelet Disorders; Blood Platelets; Bone Marrow; CD34 gene; Cell Cycle; Cell Line; Cells; Clinical; Clonal Evolution; DNA; DNA sequencing; Dasatinib; Data; Defect; Development; Disease; Disease susceptibility; Drug Exposure; Drug Screening; Dysplasia; ETV6 gene; Embryo; Engraftment; Ethylnitrosourea; Evolution; FLI1 gene; Familial Platelet Disorder; Familial disease; Family; Functional disorder; Funding; Genes; Grant; Growth Factor Receptors; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hemorrhage; Hereditary Disease; Human; Immunocompromised Host; In Vitro; Individual; Infusion procedures; Inherited; Injections; Life; Long-Term Effects; Longevity; Lymphoid Cell; Malignant - descriptor; Marrow; Megakaryocytes; Megakaryocytopoieses; Modeling; Molecular; Mus; Mutation; Myelogenous; Myeloid Cells; Myeloproliferative disease; Oncogenic; Parents; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Platelet Count measurement; Predisposing Factor; Progenitor Cell Engraftment; Proteins; RNA; Rare Diseases; Receptors, Adrenergic, beta-1; Risk; Role; Series; Somatic Mutation; Stress; System; Technology; Testing; Therapeutic; Therapeutic Studies; Therapeutic Trials; Therapeutic Use Study; Thrombocytopenia; Time; Transforming Growth Factor beta Receptors; Transforming Growth Factors; United States; Xenograft Model; Xenograft procedure; acute myeloid leukemia 1 protein; candidate identification; detection sensitivity; direct application; drug candidate; drug testing; early childhood; efficacy evaluation; hematopoietic differentiation; human disease; improved; in vivo; in vivo Model; in vivo evaluation; induced pluripotent stem cell; inhibitor therapy; insight; kinase inhibitor; leukemia; leukemic transformation; mouse model; peripheral blood; post-transplant; pre-clinical; prevent; small hairpin RNA; small molecule; src-Family Kinases; stem cell model; therapeutic evaluation; transcription factor

ABSTRACT Runt-related transcription factor 1 (RUNX1), is key to hematopoiesis. Insufficient levels of RUNX1 (RUNX1Lo) results in familial platelet (Plt) disorder associated with myeloid malignancy (FPDMM), a rare disorder with quantitative/qualitative Plt defects and an increased risk of leukemias. Understanding the basis of the Plt defects and leukemic tendency, and developing therapeutics have been hampered by a lack of models that replicate the human disease. For example, Runx1+/- mice have minimal defects in their Plts and do not demonstrate a leukemic propensity. We and others have replicated the megakaryocyte (Mks, the cells from which Plts arise) defect seen in FPDMM using human induced pluripotent stem cells (iPSCs), providing new insights into the Plt defects. My group has extended this model using short-hairpin RNA (shRNA) technology in human CD34+- hematopoietic stem and progenitor cells (HSPCs). The resulting Mks were studied in vitro and after infusion into immunocompromised mice to release Plts in a xenotransfusion model. These studies confirmed and extended the iPSC results; however, even the xenotransfusion studies are over a short window, limiting understanding of the value of potential therapeutics on the Plt defects or the basis of leukemic transformation in FPDMM. To address these needs, we are developing a RUNX1Lo human marrow xenotransplantation model in nonirradiated, immunocompromised NOD,B6.SCID/Il2rg−/−KitW41/W41 (NBSGW) mice. Initial studies show that RUNX1Lo HSPCs engraft as well as wildtype HSPCs in this model. We can detect RUNX1Lo Plts and myeloid/lymphoid cells in the peripheral blood for >20 weeks post-injection. We now propose to complete the development of this RUNX1Lo model by pursuing the following aims: Specific Aim (SA) #1: Characterize the molecular basis of FPDMM in vivo. We will characterize RUNX1Lo HSPC engraftment, lineage commitment and Mk/Plt defects. We will also examine mice for clonal evolution and malignant transformation with or without introducing a secondary oncogenic stress to define the mechanistic of the leukemic propensity in FPDMM. SA#2: Perform in vivo drug screening to reverse FPDMM pathobiology. Using the xenotransplant system developed and analyzed in SA#1, we will carry out long-term drug exposure studies on the Mks/Plts defects, HSPC engraftment, lineage commitment and on clonal evolution and leukemic transformation. At the moment, we have identified several candidate drugs that partially or fully correct the Mk defect. RepSox, a small molecule transforming growth factor receptor b1 inhibitor drug, corrects both Mk and Plt defects ,and dasatinib, a Src kinase inhibitor that may increase RUNX1-specific activity, will be the first candidate drugs to be tested. Thus, we believe that within the R03 timeframe, we will have established a RUNX1Lo xenotransplant model, developed new insights into the mechanistic basis of the observed defects in FPDMM, and examined a series of potential therapeutics. These advances will serve as preliminary data for external funding to support additional mechanistic and therapeutic studies of RUNX1Lo as well as clinical therapeutic trials in patients with FPDMM.

抽象的 Runt 相关转录因子 1 (RUNX1) 是造血的关键。 RUNX1 (RUNX1Lo) 水平不足 导致与骨髓恶性肿瘤相关的家族性血小板 (Plt) 疾病 (FPDMM)，这是一种罕见的疾病 定量/定性 Plt 缺陷和白血病风险增加。了解 Plt 缺陷的基础 和白血病倾向，并且由于缺乏复制的模型而阻碍了治疗方法的开发 人类疾病。例如，Runx1+/- 小鼠的 Plt 缺陷极小，并且没有表现出 白血病倾向。我们和其他人复制了巨核细胞（Mks，产生 Plt 的细胞） 使用人类诱导多能干细胞 (iPSC) 在 FPDMM 中观察到的缺陷，为 Plt 提供了新的见解 缺陷。我的团队使用短发夹 RNA (shRNA) 技术在人类 CD34+- 中扩展了该模型 造血干细胞和祖细胞（HSPC）。所得 Mks 在体外和输注后进行了研究 免疫功能低下的小鼠在异种输血模型中释放 Plts。这些研究证实并扩展了 iPSC 结果；然而，即使是异种输血研究也只是在很短的时间内进行，限制了对 Plt 缺陷的潜在治疗价值或 FPDMM 白血病转化的基础。到 为了满足这些需求，我们正在开发一种非辐照的 RUNX1Lo 人类骨髓异种移植模型， 免疫功能低下的 NOD，B6.SCID/Il2rg−/−KitW41/W41 (NBSGW) 小鼠。初步研究表明 RUNX1Lo HSPC 以及野生型 HSPC 均移植到该模型中。我们可以检测RUNX1Lo Plts和髓样/淋巴样 注射后细胞在外周血中持续>20周。我们现在建议完成这个开发 RUNX1Lo 模型通过追求以下目标： 具体目标 (SA) #1：表征 FPDMM 体内。我们将描述 RUNX1Lo HSPC 植入、谱系承诺和 Mk/Plt 缺陷的特征。我们 还将检查小鼠的克隆进化和恶性转化，无论是否引入二次 致癌应激来定义 FPDMM 中白血病倾向的机制。 SA#2：执行体内药物 筛查以逆转 FPDMM 病理学。使用开发和分析的异种移植系统 SA#1，我们将对Mks/Plts缺陷、HSPC植入、谱系进行长期药物暴露研究 承诺以及克隆进化和白血病转化。目前，我们已经确定了几个 部分或完全纠正 Mk 缺陷的候选药物。 RepSox，一种小分子转化生长因子 受体 b1 抑制剂药物，可纠正 Mk 和 Plt 缺陷，达沙替尼是一种 Src 激酶抑制剂，可 增加RUNX1的特异性活性，将是第一个被测试的候选药物。因此，我们认为，在 R03时间范围内，我们将建立RUNX1Lo异种移植模型，对 FPDMM 中观察到的缺陷的机制基础，并检查了一系列潜在的治疗方法。这些 进展将作为外部资金的初步数据，以支持额外的机械和治疗 RUNX1Lo 的研究以及 FPDMM 患者的临床治疗试验。