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.

摘要 Runt相关转录因子1（RUNX 1）是造血的关键。RUNX 1水平不足（RUNX 1 Lo） 导致家族性血小板（Plt）疾病与骨髓恶性肿瘤（FPDMM），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，以及开发治疗方法一直受到缺乏复制模型的阻碍。 人类疾病例如，Runx 1 +/-小鼠在其Plt中具有最小的缺陷，并且没有表现出与正常小鼠相比的显著性差异。 白血病倾向我们和其他人已经复制了巨核细胞（Mks，Plt产生的细胞） 在使用人类诱导多能干细胞（iPSC）的FPDMM中观察到的缺陷，为Plt 缺陷我的团队已经在人类CD 34 +-CD 34+细胞中使用短发夹RNA（shRNA）技术扩展了这一模型。 造血干细胞和祖细胞（HSPC）。在体外研究所得Mk，并在输注到 免疫受损小鼠在异种输血模型中释放Plt。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是在很短的时间内进行的，限制了对iPSC的理解。 FPDMM中Plt缺陷或白血病转化基础的潜在治疗价值。到 为了满足这些需求，我们正在开发一种RUNX 1 Lo人骨髓异种移植模型， 免疫受损NOD，B6.SCID/Il 2 rg −/− KitW 41/W 41（NBSGW）小鼠。初步研究表明，RUNX 1 Lo 在该模型中，HSPC移植物以及野生型HSPC。我们可以检测RUNX 1 Lo Plts和髓样/淋巴样 注射后外周血中的细胞>20周。我们现在建议完成这方面的发展， RUNX 1 Lo模型通过追求以下目标：特定目标（SA）#1：表征 体内FPDMM。我们将表征RUNX 1 Lo HSPC植入、谱系定型和Mk/Plt缺陷。我们 还将检查小鼠的克隆进化和恶性转化，有或没有引入第二个 致癌应激来定义FPDMM中白血病倾向的机制。SA #2：进行体内给药 筛选以逆转FPDMM病理学。使用在1998年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 承诺和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，纠正Mk和Plt缺陷，和达沙替尼，一种Src激酶抑制剂， 增加RUNX 1的特异性活性，将是第一批接受测试的候选药物。我们相信，在 R 03时间表，我们将建立RUNX 1 Lo异种移植模型，开发新的见解， 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 这些进展将作为外部资金的初步数据，以支持更多的机制和治疗 RUNX 1 Lo研究以及FPDMM患者的临床治疗试验。