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

摘要 矮小相关转录因子1(RUNX1)是造血的关键基因。RUNX1级别不足(RUNX1Lo) 结果家族性血小板(Plt)疾病伴髓系恶性肿瘤(FPDMM)是一种罕见的疾病，具有 数量/质量Plt缺陷和白血病风险增加。理解Plt缺陷的基础 和白血病的倾向，以及开发的治疗方法一直受到缺乏复制的模型的阻碍 人类疾病。例如，RUNX1+/-小鼠的Plt缺陷很小，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，Plt的来源细胞) 使用人类诱导多能干细胞(IPSCs)的FPDMM中发现的缺陷，为了解Plt提供了新的见解 缺陷。我的团队使用短发夹状RNA(ShRNA)技术在人类CD34+中扩展了这一模型。 造血干祖细胞(HSPC)。将所得到的MKs在体外和输注后进行研究。 免疫功能低下的小鼠在异种输血模型中释放Plts。这些研究证实并推广了 IPSC的结果；然而，即使是异种输血研究也超过了一个很短的窗口，限制了对 潜在疗法在FPDMM的Plt缺陷或白血病转化基础上的价值。至 为了满足这些需求，我们正在开发一种RUNX1LO人骨髓异种移植模型， 免疫低下的NOD，B6.SCID/IL2RG−/−KitW41/W41(NBSGW)小鼠。初步研究表明，RUNX1Lo 在该模型中，HSPC除了移植野生型HSPC外，还移植了HSPC。我们可以检测到RUNX1LO细胞和髓系/淋巴系 注射后20周外周血中的细胞。我们现在建议完成这项工作的发展 RUNX1LO模型通过追求以下目标：特定目标(SA)#1：表征分子基础 体内FPDMM。我们将描述RUNX1Lo HSPC移植、世系承诺和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，无论是否引入第二次 致癌应激以确定FPDMM中白血病倾向的机制。SA#2：体内用药 筛查以逆转FPDMM病理生物学。使用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、世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。小分子转化生长因子RepSox 受体b1抑制剂药物，纠正Mk和Plt缺陷，以及达沙替尼，一种可能 提高RUNX1的特异性活性，将成为首批被检测的候选药物。因此，我们认为，在 R03时间表，我们将建立RUNX1Lo异种移植模型，开发出新的洞察力 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，以支持更多的机制和治疗 RUNX1LO的研究以及FPDMM的临床治疗试验。

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.