Targeting Dyrk1a to Promote Donor-independent Platelet Production

以 Dyrk1a 为靶点促进不依赖供体的血小板生产

• 批准号: 10350673
• 负责人: Adam N. Goldfarb
• 金额: $ 69.92万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-20 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350673
• 关键词: Abnormal megakaryocyte; Actins; Adult; Affect; Bioreactors; Blood Platelets; Bypass; Cell Culture System; Cells; Clinical; Collection; Custom; Development; Dissection; Down Syndrome; Equilibrium; FDA approved; Foundations; Future; Gene Expression Profile; Genes; Goals; Growth; Hematopoietic stem cells; Human; Immunodeficient Mouse; Impairment; Infusion procedures; Investments; Knockout Mice; Marrow; Mediating; Megakaryocyte Proliferation; Megakaryocytes; Megakaryocytopoieses; Molecular; Molecular Target; Morphogenesis; Mus; Neonatal; Nuclear; Nuclear Translocation; Pathway interactions; Patients; Pharmacology; Phenotype; Phosphorylation; Phosphotransferases; Platelet Transfusion; Production; Productivity; RNA Binding; Regulation; Repression; Role; Safety; Signal Transduction; Somatic Cell; Stimulus; System; Techniques; Testing; Thrombopoiesis; Umbilical Cord Blood; Work; Xenograft procedure; clinical development; clinical translation; cost; cost effective; design; experimental study; fetal; improved; in vivo; induced pluripotent stem cell; inhibitor; knock-down; platelet function; progenitor; programs; scale up; self-renewal; success; transcription factor

Ex vivo production of platelets and megakaryocytes (Mk) offers solutions to the major clinical problems of donor platelet shortages and scarcity of HLA-matched products. Multiple scientific breakthroughs have paved the way toward this goal. The principal remaining roadblock consists of an intrinsic barrier to scalability. Highly proliferative fetal-type Mk progenitors yield relatively few and hypofunctional platelets due to impaired morphogenesis (e.g. enlargement and polyploidization); adult-type Mk yield more abundant and functional platelets but have minimal proliferative capacity. An ability to safely circumvent these limitations, by combining progenitor expandability with efficient platelet production, will be critical for cost-effective scale-up. Accomplishment of this goal requires a detailed understanding of the molecular mechanisms underlying the ontogenic switch, i.e. the transition from fetal to adult Mk morphogenesis. Control over this switch will enable efficient scale-up by exploiting in a sequential manner the proliferative capability of fetal Mk followed by the thrombopoietic potential of adult Mk. Our lab recently discovered a molecular basis for the Mk ontogenic switch (Elagib et al. J. Clin. Invest., 2017). Specifically, an RNA-binding factor IGF2BP3 functions as a fetal- specific master regulator by suppressing expression of the transcription factor MKL1, which orchestrates the cytoskeletal remodeling of adult-type Mk. Pharmacologic repression of IGF2BP3 with BET inhibitors induced MKL1 expression and adult morphogenesis but also caused growth arrest, compromising polyploidization. In this proposal, we identify an alternative, improved approach of circumventing IGF2BP3 repression by promoting nuclear translocation of MKL1. To accomplish this strategy, we have targeted Dyrk kinase activity, which has been implicated in cytoplasmic retention of MKL1 and in Mk abnormalities in Down syndrome. Pharmacologic Dyrk inhibition strongly enhanced cord blood Mk morphogenesis, ex vivo platelet release, and in vivo platelet production in xenotransplanted immunodeficient mice. This approach also strongly enhanced morphogenesis of iPSC (induced pluripotent stem cell derived) Mk, which normally have an early fetal phenotype. Mechanistic studies using knockout mice and knockdowns in human progenitors support a critical role for MKL1 regulation, mediated by Dyrk1a phosphorylation of Ablim2, an actin regulatory factor. The critical influence of physical milieu, e.g. stiffness and shear, on Mk morphogenesis has been attributed to MKL1 activation. Our results suggest that Dyrk1a inhibition provides a direct, potent, and tunable stimulus for Mk morphogenesis that bypasses specialized culture requirements. This approach could thus obviate cost and safety issues associated with specialized mechano-bioreactors. The proposed experiments will determine key steps in Dyrk kinase control of MKL1 in iPSC and cord blood Mk, to permit optimal design of systems with inducible Mk morphogenesis and platelet production. In addition, clinically feasible strategies for targeting this pathway will be rigorously tested for Mk morphogenesis and platelet production in cord blood progenitors.

血小板和巨核细胞（Mk）的离体生产提供了以下主要临床问题的解决方案： 供体血小板短缺和HLA匹配产品稀缺。多项科学突破为 通往这一目标的道路。剩下的主要障碍是可伸缩性的内在障碍。 高度增殖的胎儿型Mk祖细胞产生相对较少的和功能低下的血小板，这是由于受损的 形态发生（例如增大和多倍化）;成年型Mk产量更丰富和功能更强 血小板，但具有最小的增殖能力。安全地规避这些限制的能力，通过结合 具有有效血小板产生的祖细胞可扩增性对于成本有效的规模扩大将是至关重要的。 要实现这一目标，需要详细了解其分子机制。 个体发生转换，即从胎儿到成人Mk形态发生的转变。对该开关的控制将使 通过以顺序的方式利用胎儿Mk的增殖能力，随后利用 成年Mk.我们的实验室最近发现了Mk个体发育的分子基础 开关（Elagib等，J.Clin.Invest.，2017年）。具体而言，RNA结合因子IGF 2BP 3作为胎儿- 通过抑制转录因子MKL 1的表达来调节特异性主调节因子，MKL 1协调了 成年型Mk.用BET抑制剂诱导的IGF 2BP 3的药理学抑制 MKL 1表达和成体形态发生，但也引起生长停滞，损害多倍化。在 根据这一建议，我们确定了一种替代的、改进的方法， 促进MKL 1的核转位。为了实现这一策略，我们针对Dyrk激酶活性， 其与MKL 1的细胞质滞留和唐氏综合征中的Mk异常有关。 药理学Dyrk抑制强烈增强脐带血Mk形态发生，离体血小板释放， 异种移植免疫缺陷小鼠体内血小板生成。这种方法也大大提高了 iPSC（诱导多能干细胞衍生的）Mk的形态发生，其通常具有早期胚胎发育。 表型使用基因敲除小鼠和敲除人类祖细胞的机制研究支持了一个关键的 通过Dyrk 1a磷酸化肌动蛋白调节因子Ablim 2介导的MKL 1调节作用。的 物理环境（如刚度和剪切力）对Mk形态发生的关键影响归因于 MKL 1激活。我们的研究结果表明，Dyrk 1a抑制提供了一个直接的，有力的，和可调的刺激， 不需要专门培养的微生物形态发生。因此，这种方法可以节省成本， 与专门的机械生物反应器相关的安全问题。这些实验将确定关键的 iPSC和脐带血Mk中MKL 1的Dyrk激酶控制步骤，以允许系统的最佳设计， 诱导型Mk形态发生和血小板产生。此外，临床上可行的策略，针对这一点， 将严格测试Mk通路在脐带血祖细胞中的形态发生和血小板产生。