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

基本信息

批准号：
10549725
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
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-20 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10549725
关键词：
Abnormal megakaryocyte Actins Adult Affect Bioreactors Blood Platelets Bromodomains and extra-terminal domain inhibitor Bypass Cell Culture System Cell Reprogramming Cells Clinical Collection Cytoplasm Cytoskeleton Development Dissection Down Syndrome Equilibrium FDA approved Foundations Future Gene Expression Profile Genes Goals Growth Hematopoietic stem cells Human Immunodeficient Mouse Immunologic Deficiency Syndromes Impairment Infusion procedures Investments Knockout Mice Marrow Mediating Megakaryocytes Megakaryocytopoieses Molecular Molecular Target Morphogenesis Mus Neonatal Nuclear Nuclear Translocation Pathway interactions Patients Phenotype Phosphorylation Phosphotransferases Platelet Transfusion Polyploidy Production Productivity Proliferating 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 knock-down manufacturing scale-up pharmacologic platelet function progenitor programs scale up self-renewal success transcription factor

项目摘要

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) 的体外生产为主要临床问题提供了解决方案供体血小板短缺和 HLA 匹配产品稀缺。多项科学突破铺平了道路实现这一目标的方法。剩下的主要障碍是可扩展性的内在障碍。高度增殖的胎儿型 Mk 祖细胞由于受损而产生相对较少且功能低下的血小板形态发生（例如增大和多倍化）；成体型 Mk 产量更丰富、功能更丰富血小板，但增殖能力极小。通过组合安全地规避这些限制的能力祖细胞的扩展性和高效的血小板生产对于经济高效的规模化至关重要。实现这一目标需要详细了解其背后的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。控制该开关将启用通过以连续方式利用胎儿 Mk 的增殖能力，然后利用成人 Mk 的血小板生成潜力。我们的实验室最近发现了 Mk 个体发生的分子基础开关（Elagib 等人 J. Clin. Invest.，2017）。具体来说，RNA 结合因子 IGF2BP3 充当胎儿- 通过抑制转录因子 MKL1 的表达来协调特定的主调节因子成人型 Mk 的细胞骨架重塑。 BET 抑制剂诱导的 IGF2BP3 药物抑制 MKL1 表达和成体形态发生也会导致生长停滞，从而损害多倍体化。在根据该提案，我们确定了一种替代的、改进的方法来规避 IGF2BP3 抑制促进 MKL1 的核转位。为了实现这一策略，我们以 Dyrk 激酶活性为目标，这与 MKL1 的细胞质滞留和唐氏综合症的 Mk 异常有关。药理学 Dyrk 抑制强烈增强脐带血 Mk 形态发生、离体血小板释放和异种移植免疫缺陷小鼠体内血小板的产生。这种方法也有力地增强了 iPSC（诱导多能干细胞衍生）Mk 的形态发生，通常具有早期胎儿表型。使用基因敲除小鼠和人类祖细胞敲低的机制研究支持了一个关键的观点 MKL1 调节的作用，由肌动蛋白调节因子 Ablim2 的 Dyrk1a 磷酸化介导。这物理环境的关键影响，例如刚度和剪切力对 Mk 形态发生的影响归因于 MKL1 激活。我们的结果表明 Dyrk1a 抑制提供了直接、有效且可调节的刺激 Mk 形态发生绕过专门的培养要求。因此，这种方法可以避免成本和与专门的机械生物反应器相关的安全问题。拟议的实验将确定关键 Dyrk 激酶控制 iPSC 和脐带血 Mk 中 MKL1 的步骤，以允许优化系统设计诱导型 Mk 形态发生和血小板生成。此外，针对这一点的临床上可行的策略将严格测试脐带血祖细胞中 Mk 形态发生和血小板生成的途径。