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）的体内生产提供了解决主要临床问题的解决方案 HLA匹配产品的供体血小板短缺和稀缺性。多次科学突破已经铺好实现这一目标的方式。剩下的主要障碍包括可扩展性的内在障碍。高度增殖的胎儿型MK祖细胞的产生相对较少，由于受损而产生的低功能血小板形态发生（例如增大和多倍化）；成人型MK产生更丰富和功能性的血小板，但具有最小的增殖能力。通过合并来安全规避这些限制的能力祖细胞可通过有效的血小板产生的可扩展性对于成本效益的扩大至关重要。实现这一目标需要详细了解该目标的分子机制个体开关，即从胎儿到成人MK形态发生的过渡。控制此开关将启用通过以顺序利用胎儿MK的增殖能力，然后是成人MK的血小板潜力。我们的实验室最近发现了MK的分子基础 Switch（Elagib等人J.Clin。Invest。，2017年）。具体而言，RNA结合因子IGF2BP3充当胎儿特定的主调节器通过抑制转录因子MKL1的表达，该转录因子策划了成人型MK的细胞骨架重塑。 IGF2BP3的药理抑制与BET抑制剂诱导 MKL1表达和成年形态发生，但也引起生长停滞，损害多倍化。在这项建议，我们确定了一种通过促进MKL1的核易位。为了实现这一策略，我们针对DYRK激酶活动，这与唐氏综合症的MKL1和MK异常相关。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的免疫缺陷小鼠中的体内血小板产生。这种方法也强烈增强 IPSC的形态发生（诱导多能干细胞得出）MK，通常具有早期胎儿表型。使用敲除小鼠和人类祖细胞中敲除的机械研究支持了关键 MKL1调节的作用，由ABLIM2（肌动蛋白调节因子）的DyRK1a磷酸化介导。这物理环境的关键影响，例如刚度和剪切，在MK形态发生上已归因于 MKL1激活。我们的结果表明，DYRK1A抑制作用为直接，有效和可调的刺激提供了 MK形态发生，绕过专业文化要求。因此，这种方法可以消除成本，并且与专门的机械效反应器相关的安全问题。提出的实验将确定钥匙 IPSC和脐带血MK中MKL1的DYRK激酶控制的步骤，以允许使用可诱导的MK形态发生和血小板产生。此外，针对此目标的临床可行策略途径将严格测试用于脐带血祖细胞中的MK形态发生和血小板产生。