Targeting Dyrk1a to Promote Donor-independent Platelet Production

体外生产血小板和巨核细胞(Mk)为临床上的主要问题提供了解决方案 供者血小板短缺和缺乏与人类白细胞抗原相匹配的产品。多项科学突破铺平了道路 通向这一目标的道路。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祖细胞由于受损而产生的血小板相对较少且功能低下 形态发生(如放大和多倍化)；成人型Mk产量更丰富和功能更强 但具有极低的增殖能力。一种安全地绕过这些限制的能力，通过结合 祖细胞的可扩充性和高效的血小板生产将是具有成本效益的放大的关键。 要实现这一目标，需要详细了解潜在的分子机制。 个体发生转换，即从胎儿向成人Mk形态发生的转变。对此开关的控制将启用 通过顺序地利用胎儿MK的增殖能力来高效地放大，然后是 成人巨噬细胞的促血小板生成能力。我们实验室最近发现了Mk个体发生的分子基础 Switch(Elagib等人)J·克莱恩。投资，2017)。具体地说，RNA结合因子IGF2BP3作为胎儿- 通过抑制转录因子MKL1的表达来实现特定的主控调节，MKL1协调 成人型MK的细胞骨架重塑。BET抑制剂对IGF2BP3的药理抑制作用 MKL1的表达和成虫的形态发生也会引起生长停滞，危及多倍化。在……里面 这一提议，我们确定了一种替代的、改进的方法，通过以下方式规避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的胞浆滞留和唐氏综合征的Mk异常有关。 药物抑制Dyrk显著促进脐血MK形态发生、体外血小板释放和 异种移植免疫缺陷小鼠体内血小板生成。这一方法也大大增强了 诱导多能干细胞来源的巨噬细胞的形态发生 表型。使用基因敲除小鼠和人类祖细胞基因敲除的机制研究支持一个关键的 肌动蛋白调节因子Ablim2的Dyrk1a磷酸化在MKL1调节中的作用这个 物理环境，如硬度和剪切力对MK形态发生的关键影响被归因于 MKL1激活。我们的结果表明，Dyrk1a抑制提供了直接、有效和可调节的刺激 绕过专门培养要求的MK形态发生。因此，这种方法可以避免成本和 与专用机械生物反应器相关的安全问题。拟议中的实验将决定关键 在iPSC和脐带血MK中Dyrk激酶控制MKL1的步骤，以允许系统的优化设计 诱导的巨噬细胞形态发生和血小板生成。此外，针对这一点的临床可行策略 途径将严格测试MK的形态发生和脐带血祖细胞的血小板生成。

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.