Prevention of acute GvHD by inhibition of cdk2

描述（由申请人提供）：造血干细胞移植（HSCT）可以治愈多种良性和恶性疾病，但由供体T细胞介导的移植物抗宿主病（GvHD）仍然是移植相关发病率和死亡率的主要原因。尽管进行了大量的研究工作，但GvHD的预防和控制目前仍不完整，需要新的治疗方法。本探索性研究将检验抑制cdk 2是预防GvHD的新策略的假设。我们实验室的研究表明，cdk 2在T细胞免疫应答的产生中具有核心作用，并且在抗原识别期间抑制cdk 2活化对于体外诱导T细胞无反应性和体内诱导耐受性是必需的。cdk 2调节多种途径和下游功能。与细胞周期蛋白E结合，cdk 2磷酸化细胞周期抑制剂p27 kip 1，导致泛素靶向降解。Cdk 2促进Rb在特定位点上的磷酸化，从而逆转其螯合E2 F的能力，并允许Rb与组蛋白脱乙酰基酶（HDAC）和其他染色质重塑蛋白相互作用。Cdk 2直接调控NF？B、Sp1、p300/CBP和RNA聚合酶亚基。Cdk 2也磷酸化Smad 3并拮抗TGF-β诱导的其抗增殖功能，而cdk特异性位点上受损的磷酸化使得Smad 3在执行其抗增殖功能方面更有效。虽然cdk 2对于活化的T细胞的扩增是必需的，但它对于静息淋巴细胞的存活、造血或胸腺细胞发育不是关键的。这些特性使得cdk 2成为控制GvHD的有吸引力的新靶标。使用（R）-roscovitine（CYC 202），一种cdk 2-cyclin E的有效抑制剂，我们确定了cdk 2的抑制在体外和体内抑制同种异体反应性T细胞的扩增，并在同种异体骨髓移植的小鼠模型中保护免于急性致死性GvHD。我们的研究表明，抑制cdk 2阻止同种异体反应性T细胞的扩增，同时保留Treg细胞，促进T细胞凋亡逃避细胞周期阻滞，废除Smad 3磷酸化的cdk 2特异性网站，并防止激活NF？B。结合cdk 2的特性，我们的研究结果表明，cdk 2可能是一种新的治疗靶点，用于控制接受HSCT的患者中介导GvHD的同种异体反应性T细胞。为了验证这一假设，我们将进行以下研究：1）鉴定cdk 2抑制对GvHD的细胞机制的影响。2)确定cdk 2抑制对异基因BMT后免疫重建和造血的影响。3)确定cdk 2抑制对人T细胞的细胞和分子/信号传导应答的影响。 公共卫生相关性：GvHD是由HSCT后的供体T细胞介导的，对HSC受体有不利影响。GvHD的治疗控制目前仍然不完整，需要新的治疗方法。我们目前的探索性研究将检验抑制cdk 2是预防GvHD的新策略的假设。如果这种方法是成功的，它将具有重要的临床意义，因为更多的患者将受益于同种异体HSCT，并实现治愈，而没有GvHD的有害的主要副作用。

DESCRIPTION (provided by applicant): Hematopoietic stem cell transplantation (HSCT) can cure a variety of benign and malignant diseases but graft versus host disease (GvHD), mediated by donor T cells, remains a primary cause of transplant related morbidity and mortality. In spite of the intense research efforts prevention and control of GvHD remains currently incomplete and novel therapeutic approaches are required. The present exploratory study will test the hypothesis that inhibition of cdk2 is a novel strategy to prevent GvHD. Studies in our laboratory have indicated that cdk2 has a central role in the generation of T cell immune responses and inhibition of cdk2 activation during antigen recognition is mandatory for induction of T cell anergy in vitro and tolerance in vivo. Cdk2 regulates various pathways and downstream functions. In conjunction with cyclin E, cdk2 phosphorylates the cell cycle inhibitor p27kip1 resulting in ubiquitin-targeted degradation. Cdk2 promotes phosphorylation of Rb on specific sites thereby reversing its ability to sequester E2F, and allowing for interaction of Rb with histone deacetylases (HDAC) and other chromatin remodeling proteins. Cdk2 directly regulates expression of genes including NF?B, Sp1, p300/CBP, and subunits of the RNA polymerase. Cdk2 also phosphorylates Smad3 and antagonizes its antiproliferative function induced by TGF-?, whereas impaired phosphorylation on the cdk-specific sites renders Smad3 more effective in executing its antiproliferative function. While cdk2 is essential for expansion of activated T cells, it is not critical for survival of resting lymphocytes, hematopoiesis or thymocyte development. These properties make cdk2 an attractive novel target for control of GvHD. Using (R)-roscovitine (CYC202), a potent inhibitor of cdk2-cyclin E, we determined that inhibition of cdk2 suppressed expansion of alloreactive T cells in vitro and in vivo and protected from acute lethal GvHD in a mouse model of allogeneic bone marrow transplantation. Our studies revealed that inhibition of cdk2 prevented expansion of alloreactive T cells while sparing Treg cells, promoted apoptosis of T cells escaping cell cycle arrest, abrogated Smad3 phosphorylation on the cdk2-specific site and prevented activation of NF?B. Taken together with the properties of cdk2, our findings suggest that cdk2 might be a novel treatment target for control of alloreactive T cells that mediate GvHD in patients undergoing HSCT. To validate this hypothesis we will undertake the following studies to: 1) Identify the effects of cdk2 inhibition on cellular mechanisms of GvHD. 2) Determine the effects of cdk2 inhibition on immune reconstitution and hematopoiesis after allogeneic BMT. 3) Determine the effects of cdk2 inhibition on cellular and molecular/signaling responses of human T cells. PUBLIC HEALTH RELEVANCE: GvHD is mediated by donor T cells after HSCT and has detrimental effects on the recipient of HSC. Therapeutic control of GvHD remains currently incomplete and novel therapeutic approaches are warranted. Our present exploratory studies will test the hypothesis that inhibition of cdk2 is a novel strategy to prevent GvHD. If such approach is successful, it will have significant clinical implications because more patients will benefit from allogeneic HSCT and achieve cure without the detrimental major side effect of GvHD.