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。尽管研究力度很大，但目前GvHD的预防和控制仍然不完整，需要新的治疗方法。目前的探索性研究将验证抑制cdk2是一种预防GvHD的新策略的假设。我们实验室的研究表明，cdk2在T细胞免疫反应的产生中起着核心作用，在抗原识别过程中抑制cdk2的激活对于诱导T细胞在体外的能量和体内的耐受性是必需的。Cdk2调节多种途径和下游功能。cdk2与细胞周期蛋白E一起磷酸化细胞周期抑制剂p27kip1，导致泛素靶向降解。Cdk2促进Rb在特定位点的磷酸化，从而逆转其隔离E2F的能力，并允许Rb与组蛋白去乙酰化酶（HDAC）和其他染色质重塑蛋白相互作用。Cdk2直接调控包括NF？B, Sp1， p300/CBP和RNA聚合酶亚基。Cdk2还能磷酸化Smad3，并拮抗其由TGF-诱导的抗增殖功能。而cdk特异性位点磷酸化受损则使Smad3更有效地执行其抗增殖功能。虽然cdk2对活化T细胞的扩增至关重要，但它对静止淋巴细胞的存活、造血或胸腺细胞的发育并不重要。这些特性使cdk2成为控制GvHD的一个有吸引力的新靶点。使用cdk2-cyclin E的有效抑制剂(R)-罗斯科维汀（CYC202），我们在体外和体内确定了抑制cdk2抑制同种异体T细胞的扩增，并在同种异体骨髓移植小鼠模型中保护急性致死性GvHD。我们的研究表明，抑制cdk2抑制了异位反应性T细胞的扩增，同时保留了Treg细胞，促进了逃避细胞周期停滞的T细胞的凋亡，消除了cdk2特异性位点上Smad3的磷酸化，并阻止了NF - B的活化。结合cdk2的特性，我们的研究结果表明，cdk2可能是控制造血干细胞移植患者介导GvHD的同种异体反应性T细胞的新治疗靶点。为了验证这一假设，我们将进行以下研究：1)确定cdk2抑制对GvHD细胞机制的影响。2)确定cdk2抑制对同种异体骨髓移植后免疫重建和造血的影响。3)确定cdk2抑制对人T细胞细胞和分子/信号反应的影响。

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.