CDK6 in T cell development and cancer

描述（由申请人提供）：Cdk 4和cdk 6被广泛认为是响应生长调节信号启动细胞周期所必需的，但却是多余的。与这一概念相矛盾的是，最近的遗传学证据表明cdk 4和cdk 6对早期发育和培养中原代细胞的增殖都不是必需的。然而，这种对cdk 4/6损失的不敏感性可能归因于补偿，因为cdk 4/6敲除细胞中的“游离”D型细胞周期蛋白似乎能够激活cdk 2以允许增殖。此外，现在有相当多的证据支持cdk 4/6在转化细胞和肿瘤细胞中的关键作用。例如，cdk 4敲除的成纤维细胞抵抗转化，缺乏细胞周期蛋白D1激活cdk 4/6的能力的小鼠对ErbB 2诱导的乳腺肿瘤具有抗性。cdk 6在肿瘤发生中的作用知之甚少，然而最近的研究揭示了cdk 6在某些细胞类型分化中的新功能。例如，缺乏cdk 6的小鼠显示T细胞增殖缺陷。重要的是，传统的基因敲除实验可能低估了特定亚基对发育和癌症的影响，这是由于D细胞周期蛋白亚基的混杂性，当它们的“适当”伴侣丢失时，这些亚基激活不同的cdk。为了阐明cdk 6在细胞周期控制和肿瘤发生中的作用，我们使用精细的遗传工具，产生了cdk 6靶向突变的小鼠。这些敲入等位基因产生过度活化或失活的激酶亚基，分别可以更好地模拟肿瘤细胞或模型药物干预中cdk 6的过度活化。我们已经发现，cdk 6的损失或特异性失活的cdk 6激酶功能大大减少胸腺细胞的数量，并显着减少DN 2细胞的增殖，同时伴随着增加DN 3分数。相反，INK 4不敏感、过度活跃的CDK 6 R31 C等位基因增加胸腺细胞构成，与大多数T细胞亚群的增殖增加一致，最显著的是DN 2和CD 8+部分。然而，这种增殖的增加被细胞凋亡的增加所抵消，这可能限制胸腺细胞的扩增和肿瘤发生。值得注意的是，LCK-myrAKT; cdk 6-/-小鼠的初步分析显示完全没有肿瘤，与LCK-myrAKT; cdk 6 WT小鼠相反，后者迅速死于大规模T细胞肿瘤。总之，这些数据表明cdk 6是T细胞增殖和发育的重要调节因子，并且在某些形式的T细胞白血病和淋巴瘤中可能是至关重要的。因此，Cdk 6可能被证明是治疗T细胞肿瘤和免疫疾病的有价值的治疗候选物。为了进一步研究cdk 6突变体在小鼠T细胞发育中的作用，特别是早期祖细胞功能和T细胞癌的形成，我们建议：（1）评估Cdk 6在胸腺外T细胞祖细胞中的作用，并确定Cdk 6突变胸腺细胞中CD 25和CD 44表达改变的来源;（2）确定cdk 6缺失或突变对胸腺中AKT诱导的肿瘤的影响;和（3）确定R31 C胸腺细胞中p53的缺失是否与cdk 6过度活性协作，并研究cdk 6功能缺失对p53突变小鼠中肿瘤形成的影响。 公共卫生相关性：越来越清楚的是，控制细胞分裂能力的酶在干细胞和祖细胞中起关键作用，干细胞和祖细胞在细胞分化中起作用，细胞在成体组织中获得其特定功能的过程，以及肿瘤形成。在这里，我们描述了我们对cdk 6的广泛的初步研究，cdk 6是一种细胞分裂控制酶，我们发现它是胸腺中正常T细胞形成的关键，我们发现这种酶的缺失可以防止T细胞肿瘤的形成。我们在此提出的研究旨在使用我们已经产生的独特的遗传改变的小鼠来了解cdk 6在T细胞发育和肿瘤发生中的作用，以努力学习如何用未来的治疗方法最好地靶向这种酶。

DESCRIPTION (provided by applicant): Cdk4 and cdk6 have been widely thought to be essential but redundant for initiation of the cell cycle in response to growth regulatory signals. In contradiction to this concept, recent genetic evidence indicates that cdk4 and cdk6 are not essential for early development, nor for proliferation of primary cells in culture. However, this insensitivity to cdk4/6 loss may be ascribed to compensation, since "free" D-type cyclins in cdk4/6 knockout cells appear able to activate cdk2 to allow proliferation. Further, considerable evidence now exists to support a crucial role of cdk4/6 in transformed and tumor cells. For example, cdk4 knockout fibroblasts resist transformation and mice lacking the ability of cyclin D1 to activate cdk4/6 are resistant to ErbB2-induced mammary tumors. The role of cdk6 in tumorigenesis is poorly understood, however recent studies have revealed a novel function of cdk6 in the differentiation of some cell types. For example, mice lacking cdk6 show defects in T cell proliferation. Importantly, conventional knockout experiments may underestimate the impact of specific subunits on development and cancer, due to promiscuity of D cyclin subunits that activate different cdks when their "proper" partners are lost. To clarify the role of cdk6 in cell cycle control and tumorigenesis using refined genetic tools, we have generated mice with targeted mutations in Cdk6. These knockin alleles generate hyperactive or inactive kinase subunits that may better mimic hyperactivation of cdk6 in tumor cells or model pharmaceutical intervention, respectively. We have found that loss of cdk6 or specific inactivation of cdk6 kinase function greatly reduces thymocyte numbers and significantly reduces proliferation in DN2 cells while concomitantly increasing the DN3 fraction. Conversely, the INK4-insensitive, hyperactive CDK6R31C allele increases thymic cellularity, consistent with increased proliferation of most T cell subsets, most notably the DN2 and CD8+ fractions. However, this increase in proliferation is countered by increased apoptosis, which may limit thymocyte expansion and tumorigenesis. Notably, preliminary analysis of LCK-myrAKT;cdk6-/- mice shows a complete absence of tumors, in contrast to LCK-myrAKT;cdk6WT mice, which quickly succumb to massive T cell tumors. Together, these data demonstrate that cdk6 is an important regulator of T cell proliferation and development, and may be crucial in some forms of T cell leukemia and lymphoma. Cdk6 may thus prove to be a valuable therapeutic candidate for treatment of both T cell tumors and immune disorders. To further characterize the role of cdk6 mutants in T cell development in mice, with particular emphasis on early progenitor function and formation of T cell cancers, we propose to (1) Assess the role of Cdk6 in extra-thymic T cell progenitors and determine the source of alterations in CD25 and CD44 expression in Cdk6-mutant thymocytes; (2) Determine the impact of cdk6 loss or mutation on AKT-induced tumors in the thymus; and (3) Determine if loss of p53 collaborates with cdk6 hyperactivity in R31C thymocytes and study the effects of cdk6 loss of function on tumor formation in p53 mutant mice. PUBLIC HEALTH RELEVANCE: It has become increasingly clear that the enzymes that control the ability of cells to divide play key roles in stem and progenitor cells that act in cellular differentiation, the process whereby cells acquire their specific functions in adult tissue, and also in tumor formation. Here we describe our extensive preliminary studies of cdk6, a cell division control enzyme that we show is key to the formation of normal T cells in the thymus and we find that loss of this enzyme prevents T cell tumor formation. The studies we propose herein are aimed at using unique, genetically altered mice that we have produced to understand the role of cdk6 in T cell development and tumorigensis in an effort to learn how to best target this enzyme with future therapeutics.