CDK6 in T cell development and cancer

描述（由申请人提供）：Cdk4和cdk6被广泛认为是必要的，但在响应生长调节信号的细胞周期启动中是多余的。与这一概念相反，最近的遗传证据表明，cdk4和cdk6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cdk4/6缺失的不敏感性可能归因于补偿，因为cdk4/6敲除细胞中的“游离”d型细胞周期蛋白似乎能够激活cdk2以允许增殖。此外，现在有大量证据支持cdk4/6在转化细胞和肿瘤细胞中的关键作用。例如，敲除cdk4的成纤维细胞会抵抗转化，缺乏cyclin D1激活cdk4/6能力的小鼠对erbb2诱导的乳腺肿瘤具有抗性。cdk6在肿瘤发生中的作用尚不清楚，但最近的研究揭示了cdk6在某些细胞类型分化中的新功能。例如，缺乏cdk6的小鼠在T细胞增殖方面表现出缺陷。重要的是，传统的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“合适”伴侣丢失时，它们会激活不同的cdks。为了利用精细的遗传工具阐明cdk6在细胞周期控制和肿瘤发生中的作用，我们培育了cdk6靶向突变的小鼠。这些敲入基因等位基因产生高活性或低活性的激酶亚基，可能更好地模拟肿瘤细胞中cdk6的高活性或模拟药物干预。我们发现cdk6的缺失或cdk6激酶功能的特异性失活会大大减少胸腺细胞数量，并显著降低DN2细胞的增殖，同时增加DN3的比例。相反，ink4不敏感、过度活跃的CDK6R31C等位基因会增加胸腺细胞的数量，这与大多数T细胞亚群（最明显的是DN2和CD8+部分）的增殖增加一致。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张和肿瘤的发生。值得注意的是，LCK-myrAKT的初步分析；与LCK-myrAKT相比，cdk6-/-小鼠显示完全没有肿瘤；cdk6WT小鼠，这种小鼠很快就会屈服于大量的T细胞肿瘤。总之，这些数据表明cdk6是T细胞增殖和发育的重要调节因子，并且可能在某些形式的T细胞白血病和淋巴瘤中起关键作用。因此，Cdk6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者。为了进一步表征cdk6突变体在小鼠T细胞发育中的作用，特别强调早期祖细胞功能和T细胞癌的形成，我们提出：(1)评估cdk6在胸腺外T细胞祖细胞中的作用，并确定cdk6突变胸腺细胞中CD25和CD44表达改变的来源；(2)确定cdk6缺失或突变对akt诱导的胸腺肿瘤的影响；(3)确定p53缺失是否与R31C胸腺细胞中cdk6的高活性协同作用，研究cdk6功能缺失对p53突变小鼠肿瘤形成的影响。

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.