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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Special Emphasis Panel (ZRG1-ONC-H (02))
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Hildesheim, Jeffrey
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Tufts University
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Hu, Miaofen G; Deshpande, Amit; Enos, Miriam et al. (2009) A requirement for cyclin-dependent kinase 6 in thymocyte development and tumorigenesis. Cancer Res 69:810-8
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