Chemical-genetic analysis of cyclin dependent kinase 2 function reveals an important role in cellular transformation by multiple oncogenic pathways.

A family of conserved serine/threonine kinases known as cyclin-dependent kinases (CDKs) drives orderly cell cycle progression in mammalian cells. Prior studies have suggested that CDK2 regulates S-phase entry and progression, and frequently shows increased activity in a wide spectrum of human tumors. Genetic KO/knockdown approaches, however, have suggested that lack of CDK2 protein does not prevent cellular proliferation, both during somatic development in mice as well as in human cancer cell lines. Here, we use an alternative, chemical-genetic approach to achieve specific inhibition of CDK2 kinase activity in cells. We directly compare small-molecule inhibition of CDK2 kinase activity with siRNA knockdown and show that small-molecule inhibition results in marked defects in proliferation of nontransformed cells, whereas siRNA knockdown does not, highlighting the differences between these two approaches. In addition, CDK2 inhibition drastically diminishes anchorage-independent growth of human cancer cells and cells transformed with various oncogenes. Our results establish that CDK2 activity is necessary for normal mammalian cell cycle progression and suggest that it might be a useful therapeutic target for treating cancer.

MYC pathway activation in triple-negative breast cancer is synthetic lethal with CDK inhibition.

Estrogen, progesterone, and HER2 receptor-negative triple-negative breast cancers encompass the most clinically challenging subtype for which targeted therapeutics are lacking. We find that triple-negative tumors exhibit elevated MYC expression, as well as altered expression of MYC regulatory genes, resulting in increased activity of the MYC pathway. In primary breast tumors, MYC signaling did not predict response to neoadjuvant chemotherapy but was associated with poor prognosis. We exploit the increased MYC expression found in triple-negative breast cancers by using a synthetic-lethal approach dependent on cyclin-dependent kinase (CDK) inhibition. CDK inhibition effectively induced tumor regression in triple-negative tumor xenografts. The proapoptotic BCL-2 family member BIM is up-regulated after CDK inhibition and contributes to this synthetic-lethal mechanism. These results indicate that aggressive breast tumors with elevated MYC are uniquely sensitive to CDK inhibitors.

A genetic library screen for signaling proteins that interact with phosphorylated T cell costimulatory receptors.

In the past decade, the fundamental importance and therapeutic potential of costimulatory signals for lymphocyte activation have spurred a large amount of work in immunology, infection, cancer, autoimmune diseases, etc. However, the mechanisms behind T cell costimulation remain unclear, partly due to the lack of suitable techniques. There is an urgent need for functional genomic research to develop comprehensive approaches to direct identification of protein-protein interactions that are dependent on the posttranslational modification of one component of the complex, particularly in the field of T cell immunology. Using inducible costimulator (ICOS) as a model, we failed to find any proteins that associated with the cytoplasmic tail of ICOS by the yeast two-hybrid approach. Therefore, we have developed a new yeast three-hybrid system that facilitates the rapid screening of cDNA libraries to find signaling molecules that interact with phosphorylated T cell costimulatory receptors. We demonstrate the utility of this technique to detect the interaction between ICOS and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). The p85 unit of PI3K is the only signaling molecule identified so far that interacts with ICOS. This system may be of great help in dissecting the mechanisms of T cell costimulation and could be applied to other receptors.

Subnuclear shuttling of human telomerase induced by transformation and DNA damage.

The telomerase ribonucleoprotein complex caps chromosome ends by adding telomeric repeats. Here we show that catalytically active human telomerase has a regulated intranuclear localization that is dependent on the cell-cycle stage, transformation and DNA damage. In primary cell lines, low expression of a fusion protein of green fluorescent protein and telomerase reverse transcriptase (GFP-hTERT) increases telomerase activity and stabilizes the maintenance of telomere length. Confocal microscopy shows that the release of telomerase to the nucleoplasm from sequestration at nucleolar sites is enhanced at the expected time of telomere replication. By contrast, in tumour and transformed cells, there is an almost complete dissociation of telomerase from nucleoli at all stages of the cell cycle. Transfection of the simian virus 40 genome into a primary cell line is sufficient to mobilize telomerase from nucleoli to the nucleoplasm. Conversely, ionizing radiation induces the reassociation of telomerase with nucleoli in both primary and transformed cells. These findings show that transformation and DNA damage have opposite effects on the cellular regulation of active telomerase, affecting the enzyme's access to both telomeric and nontelomeric substrates.

Changes in monocyte/macrophage neurotoxicity in the era of HAART: implications for HIV-associated dementia.

OBJECTIVE: To determine changes in CD14/CD69 cells of HIV seropositive patients with HIV-1 associated dementia (HAD) on highly active antiretroviral therapy (HAART) and to determine the effect of soluble factors from cultured monocyte/macrophage (M/M phi) on neural cell functional proteins. DESIGN AND METHODS: Whole blood from patients with HAD, HIV seronegative subjects, and HIV seropositive subjects with no dementia (HIV-ND) was analyzed for CD14/CD69 cells using flow cytometry. M/M phi were isolated and cultured, and supernatants tested for neurotoxicity. Modulation of neural cell proteins and mitogen-activated protein kinases in response to supernatant exposure was examined by Western blot. RESULTS: CD14/CD69 levels from HAART-treated HAD subjects were significantly elevated over those from HIV-ND subjects and controls. However, levels were significantly lower than those reported in similarly selected HAD subjects tested prior to the HAART era. Treatment of neural cells with M/M phi-derived culture supernatants from HAART-treated HAD subjects was not associated with cell death, but resulted in a trend towards lower activation of the JNK, AKT, and ERK kinases, decreased expression of SNAP-25, and increased expression of neurofilament light than was observed after treatment with HIV-ND M/M phi supernatants. CONCLUSIONS: The clinical phenotype of HAD appears to be evolving from a subacute dementing disease to a more protracted disorder. Decreased CD14/CD69 levels may reflect changes in some aspects of the pathophysiology of brain injury in the era of HAART. Changes in neural cell signaling, structural and functional proteins may represent more subtle neurotoxicity, manifested in cell dysfunction rather than frank cell death.