Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDKactivating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a ]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC 50 of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 Mmol/L, with CDK2 being inhibited 35-fold less potently (IC 50 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent. [Cancer Res 2009;69(15):6208-15]

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC₅₀= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G₂/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI₅₀= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

2Protective CD8 + T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named Lymphocyte Expansion Molecule (LEM) that promoted antigen-dependent CD8 + T cell proliferation, effector function and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results.Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration resulting in the production of pro-proliferative mitochondrial Reactive Oxygen Species (mROS).LEM provides a link between immune activation and the expansion of protective CD8 + T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified CTL. 3Cytotoxic CD8 + T cells (CTL) are a central arm of the immune system responsible for protection from intracellular viruses and cancer because they kill infected or transformed cells (1). Since chronic virus infection (2) and cancer (3) are wide spread diseases it is clear that CTL-immunity often fails. A major reason for this failure is because high viral (4, 5) or tumor (6-8) load results in either deletion or functional inactivation (known as immune exhaustion) of CTL. The result is failure in both short-term CTL immunity and immunological memory because memory CD8 T cell development is blocked (9). Impaired expansion is an important cause of deletion and immune exhaustion and results in the failure to produce sufficient numbers of protective CTL and memory cells (5). Retro mutant mice have increased immunity to chronic viral infectionInfection of wild-type C57BL/6 mice with the clone 13 variant of lymphocytic choriomeningitis virus (LCMV C13) is an established model for human chronic viral infection resulting in a massive viral load that causes both deletion and immune exhaustion of CTL and a block in memory CD8 T cell development (10).We examined the CTL response to LCMV C13 infection after germ-line mutagenesis to identify mutants with enhanced immunity. To this end, 430 third-generation (G3) ethyl-N-nitrosourea (ENU)-induced germ-line mutants were produced in a C57BL/6J background (11). G3 mice were infected with LCMV C13 and after 8 days the level of LCMV-specific CD8 T cells measured in the spleen by staining with a tetramer for the np396 LCMV peptide and flow-cytometry (12). Three independent germ-line transmissible modifications, which resulted in increased levels of LCMV-specific 4 CD8 T cells were isolated, of which one (a semi-dominant) was bred to homozygosity (Fig. S1a). We named this strain Retro.Homozygous Retro mutant mice showed a 10-fold increase in CD8 T cells specific for LCMV np396 peptide compared to wild-type (WT) and a smaller but significant increase in the number of CTL specific for the gp33-LCMV peptide ( Fig. 1 a, b).Compared to WT mice, a smaller percentage of Retr...

Regulatory T-cells (Tregs) play a pivotal role in the maintenance of immune tolerance and hold great promise as cell therapy for a variety of immune-mediated diseases. However, the cellular mechanisms that regulate Treg maintenance and homeostasis have yet to be fully explored. While Tregs express Granzyme-B (GrB) to suppress effector T-cells via direct-killing, the mechanisms by which they protect themselves from GrB-mediated self-inflicted damage are unknown. We show, for the first time, that both iTregs and nTregs increase their intracellular expression of GrB and its endogenous inhibitor, Serine Protease Inhibitor-6 (Spi6) upon activation. Sub-cellular fractionation and measurement of GrB activity in the cytoplasm of Tregs show that activated Spi6−/− Tregs had significantly higher cytoplasmic GrB activity. We observed an increase in GrB-mediated apoptosis in Spi6−/− nTregs and impaired suppression of alloreactive T-cells in vitro. Spi6−/− Tregs were rescued from apoptosis by the addition of a GrB inhibitor (Z-AAD-CMK) in vitro. Furthermore, adoptive transfer experiments showed that Spi6−/− nTregs were less effective than WT nTregs in suppressing Graft-versus-host-disease (GVHD) due to their impaired survival, as shown in our in vivo bioluminescence imaging. Finally, Spi6-deficient recipients rejected MHC class II-mismatch heart allografts at a much faster rate and showed a higher rate of apoptosis among Tregs, as compared to WT recipients. Our data demonstrate, for the first time, a novel role for Spi6 in Treg homeostasis by protecting activated Tregs from GrB-mediated injury. These data could have significant clinical implications for Treg-based therapy in immune-mediated diseases.