When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia [corrected] and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.

Purpose: Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers.To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis. Experimental Design: The first compound in this series, suitable for i.v. administration, has entered clinical development.To fully explore the potential of Polo-like kinase1inhibition in oncology, we have profiled additional compounds and now describe a novel clinical candidate. Results: BI 6727 is a highly potent (enzyme IC 50 = 0.87 nmol/L, EC 50 = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high volume of distribution and a long terminal half-life in mice (V ss = 7.6 L/kg, t 1/2 = 46 h) and rats (V ss = 22 L/kg, t 1/2 = 54 h). Second, BI 6727 has physicochemical and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer.With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. Conclusion: These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clinical studies have been initiated.

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREB␣ and ⌬ isoforms and to an overexpression of the CREB␤ isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREB␣, ␤, and ⌬) inactivated. In contrast to the CREB␣⌬ mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the ␣␤ lineage, which is not affected in CREB␣⌬ mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the ␣␤ T cell lineage. In contrast ␥␦ T cell differentiation is normal in CREB mutant mice.Many of the transcriptional effects of cAMP are mediated by the cAMP response element binding protein CREB, which binds to the CRE element in the upstream region of a variety of genes. CREB is a member of the CREB͞ATF family of transcription factors and is phosphorylated by protein kinase A after an intracellular increase in cAMP (1). More recently it has become apparent that CREB is an in vivo substrate for a variety of other kinases including calmodulin kinases II and IV (2) or RSK2 (3), implying that CREB activates transcription in response to cAMP, Ca 2ϩ , and growth factor stimulation (4-6).A targeted mutation of the CREB gene has been reported (7). Unexpectedly, these mice were healthy, fertile, and did not show any developmental abnormalities. However, they showed abnormalities in studies of learning and memory (8) and of behavioral responses to opiate withdrawal (9). Interestingly, a detailed analysis of these mice led to the identification of the isoform CREB␤ (10), which was strongly up-regulated in CREB mutant mice. This suggests that its presence in CREB mutant mice, in addition to an up-regulation of the cAMP response element modulator (CREM) (7) could explain the mild phenotype observed in these mice (CREB␣⌬ mice).To evaluate the in vivo significance of the newly identified CREB␤ isoform, we have generated CREB null mice by homologous recombination in embryonic stem cells. In contrast to CREB␣⌬ mice, CREB null mice die perinatally. Because CREM is also up-regulated in CREB null mice, CREB␤ is responsible for viability of CREB␣⌬ mice. In this report, we describe the phenotype of the CREB null mice, which gives insights into the developmental role of CREB.Functionally important CRE elements are found in the regulatory regions of a variety of T cell-specific genes (11, 12), implicating members of the CREB͞ATF family in the regulation of gene expression during T cell differentiation and activation. However, the physiological importance of these findings remains to be determined. It has re...

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

Activating transcription factor 1 (ATF1), CREB, and the cyclic AMP (cAMP) response element modulatory protein (CREM), which constitute a subfamily of the basic leucine zipper transcription factors, activate gene expression by binding as homo-or heterodimers to the cAMP response element in regulatory regions of target genes. To investigate the function of ATF1 in vivo, we inactivated the corresponding gene by homologous recombination. In contrast to CREB-deficient mice, which suffer from perinatal lethality, mice lacking ATF1 do not exhibit any discernible phenotypic abnormalities. Since ATF1 and CREB but not CREM are strongly coexpressed during early mouse development, we generated mice deficient for both CREB and ATF1. ATF1 CREB؊/؊ embryos die before implantation due to developmental arrest. ATF1 ؉/؊ CREB ؊/؊ embryos display a phenotype of embryonic lethality around embryonic day 9.5 due to massive apoptosis. These results indicate that CREB and ATF1 act in concert to mediate signals essential for maintaining cell viability during early embryonic development.The activating transcription factor 1 (ATF1), CREB, and the cyclic AMP response element modulatory protein (CREM) share high sequence homology and mediate the transcriptional response to various extracellular signals, including peptide hormones (15, 27), growth factors (10, 11, 26), neurotransmitters, and Ca 2ϩ (17,23). Activation of the CREB/CREM/ATF1 proteins is mediated via different signaling pathways which converge to phosphorylate a distinct serine residue (22). Phosphorylation of CREB by mitogen-activated protein kinase and Akt/protein kinase B has been implicated as important for cellular survival in cultured cells (4, 9). CREB is also thought to act as an antiapoptotic factor in sympathetic neurons (19). In human clear cell sarcoma, the ATF1 gene is fused to the genes encoding Ewing's sarcoma protein and seems to be responsible for maintaining tumor viability (5). It was also suggested that ATF1 is upregulated in human metastatic melanoma cells. Disruption of ATF1 activity in these cells by using an inhibitory anti-ATF1 antibody fragment suppressed their tumorigenicity and metastatic potential in nude mice (14).As previously reported, the corresponding genes of CREB and CREM have been inactivated by gene targeting in mice. Mice lacking the CREM gene exhibit an arrest in spermatogenesis (3, 16), whereas CREB-deficient mice die perinatally due to atelectasis of the lung (21). We show here that loss of ATF1 function after inactivation of the ATF1 gene in mice does not cause any obvious phenotypic abnormalities. Our expression studies showed that ATF1 and CREB but not CREM are strongly coexpressed during early mouse development. To identify the role of both proteins during early development and to circumvent possible compensatory effects, we therefore generated mice deficient for both ATF1 and CREB. Interestingly, complete inactivation of both proteins, ATF1 and CREB, results in embryonic death before implantation. Embryos with only one functional ATF1...

DR5 (also called TRAIL receptor 2 and KILLER) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called TRAIL and Apo2 ligand). DR5 is a transcriptional target of p53, and its overexpression induces cell death in vitro. However, the in vivo biology of DR5 has remained largely unexplored. To better understand the role of DR5 in development and in adult tissues, we have created a knockout mouse lacking DR5. This mouse is viable and develops normally with the exception of having an enlarged thymus. We show that DR5 is not expressed in developing embryos but is present in the decidua and chorion early in development. DR5-null mouse embryo fibroblasts expressing E1A are resistant to treatment with TRAIL, suggesting that DR5 may be the primary proapoptotic receptor for TRAIL in the mouse. When exposed to ionizing radiation, DR5-null tissues exhibit reduced amounts of apoptosis compared to wild-type thymus, spleen, Peyer's patches, and the white matter of the brain. In the ileum, colon, and stomach, DR5 deficiency was associated with a subtle phenotype of radiation-induced cell death. These results indicate that DR5 has a limited role during embryogenesis and early stages of development but plays an organ-specific role in the response to DNA-damaging stimuli.

Scaffold modification based on Wang's pioneering MDM2-p53 inhibitors led to novel, chemically stable spiro-oxindole compounds bearing a spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-one scaffold that are not prone to epimerization as observed for the initial spiro[3H-indole-3,3'-pyrrolidin]-2(1H)-one scaffold. Further structure-based optimization inspired by natural product architectures led to a complex fused ring system ideally suited to bind to the MDM2 protein and to interrupt its protein-protein interaction (PPI) with TP53. The compounds are highly selective and show in vivo efficacy in a SJSA-1 xenograft model even when given as a single dose as demonstrated for 4-[(3S,3'S,3'aS,5'R,6'aS)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-(cyclopropylmethyl)-2-oxo-1,2,3',3'a,4',5',6',6'a-octahydro-1'H-spiro[indole-3,2'-pyrrolo[3,2-b]pyrrole]-5'-yl]benzoic acid (BI-0252).

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immunedeficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.