Both in vitro and in vivo evidence indicate that cAMP-dependent protein kinase (PKA) mediates some of the acute and chronic cellular responses to alcohol. However, it is unclear whether PKA regulates voluntary alcohol consumption. We therefore studied alcohol consumption by mice that completely lack the regulatory IIbeta (RIIbeta) subunit of PKA as a result of targeted gene disruption. Here we report that RIIbeta knockout mice (RIIbeta-/-) showed incr eased consumption of solutions containing 6, 10, and 20% (v/v) ethanol when compared with wild-type mice (RIIbeta+/+). On the other hand, RIIbeta-/- mice showed normal consumption of solutions containing either sucrose or quinine. When compared with wild-type mice, the RIIbeta-/- mice were found to be less sensitive to the sedative effects of ethanol as measured by more rapid recovery from ethanol-induced sleep, even though plasma ethanol concentrations did not differ significantly from those of controls. Finally, both RIbeta- and catylatic subunit beta1-deficient mice showed normal voluntary consumption of ethanol, indicating that increased ethanol consumption is not a general characteristic associated with deletion of PKA subunits. These data demonstrate a role for the RIIbeta subunit of PKA in regulating voluntary consumption of alcohol and sensitivity to the intoxication effects that are produced by this drug.

Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. Here we describe a novel role for BRD4 in DNA damage response (DDR). BRD4 associates with and regulates the function of pre-replication factor CDC6 and plays an indispensable part in DNA replication checkpoint signaling. Inhibition of BRD4 by JQ1 or AZD5153 resulted in a rapid, time-dependent reduction in CHK1 phosphorylation and aberrant DNA replication re-initiation. Furthermore, BRD4 inhibition sensitized cancer cells to various replication stress-inducing agents, and synergized with ATR inhibitor AZD6738 to induce cell killing across a number of cancer cell lines. The synergistic interaction between AZD5153 and AZD6738 is translatable to in vivo ovarian cell-line and patient-derived xenograft models. Taken together, our study uncovers a new biological function of BRD4 and provides mechanistic rationale for combining BET inhibitors with DDR-targeted agents for cancer therapy.

Using the cre-loxP recombination system, we generated a line of mice expressing a constitutively active catalytic subunit of Protein Kinase A (PKA) in a temporally and spatially regulated fashion. In the absence of cre recombinase the modified catalytic subunit allele is functionally silent, but after recombination the mutant allele is expressed, resulting in enhanced PKA effects at basal cAMP levels. Mice expressing the modified protein in hepatocytes using albumin-cre transgenics show defects in glucose homeostasis, glycogen storage, fructose 2,6-bisphosphate levels, and induction of glucokinase mRNA during feeding. Similar to animals lacking glucokinase in the liver (Postic et al.: J Biol Chem 274:305-315, 1999), these mice also have defects in glucose-stimulated insulin secretion, a hallmark of Type II diabetes. The widespread expression of PKA and the involvement of this kinase in a myriad of signaling pathways suggest that these animals will provide critical tools for the study of PKA function in vivo.

We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology.

The V600E mutation of B-Raf kinase results in constitutive activation of the MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-based design, a novel series of pyrazolopyridine inhibitors of B-Raf(V600E) was developed. Optimization led to the identification of 3-methoxy pyrazolopyridines 17 and 19, potent, selective, and orally bioavailable agents that inhibited tumor growth in a mouse xenograft model driven by B-Raf(V600E) with no effect on body weight. On the basis of their in vivo efficacy and preliminary safety profiles, 17 and 19 were selected for further preclinical evaluation.