Genomic Aberrations that Activate D-type Cyclins Are Associated with Enhanced Sensitivity to the CDK4 and CDK6 Inhibitor Abemaciclib.

Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3'UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.

LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti-tumor activities in mouse xenograft models.

The HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min(-1) and t1/2 of 525 min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037).

Changes in osteoblast, chondrocyte, and adipocyte lineages mediate the bone anabolic actions of PTH and small molecule GSK-3 inhibitor.

Parathyroid hormone (PTH) and glycogen synthase kinase-3 (GSK-3) inhibitor 603281-31-8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281-31-8 in rat osteopenia model. Female 6-month-old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1-38) at 10 microg/kg/day s.c. or 603281-31-8 at 3 mg/kg/day p.o. for 60 days. Twenty-four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P<0.05) were subjected to pathway analysis to delineate relevant bio-processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK-3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three-fold increase in marrow adiposity. PTH and GSK-3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK-3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU-fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK-3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes.

Optimization and validation of small quantity RNA profiling for identifying TNF responses in cultured human vascular endothelial cells.

INTRODUCTION: Affymetrix oligonucleotide microarrays are widely used in basic and applied research (Lander, E.S., (1999). Array of hope. Nature Genetics 21, 3-4; Lockhart, D.J. & Winzeler, E.A. (2000) Genomics, gene expression and DNA arrays. Nature 405, 827-836.) The need for a significant amount of starting RNA has limited its use in applications where the amount of RNA is limiting, such as with Laser Captured Microdissection (LCM), small biopsies, or peripheral blood in rodent models. To overcome this limitation, various RNA amplification and labeling methods have been described, however, further optimization and validation of these methods are needed. METHODS: Here we reported using the Arcturus technology to optimize amplification and labeling of small amounts of RNA for Affymetrix microarray studies. We assessed the technical feasibility and variation introduced by differences in starting RNA quantity and differences in technical performance by microarray hybridization. RESULTS: We demonstrated that the current approach is reliable to amplify as little as 40 ng total RNA, and it is suitable for Affymetrix studies yielding satisfactory quantitative chip performance. We also showed that differences in labeling methods contribute more to variation than the differences in starting RNA quantity per se. As a model, we studied the well-documented TNF-induced inflammatory responses in cultured human vascular endothelial cells. We were able to recapitulate the TNF-induced responses using small RNA sample profiling.

Genetic polymorphisms in human proton-dependent dipeptide transporter PEPT1: implications for the functional role of Pro586.

The human proton-dependent dipeptide transporter (PEPT1, gene SLC15A1) is important for intestinal absorption of di- and tripeptides and a variety of peptidomimetic compounds. Using a DNA polymorphism discovery panel of 44 ethnically diverse individuals, nine nonsynonymous and four synonymous coding-region single-nucleotide polymorphisms (SNPs) were identified in PEPT1. HeLa cells were transiently transfected with plasmids constructed by site-directed mutagenesis for each of the nine nonsynonymous variants. Quantitative polymerase chain reaction showed that the mRNA transcription level of all of the mutants was comparable with the mRNA transcription level of the reference sequence in transfected HeLa cells. Functional analysis in transiently transfected HeLa cells revealed that all nonsynonymous variants retained similar pH-dependent activity and K(t) values for [glycyl-1,2-(14)C]glycylsarcosine (Gly-Sar) uptake as the reference PEPT1. In addition, a group of seven peptide-like drugs showed inhibitory effect on Gly-Sar uptake by these variants comparable with the reference, suggesting conserved drug recognition. Of the nine nonsynonymous SNPs, a single SNP (P586L) demonstrated significantly reduced transport capacity as evidenced by a much lower V(max) value. This was consistent with lower immunoactive protein level (Western analysis) and lower plasma membrane expression (immunocytochemical analysis). Therefore, Pro(586) may have profound effect on PEPT1 translation, degradation, and/or membrane insertion.

Influence of ADH1B polymorphism on alcohol use and its subjective effects in a Jewish population.

Class I alcohol dehydrogenases (ADHs) are the principal enzymes responsible for ethanol metabolism in humans. Genetic polymorphism at the ADH1B locus (old nomenclature ADH2) results in isozymes with quite different catalytic properties. The frequency of the ADH1B*2 allele varies among ethnic groups. ADH1B*2 is most often observed in Asian populations, and has been shown to be protective against alcoholism. The Jewish population has a higher frequency of the ADH1B*2 allele and lower rates of alcohol-related problems as compared to other Caucasian populations. Thus, it would be of interest to determine whether the ADH1B*2 allele is associated with alcohol consumption and its subjective effects in this group. Four groups of Jewish subjects (male and female college-age samples, and male and female general samples) were recruited from the same region of the United States. All subjects completed a questionnaire to delineate alcohol consumption and its subjective consequences. Genotype at the ADH1B locus was determined for each participant. ADH1B*2 allele frequencies were similar for the Jewish college-age and general population samples. Men in both the college-age and general population in the ADH1B*2 group reported more unpleasant reactions following alcohol consumption than men in the ADH1B*1 group. Men in the general population in the ADH1B*2 group drank alcohol less frequently than men who were homozygous ADH1B*1; there was a similar trend among the women. The ADH1B polymorphism is associated with unpleasant reactions after alcohol consumption, and frequency of alcohol consumption in these Jewish samples.