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Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component., Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148., Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed., Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial.

Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.

Purpose: Preoperative chemotherapy has demonstrated a survival benefit for patients with potentially resectable esophageal cancer; however, currently it is not possible to predict the benefit of this treatment for an individual patient. This prospective study was designed to correlate gene expression profiles with clinical outcome in this setting., Patients and Methods: Eligible patients were deemed to have resectable disease after staging by computed tomography, endoscopic ultrasound, and laparoscopy as indicated and following discussion at the multidisciplinary team meeting. All patients received neoadjuvant platinum and fluoropyrimidine-based chemotherapy; and clinical data were entered prospectively onto a study-specific database. Total RNA was isolated from pretreatment tumor biopsies obtained at baseline endoscopy and analyzed using a cDNA array consisting of 22,000 cDNA clones., Results: Of the patients with adequate follow-up accrued between 2002 and 2005, 35 satisfied the quality control measures for the microarray profiling. Median follow-up was 938 days. Supervised hierarchical clustering of normalized data revealed 165 significantly differentially expressed genes based on overall survival (OS; P < .01) with 2 distinct clusters: a poor outcome group: N = 17 (1 year OS 46.2%) and a good outcome group: N = 18 (1 year OS 100%). Genes identified included those previously associated with esophageal cancer and, interestingly, a group of genes encoding proteins involved in the regulation of the TOLL receptor-signaling pathway., Conclusion: This initial study has highlighted groups of tumors with distinct gene expression profiles based on survival and warrants further validation in a larger cohort. This approach may further our understanding of individual tumor biology and thus facilitate the development of tailored treatment.

The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials.

HM74 and HM74a have been identified as receptors for niacin. HM74a mediates the pharmacological anti-lipolytic effects of niacin in adipocytes by reducing intracellular cyclic AMP (cAMP) and inhibiting release of free fatty acids into the circulation. In macrophages, niacin induces peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent and cAMP-dependent expression of genes mediating reverse cholesterol transport, although via an unidentified receptor. We describe constitutive expression of HM74a mRNA and hypoxia- and IFNgamma-inducible expression of HM74 and HM74a in human monocytic cell lines and primary cells in culture. In U937 cells niacin-induced expression of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent endogenous ligand of PPARgamma. Both niacin and the structurally distinct HM74/HM74a ligand acifran-induced nuclear expression of PPARgamma protein and enhanced PPARgamma transcriptional activity. Niacin-induced PPARgamma transcriptional activity was pertussis toxin sensitive and required activity of phospholipase A(2) (EC 3.1.1.4), cyclo-oxygenase (EC 1.14.99.1) and prostaglandin D(2) synthase (EC 5.3.99.2). Niacin also induced PPARgamma transcriptional activity in HM74 and HM74a CHO cell transfectants, although not in vector-only control cells. This was sensitive to pertussis toxin and to inhibition of phoshoplipase A(2) and cyclo-oxygenase activity. Additionally, niacin increased intracellular cAMP in U937 via a pertussis toxin and cyclo-oxygenase-sensitive mechanism. These results indicate that HM74 and HM74a can mediate macrophage responses to niacin via activation of the prostaglandin synthesis pathway and induction and activation of PPARgamma. This suggests a novel mechanism(s) mediating the clinical effects of pharmacological doses of niacin.

Often the use of cytotoxic drugs in cancer therapy results in stable disease rather than regression of the tumor, and this is typically seen as a failure of treatment. We now show that DNA damage is able to induce senescence in tumor cells expressing wild-type p53. We also show that cytotoxics are capable of inducing senescence in tumor tissue in vivo. Our results suggest that p53 and p21 play a central role in the onset of senescence, whereas p16(INK4a) function may be involved in maintaining senescence. Thus, like apoptosis, senescence appears to be a p53-induced cellular response to DNA damage and an important factor in determining treatment outcome.

In this study the effects of SN-38 on colon adenocarcinoma cell lines expressing wild-type p53 (LS174T) or mutant non-functional p53 (HT29) have been investigated. On exposure to SN-38, HT29 cells rapidly progressed through G1 and S and arrested in G2/M. Release and concomitant increase in apoptosis after 48 h was concentration- and time-dependent (P < 0.001), being more rapid at higher concentrations, but reaching plateau at 10 ng ml(-1) with prolonged exposure. LS174T cells showed only a small increase in apoptosis, and only at high concentrations (50-100 ng ml(-1)). The main effect of SN-38 in LS174T cells was prolonged cell cycle arrest, which was independent of concentration. Arrest occurred in all phases of the cell cycle, with the distribution depending on concentration (P < 0.001) and not duration (P > 0.05). With increasing concentration, LS174T cells arrested in G2/M, S and G1. Cell cycle arrest was coincident with increased p53 expression in each phase of the cell cycle. Expression in G1 increased with time and concentration (P < 0.001, P = 0.01 respectively)whereas in S and G2/M p53 expression increased only with time (P< 0.001). Dose-dependent p53-associated G1 arrest, in the absence of DNA synthesis indicates an additional cytotoxic mechanism for SN-38, which requires higher concentrations than the S phase mechanism, and detection of which seems to involve p53. For incubations with the same ED (exposure x duration), apoptosis in HT29 cells was significantly higher for prolonged exposure to lower concentrations, whereas in LS174T cells there was a trend towards increased apoptosis with shorter exposures to higher concentrations, indicating a schedule effect of SN-38. Although expression of wild-type p53 leads to a more rapid induction of apoptosis, SN-38 cytotoxicity was generally greater in cells with mutant p53, as wild-type cells escaped apoptosis by p53 associated prolonged cell cycle arrest. Thus, pulsed schedules with higher doses may be more effective in cells expressing wild-type p53, whereas continued exposure with protracted schedules may be more active in cells expressing mutant p53.

Most modern chemo- and radiotherapy treatments of human cancers use the DNA damage pathway, which induces a p53 response leading to either G1 arrest or apoptosis. However, such treatments can induce mutations and translocations leading to secondary malignancies or recurrent disease, which often have a poor prognosis because of resistance to therapy. Here we report that 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK7 TFIIH-associated kinase, CKI and CKII kinases, blocking RNA polymerase II in the early elongation stage, triggers p53-dependent apoptosis in human colon adenocarcinoma cells in a transcription independent manner. The fact that DRB kills tumour-derived cells without employment of DNA damage gives rise to the possibility of the development of a new alternative chemotherapeutic treatment of tumours expressing wild type p53, with a decreased risk of therapy-related, secondary malignancies.

Bovine pancreatic ribonuclease (RNase A) is a member of a homologous group of extensively studied proteins. It is a small, basic protein, containing 124 amino acid residues and four stabilizing disulfide bridges. Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids. Since this degradation of RNA interferes with normal cell functions, the signal peptide of alkaline phosphatase (phoA, Escherichia coli) was cloned onto the gene coding for RNase A, directing the protein to the periplasm. Several expression systems have been evaluated which use T7, trc, or PR promoters to transcribe the RNase A gene. Also, variation in host strains was tested to optimize the protein yield. It was found that the PR system gave better expression than the two other systems. E. coli strain BL21 was shown to be the strain in which export to the periplasm was most effective and recombinant RNase A could be isolated from the periplasmic fraction of these cells. The system provides a stable yield of active recombinant bovine pancreatic RNase of about 45-50 mg/liter of cell culture.