Development of a prognostic model for anoikis and identifies hub genes in hepatocellular carcinoma.

Considering the high fatality of hepatocellular carcinoma (HCC), current prognostic systems are insufficient to accurately forecast HCC patients' outcomes. In our study, nine anoikis­related genes (PTRH2, ITGAV, ANXA5, BIRC5, BDNF, BSG, DAP3, SKP2, and EGF) were determined to establish a risk scoring model using LASSO regression, which could be validated in ICGC dataset. Kaplan-Meier curves and time-dependent receiver operating characteristic (ROC) curve analysis confirmed the risk score possessed an accurate predictive value for the prognosis of HCC patients. The high-risk group showed a higher infiltration of aDCs, macrophages, T-follicular helper cells, and Th2 cells. Besides, PD-L1 was significantly higher in the high-risk group compared to the low-risk group. Several anoikis­related genes, such as ANX5, ITGAV, BDNF and SKP2, were associated with drug sensitivity in HCC. Finally, we identified BIRC5 and SKP2 as hub genes among the nine model genes using WGCNA analysis. BIRC5 and SKP2 were over-expressed in HCC tissues, and their over-expression was associated with poor prognosis, no matter in our cohort by immunohistochemical staining or in the TCGA cohort by mRNA-Seq. In our cohort, BIRC5 expression was highly associated with the T stage, pathologic stage, histologic grade and AFP of HCC patients. In general, our anoikis-related risk model can enhance the ability to predict the survival outcomes of HCC patients and provide a feasible therapeutic strategy for immunotherapy and drug resistance in HCC. BIRC5 and SKP2 are hub genes of anoikis­related genes in HCC.

Mining of long non-coding RNAs with target genes in response to rust based on full-length transcriptome in Kentucky bluegrass.

Kentucky bluegrass (Poa pratensis L.) is an eminent turfgrass species with a complex genome, but it is sensitive to rust (Puccinia striiformis). The molecular mechanisms of Kentucky bluegrass in response to rust still remain unclear. This study aimed to elucidate differentially expressed lncRNAs (DELs) and genes (DEGs) for rust resistance based on the full-length transcriptome. First, we used single-molecule real-time sequencing technology to generate the full-length transcriptome of Kentucky bluegrass. A total of 33,541 unigenes with an average read length of 2,233 bp were obtained, which contained 220 lncRNAs and 1,604 transcription factors. Then, the comparative transcriptome between the mock-inoculated leaves and rust-infected leaves was analyzed using the full-length transcriptome as a reference genome. A total of 105 DELs were identified in response to rust infection. A total of 15,711 DEGs were detected (8,278 upregulated genes, 7,433 downregulated genes) and were enriched in plant hormone signal transduction and plant-pathogen interaction pathways. Additionally, through co-location and expression analysis, it was found that lncRNA56517, lncRNA53468, and lncRNA40596 were highly expressed in infected plants and upregulated the expression of target genes AUX/IAA, RPM1, and RPS2, respectively; meanwhile, lncRNA25980 decreased the expression level of target gene EIN3 after infection. The results suggest that these DEGs and DELs are important candidates for potentially breeding the rust-resistant Kentucky bluegrass.

Glutamine synthetase gene PpGS1.1 negatively regulates the powdery mildew resistance in Kentucky bluegrass.

Excessive nitrogen (N) application may induce powdery mildew (PM) in perennial grasses, but the resistance mechanisms to PM remain unclear. This study evaluated the physiological and molecular mechanisms of PM resistance affected by N supplies in Kentucky bluegrass (Poa pratensis L.). Cultivar 'Bluemoon' (N tolerant) and 'Balin' (N sensitive) were treated with low N (0.5 mM), normal N (15 mM), and high N (30 mM) for 21 d in a greenhouse. With increasing N levels, the disease growth was more severe in 'Balin' than in 'Bluemoon'. RNA-seq and weighted gene coexpression network analysis revealed that the PpGS1.1 gene encoding glutamine synthetase was a potential hub gene for PM resistance after comparisons across cultivars and N treatments. The N metabolism pathway was connected with the plant-pathogen interaction pathway via PpGS1.1. The expression of PpGS1.1 in rice protoplasts indicated that the protein was located in the nucleus and cytoplasm. Overexpression of PpGS1.1 in wild-type Kentucky bluegrass increased carbon and N contents, and the transgenic plants became more susceptible to PM with a lower wax density. The most differentially expressed genes (DEGs) for N metabolism were upregulated and DEGs for fatty acid metabolism pathway were downregulated in the overexpression lines. The results elucidated mechanisms of PM resistance in relation to N metabolism in Kentucky bluegrass.

Identification of Golovinomyces artemisiae Causing Powdery Mildew, Changes in Chlorophyll Fluorescence Parameters, and Antioxidant Levels in Artemisia selengensis.

Artemisia selengensis Turcz. is a valuable edible and medicinal vegetable crop widely cultivated in Northeast China. Powdery mildew (PM) disease occurs during field and greenhouse cultivation, resulting in production losses and quality deterioration. The pathogen in A. selengensis was Golovinomyces artemisiae identified using optical microscopic and scanning electron microscopic observations, morphological identification, and molecular biological analyses. Parameters of chlorophyll fluorescence (ChlF) and antioxidant system responses as well as callose and lignin contents in A. selengensis were analyzed with inoculating G. artemisiae. Obvious of PM-infected leaves were confirmed with significantly lower values in electron transport rate (ETR), non-photochemical quenching (NPQ), photochemical quenching (qP), and actual photochemical efficiency [Y(II)], but higher values in non-adjusting energy dissipation yield [Y(NO)], supposed that maximal photosystem II quantum yield (Fv/Fm) value and images could be used to monitor PM degree on infectedA. selengensis. In addition, malondialdehyde (MDA), superoxide anion (O2 -), callose, lignin contents, and peroxidase (POD) activity increased, while superoxide dismutase (SOD) activity, catalase (CAT) activity, and ascorbic acid (AsA) content decreased significantly in infected leaves compared to mock-inoculated leaves, indicated that lignin and protective enzymes are the key indicators for detecting PM resistant in A. selengensis. These results suggest that PM caused by G. artemisiae disrupted the photosynthetic capacity and induced imbalance of antioxidant system inA. selengensis. The findings were of great significance for designing a feasible approach to effectively prevent and control the PM disease in A. selengensis as well as in other vegetable crops.

Control of coronary vascular resistance by eicosanoids via a novel GPCR.

Arachidonic acid metabolites epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs) are important regulators of myocardial blood flow and coronary vascular resistance (CVR), but their mechanisms of action are not fully understood. We applied a chemoproteomics strategy using a clickable photoaffinity probe to identify G protein-coupled receptor 39 (GPR39) as a microvascular smooth muscle cell (mVSMC) receptor selective for two endogenous eicosanoids, 15-HETE and 14,15-EET, which act on the receptor to oppose each other's activity. The former increases mVSMC intracellular calcium via GPR39 and augments coronary microvascular resistance, and the latter inhibits these actions. Furthermore, we find that the efficacy of both ligands is potentiated by zinc acting as an allosteric modulator. Measurements of coronary perfusion pressure (CPP) in GPR39-null hearts using the Langendorff preparation indicate the receptor senses these eicosanoids to regulate microvascular tone. These results implicate GPR39 as an eicosanoid receptor and key regulator of myocardial tissue perfusion. Our findings will have a major impact on understanding the roles of eicosanoids in cardiovascular physiology and disease and provide an opportunity for the development of novel GPR39-targeting therapies for cardiovascular disease.

Cloning of TaeRF1 gene from Caucasian clover and its functional analysis responding to low-temperature stress.

Low temperature (LT) is an important threat to the normal growth of plants. In this study, based on the full-length transcriptome sequencing results, the cold resistance genes were cloned from Caucasian clover with strong cold resistance. We cloned the CDS of TaeRF1, which is 1311 bp in length and encodes 436 amino acids. The molecular weight of the protein is 48.97 kDa, which had no transmembrane structure, and its isoelectric point (pI) was 5.42. We predicted the structure of TaeRF1 and found 29 phosphorylation sites. Subcellular localization showed that TaeRF1 was localized and expressed in cell membrane and chloroplasts. The TaeRF1 gene was induced by stress due to cold, salt, alkali and drought and its expression level was higher in roots and it was more sensitive to LT. Analysis of transgenic A. thaliana plants before and after LT treatment showed that the TaeRF1 gene enhanced the removal of excess H2O2, and increased the activity of antioxidant enzymes, thus improving the plant's ability to resist stress. Additionally, the OE lines showed increased cold tolerance by upregulating the transcription level of cold-responsive genes (CBF1, CBF2, COR15B, COR47, ICE1, and RD29A). This study demonstrates that TaeRF1 is actively involved in the responses of plants to LT stress. We also provide a theoretical basis for breeding and a potential mechanism underlying the responses of Caucasian clover to abiotic stress.

Prediction of acute coronary syndrome within 3 years using radiomics signature of pericoronary adipose tissue based on coronary computed tomography angiography.

OBJECTIVES: To evaluate whether radiomics signature of pericoronary adipose tissue (PCAT) based on coronary computed tomography angiography (CCTA) could improve the prediction of future acute coronary syndrome (ACS) within 3 years. METHODS: We designed a retrospective case-control study that patients with ACS (n = 90) were well matched to patients with no cardiac events (n = 1496) during 3 years follow-up, then which were randomly divided into training and test datasets with a ratio of 3:1. A total of 107 radiomics features were extracted from PCAT surrounding lesions and 14 conventional plaque characteristics were analyzed. Radiomics score, plaque score, and integrated score were respectively calculated via a linear combination of the selected features, and their performance was evaluated with discrimination, calibration, and clinical application. RESULTS: Radiomics score achieved superior performance in identifying patients with future ACS within 3 years in both training and test datasets (AUC = 0.826, 0.811) compared with plaque score (AUC = 0.699, 0.640), with a significant difference of AUC between two scores in the training dataset (p = 0.009); while the improvement of integrated score discriminating capability (AUC = 0.838, 0.826) was non-significant. The calibration curves of three predictive models demonstrated a good fitness respectively (all p > 0.05). Decision curve analysis suggested that integrated score added more clinical benefit than plaque score. Stratified analysis revealed that the performance of three predictive models was not affected by tube voltage, CT version, different sites of hospital. CONCLUSION: CCTA-based radiomics signature of PCAT could have the potential to predict the occurrence of subsequent ACS. Radiomics-based integrated score significantly outperformed plaque score in identifying future ACS within 3 years. KEY POINTS: • Plaque score based on conventional plaque characteristics had certain limitations in the prediction of ACS. • Radiomics signature of PCAT surrounding plaques could have the potential to improve the predictive ability of subsequent ACS. • Radiomics-based integrated score significantly outperformed plaque score in the identification of future ACS within 3 years.

Turf performance and physiological responses of native Poa species to summer stress in Northeast China.

Rapid rise in temperature in summer causes severe injury to cool-season turfgrass of both native species and introduced ones in Heilongjiang of Northeast China. The objectives of this study were to compare physiological responses to seasonal heat stresses and turf performances between native and introduced commercial Poa accessions. Three Chinese native Poa species (i.e., P. pratensis, P. sibirica and P. sphondylodes) and three USA Kentucky bluegrass cultivars (ie. 'Midnight', 'Moonlight' and 'BlueChip') were evaluated under field conditions in 2017 and 2018. All accessions showed unique characteristics and considerable seasonal differences in response to temperatures. However, performances over all accessions were largely similar in early spring and autumn. In summer, native P. pratensis performed similar to 'Midnight', 'Moonlight' or 'BlueChip', with respect to such traits or parameters as quality, coverage, color intensity, growth rate, osmolytes, ROS and anti-oxidant production. Native P. pratensis could be used as a new turf resource for further improvement and application under the specific climatic conditions in Heilongjiang; native P. sphondylodes may be used in repairing damaged environments or for alternative seasonal greenness.

Differential Metabolomic Responses of Kentucky Bluegrass Cultivars to Low Nitrogen Stress.

Kentucky bluegrass (Poa pratensis L.) is a cool-season turfgrass species that responds strongly to nitrogen (N), but the metabolomic responses of this grass species to N supply is unknown. The N-tolerant cultivar Bluemoon and N-sensitive cultivar Balin were exposed to normal N (15 mM) and low N (0.5 mM) for 21 days for identification of differentially expressed metabolites (DEMs) between normal N and low N treatments. Balin had more reductions of chlorophyll and total soluble protein concentrations and a higher accumulation of superoxide radicals under low N stress. A total of 99 known DEMs were identified in either cultivar or both including 22 amino acids and derivatives, 16 carbohydrates, 29 organic acids, and 32 other metabolites. In Bluemoon, ß-alanine metabolism was most enriched, followed by alanine, aspartate, and glutamate metabolism, biosynthesis of valine, leucine, and isoleucine biosynthesis, and glycine, serine, and threonine metabolism. In Balin, alanine, aspartate, and glutamate metabolism were most enriched, followed by the tricarboxylic acid (TCA), glyoxylate and decarbohydrate metabolism, and carbon fixation. Bluemoon generally maintained higher TCA cycle capacity and had more downregulated amino acids, while changes in more organic acids occurred in Balin under low N stress. Some metabolite changes by low-N stress were cultivar-specific. The results suggested that regulation of metabolites related to energy production or energy saving could contribute to low N tolerance in Kentucky bluegrass.

Assessment of differences in morphological and physiological leaf lodging characteristics between two cultivars of Hippeastrum rutilum.

BACKGROUND: Environmental lodging stress, which is a result of numerous factors, is characterized by uncertainty. However, several studies related to lodging in cereal crops have reported that lodging in the Hippeastrum rutilum environment is very rare. Hippeastrum rutilum is a garden flower with high ornamental value and abundant germplasm resources. Under past cultivation practices, it was found that the plant types of 'Red Lion', with red flowers, and 'Apple Blossom', with pink flowers, are quite different. The leaves of 'Red Lion' are upright, while the leaves of 'Apple Blossom' show lodging, which seriously affects its ornamental value. The aims of this study were to compare the differences between the two varieties with leaf lodging and upright leaves according to morphological and physiological attributes. In this study, karyotype analysis and phenotypic morphological and physiological characteristics were compared to explore the differences between the two plant types. RESULTS: The karyotype analysis of the two cultivars showed that their chromosome types were both tetraploid plants. The results showed that the lignin content in the leaves of 'Red Lion' was high, the cross-sectional structure of the leaf vascular bundle was more stable, and the chlorophyll content was high. In addition, significantly less energy was transferred to the electron transport chain (ETR) during the photoreaction. Similarly, the results regarding the maximum photosynthetic rate (Fv/Fm), nonphotochemical quenching (NPQ) and effective quantum yield of photosystem II photochemistry (△F/Fm') all indicated that the photosynthetic capacity of "Red Lion" was greater than that of "Apple Blossom", which was affected by leaf lodging. The size of the leaves was significantly smaller, and the leaf sag angle, leaf width, and leaf tip angle presented significantly lower values in 'Red Lion' than in 'Apple Blossom', which exhibits leaf sag. The difference in these factors may be the reason for the different phenotypes of the two cultivars. CONCLUSION: The results of this study proved that lodging affects the photosynthetic capacity of Hippeastrum rutilum and revealed some indexes that might be related to leaf lodging, laying a theoretical foundation for cultivating and improving new varieties.

Beneficial Effects of Mixing Kentucky Bluegrass With Red Fescue via Plant-Soil Interactions in Black Soil of Northeast China.

Continuous monoculture of cool-season turfgrass causes soil degradation, and visual turf quality decline is a major concern in black soil regions of Northeast China. Turf mixtures can enhance turfgrass resistance to biotic and abiotic stresses and increase soil microbial diversity. Understanding mechanism by plant-soil interactions and changes of black soil microbial communities in turf mixture is beneficial to restoring the degradation of urbanized black soils and maintaining sustainable development of urban landscape ecology. In this study, based on the previous research of different sowing models, two schemes of turf monoculture and mixture were conducted in field plots during 2016-2018 in a black soil of Heilongjiang province of Northeast China. The mixture turf was established by mixing 50% Kentucky bluegrass "Midnight" (Poa pratensis L.) with 50% Red fescue "Frigg" (Festuca rubra L.); and the monoculture turf was established by sowing with pure Kentucky bluegrass. Turf performance, soil physiochemical properties, and microbial composition from rhizosphere were investigated. Soil microbial communities and abundance were analyzed by Illumina MiSeq sequencing and quantitative PCR methods. Results showed that turfgrass quality, turfgrass biomass, soil organic matter (SOM), urease, alkaline phosphatase, invertase, and catalase activities increased in PF mixture, but disease percentage and soil pH decreased. The microbial diversity was also significantly enhanced under turf mixture model. The microbial community compositions were significantly different between the two schemes. Turf mixtures obviously increased the abundances of Beauveria, Lysobacter, Chryseolinea, and Gemmatimonas spp., while remarkably reduced the abundances of Myrothecium and Epicoccum spp. Redundancy analysis showed that the compositions of bacteria and fungi were related to edaphic parameters, such as SOM, pH, and enzyme activities. Since the increasing of turf quality, biomass, and disease resistance were highly correlated with the changes of soil physiochemical parameters and microbial communities in turf mixture, which suggested that turf mixture with two species (i.e., Kentucky blue grass and Red fescue) changed soil microbial communities and enhanced visual turfgrass qualities through positive plant-soil interactions by soil biota.

Effects of Exogenous Putrescine on Delaying Senescence of Cut Foliage of Nephrolepis cordifolia.

Senescence is the main limitation for cut foliage display in vase. Naturally occurring polyamines such as putrescine (Put) have been considered effective anti-senescence agents. However, effect of Put on cut foliage in vase in a realistic indoor environment has not yet been revealed. In the present study, effects of Put spraying on the postharvest performance of cut foliage of Nephrolepis cordifolia L. were investigated. Cut fronds sprayed with deionized water (Put0) showed visible injuries after 10 days in vase. Meanwhile, chlorophyll (Chl), soluble protein (Sp), and proline (Pro) content were decreased by 60.15, 57.93, and 73.09% respectively, photochemical activity reflected by Chl fluorescence parameters was inhibited, whereas electrolyte leakage (EL), contents of soluble sugar (Ss), malondialdehyde (MDA), and hydrogen peroxide (H2O2) were increased (+194.29, +44.83, +34.06, and +178.01%, respectively). Put spraying extended the vase life of the cut foliage and the 2.0 mM Put had a longer vase life (21 days) than 0.2 mM (15 days). Leaf spraying of 2.0 mM Put for 10 days significantly ameliorated the losses of Chl, Sp, and Pro content (-10.72, -26.29, and -42.64%, respectively), followed by 0.2 mM Put (-27.36, -36.24, and -60.55%, respectively). Put spraying also improved photochemical capability and prevented membrane impairment as well as visible injury in comparison with Put0. In addition, 2.0 mM Put had a better mitigating ability than that of 0.2 mM. Leaf spraying of 2.0 mM Put greatly reduced the decline of the effective quantum yield of photochemical energy conversion in PSII (ΦPSII), the maximal quantum yield of PSII photochemistry measured in the dark-adapted state (Fv/Fm) and electron transport rate (ETR) (-7.89, -12.91, and -10.06%, respectively), and also inhibited the increases of EL, MDA, Ss, and H2O2 (+31.87, +6.43, +16.22, and +49.40%, respectively). Overall, Put played important roles in deterring the degradation of Chl, Ss, and Pro, detoxifying the H2O2, weakening the sugar signaling, mitigating the decline of photochemical activity, and eventually postponing the leaf senescence. The present study gives new insights into effects of Put on leaf senescence and provides a strategy for preserving post-harvest cut foliage.

Mechanistic insights into the activation of ester prodrugs of 666-15.

cAMP-response element binding protein (CREB) is an oncogenic transcription factor implicated in many different types of cancer. We previously reported the discovery of 666-15 as a potent inhibitor of CREB-mediated gene transcription. In an effort to improve the aqueous solubility of 666-15, amino ester prodrugs 1 and 4 were designed and synthesized. Detailed chemical and biological studies of 1 and 4 revealed that a small portion of the prodrugs were converted into 666-15 through intermediate 3 instead of a long-range O,N-acyl transfer reaction that was initially proposed. These results provide unique insights into the activation of these ester prodrugs.

Discovery of a Synergistic Inhibitor of cAMP-Response Element Binding Protein (CREB)-Mediated Gene Transcription with 666-15.

CREB is a transcription factor implicated in the pathogenesis of multiple cancers. Targeting CREB is a promising strategy to develop potential cancer therapeutics. Previously, we identified 666-15 as a potent CREB inhibitor. Herein, we designed an ester prodrug of 666-15 through a long-range O,N-acyl transfer reaction for improved aqueous solubility. Unexpectedly, we discovered a small molecule 11 (653-47) that can potentiate the CREB inhibitory activity of 666-15 although 653-47 alone does not inhibit CREB.

Biosynthesis and Signal Transduction of ABA, JA, and BRs in Response to Drought Stress of Kentucky Bluegrass.

Kentucky bluegrass (KB, Poa pratensis) is one of the most widely used cool-season turfgrass species, but it is sensitive to drought stress. Molecular studies in KB are hindered by its large and complex genome structure. In this study, a comparative transcriptomic study was conducted between a short and long period of water deficiency. Three transcriptome libraries were constructed and then sequenced by using leaf RNA samples of plants at 0, 2, and 16 h after PEG6000 treatment. A total of 199,083 differentially expressed genes (DEGs) were found. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that DEGs were enriched in "Plant hormone signal transduction" and "MAPK signaling pathway-Plant". Some key up-regulated genes, including PYL, JAZ, and BSK, were involved in hormone signaling transduction of abscisic acid, jasmonic acid, and brassinosteroid and possibly these genes play important roles in coping with drought stress in KB. Furthermore, our results showed that the concentrations of ABA, JA and BR increased significantly with the extension of the drought period. The specific DEGs encoding functional proteins, kinase and transcription factors, could be valuable information for genetic manipulation to promote drought tolerance of KB in the future.

Design, synthesis and biological evaluation of regioisomers of 666-15 as inhibitors of CREB-mediated gene transcription.

cAMP-response element binding protein (CREB) is a nuclear transcription factor that has been implicated in the pathogenesis and maintenance of various types of human cancers. Identification of small molecule inhibitors of CREB-mediated gene transcription has been pursued as a novel strategy for developing cancer therapeutics. We recently discovered a potent and cell-permeable CREB inhibitor called 666-15. 666-15 is a bisnaphthamide and has been shown to possess efficacious anti-breast cancer activity without toxicity in vivo. In this study, we designed and synthesized a series of analogs of 666-15 to probe the importance of regiochemistry in naphthalene ring B. Biological evaluations of these analogs demonstrated that the substitution pattern of the alkoxy and carboxamide in naphthalene ring B is very critical for maintaining potent CREB inhibition activity, suggesting that the unique bioactive conformation accessible in 666-15 is critically important.

Functional screening for G protein-coupled receptor targets of 14,15-epoxyeicosatrienoic acid.

Epoxyeicosatrienoic acids (EETs) are potent vasodilators that play important roles in cardiovascular physiology and disease, yet the molecular mechanisms underlying the biological actions of EETs are not fully understood. Multiple lines of evidence suggest that the actions of EETs are in part mediated via G protein-coupled receptor (GPCR) signaling, but the identity of such a receptor has remained elusive. We sought to identify 14,15-EET-responsive GPCRs. A set of 105 clones were expressed in Xenopus oocyte and screened for their ability to activate cAMP-dependent chloride current. Several receptors responded to micromolar concentrations of 14,15-EET, with the top five being prostaglandin receptor subtypes (PTGER2, PTGER4, PTGFR, PTGDR, PTGER3IV). Overall, our results indicate that multiple low-affinity 14,15-EET GPCRs are capable of increasing cAMP levels following 14,15-EET stimulation, highlighting the potential for cross-talk between prostanoid and other ecosanoid GPCRs. Our data also indicate that none of the 105 GPCRs screened met our criteria for a high-affinity receptor for 14,15-EET.

Systemic Inhibition of CREB is Well-tolerated in vivo.

cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics.

Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity.

Recent studies have shown that nuclear transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is overexpressed in many different types of cancers. Therefore, CREB has been pursued as a novel cancer therapeutic target. Naphthol AS-E and its closely related derivatives have been shown to inhibit CREB-mediated gene transcription and cancer cell growth. Previously, we identified naphthamide 3a as a different chemotype to inhibit CREB's transcription activity. In a continuing effort to discover more potent CREB inhibitors, a series of structural congeners of 3a was designed and synthesized. Biological evaluations of these compounds uncovered compound 3i (666-15) as a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 µM). 666-15 also potently inhibited cancer cell growth without harming normal cells. In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity. These results further support the potential of CREB as a valuable cancer drug target.

The ITIM-containing receptor LAIR1 is essential for acute myeloid leukaemia development.

Conventional strategies are not particularly successful in the treatment of leukaemia, and identification of signalling pathways crucial to the activity of leukaemia stem cells will provide targets for the development of new therapies. Here we report that certain receptors containing the immunoreceptor tyrosine-based inhibition motif (ITIM) are crucial for the development of acute myeloid leukaemia (AML). Inhibition of expression of the ITIM-containing receptor LAIR1 does not affect normal haematopoiesis but abolishes leukaemia development. LAIR1 induces activation of SHP-1, which acts as a phosphatase-independent signalling adaptor to recruit CAMK1 for activation of downstream CREB in AML cells. The LAIR1-SHP-1-CAMK1-CREB pathway sustains the survival and self-renewal of AML stem cells. Intervention in the signalling initiated by ITIM-containing receptors such as LAIR1 may result in successful treatment of AML.