SUMMARY The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells.

The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.A defining pathology of type 2 diabetes is impaired insulinstimulated glucose uptake in skeletal muscle. Skeletal muscle is the largest tissue in the human body by mass and is the chief site of insulin-stimulated glucose disposal. Insulin stimulation causes translocation of GLUT4 glucose transporters from intracellular regions to the plasma membrane and t-tubule system where they function to import glucose. In individuals with type 2 diabetes, insulin fails to stimulate adequate GLUT4 translocation, resulting in impaired glucose uptake and poor glucose tolerance.Skeletal muscle is unique as an insulin-sensitive tissue because voluntary contraction during exercise causes GLUT4 translocation completely independent of insulin signaling (1, 2). Contraction-stimulated glucose uptake is preserved in the muscle of individuals with type 2 diabetes, thus demonstrating the existence of signaling pathways that circumvent defective components of the insulin signaling pathway (3). If and where insulin-and contraction-stimulated glucose uptake pathways converge have been topics of considerable interest. Recently, the Akt substrate of 160 kDa (AS160) 2 was identified as a mediator of both insulin-and contraction-stimulated glucose uptake and, therefore, a potential nexus for convergent signaling (4, 5).A...

Host proteins are essential for entry and replication of HIV and provide important non-viral therapeutic targets. Large-scale RNAi-based screens have identified nearly a thousand candidate host factors, but with little agreement among studies and few validated factors. Here, we demonstrate that a genome-wide CRISPR-based screen identifies bona fide host factors in a physiologically relevant cell system. We identify five factors, including CD4 and CCR5, that are required for HIV infection yet dispensable for cellular proliferation and viability. TPST2 and SLC35B2 act in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating recognition by HIV envelope. ALCAM mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validate these pathways in primary human CD4+ T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest focusing on these pathways for therapeutic intervention.

5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.

Results from the Diabetes Prevention Program highlight the effectiveness of metformin or regular physical activity in the prevention of type 2 diabetes. Independently, metformin and exercise increase insulin sensitivity, but they have not been studied in combination. To assess the combined effects, insulin-resistant subjects ( n = 9) matched for weight, body fat, and aerobic fitness were studied before any treatment (B), after 2–3 wk of 2,000 mg/day metformin (MET), and after metformin plus 40 min of exercise at 65% V̇o2peak (MET + Ex). A second group ( n = 7) was studied at baseline and after an identical bout of exercise with no metformin (Ex). Biopsies of the vastus lateralis were taken at B, after MET, immediately after MET + Ex ( group 1), or immediately after Ex ( group 2). Insulin sensitivity was assessed 4 h postexercise with a euglycemic hyperinsulinemic (40 mU·m2·min−1) clamp enriched with [6,6-2H]glucose. Insulin sensitivity was 54% higher after Ex ( P < 0.01), but there was no change with Met + Ex. Skeletal muscle AMPKα2 activity was elevated threefold ( P < 0.01) after Ex, but there was no increase with MET + Ex. These findings suggest that the combination of short-term metformin treatment and an acute bout of exercise does not enhance insulin sensitivity, and the addition of metformin may attenuate the well-documented effects of exercise alone.

OBJECTIVETBC1D1 is a member of the TBC1 Rab-GTPase family of proteins and is highly expressed in skeletal muscle. Insulin and contraction increase TBC1D1 phosphorylation on phospho-Akt substrate motifs (PASs), but the function of TBC1D1 in muscle is not known. Genetic linkage analyses show a TBC1D1 R125W missense variant confers risk for severe obesity in humans. The objective of this study was to determine whether TBC1D1 regulates glucose transport in skeletal muscle.RESEARCH DESIGN AND METHODSIn vivo gene injection and electroporation were used to overexpress wild-type and several mutant TBC1D1 proteins in mouse tibialis anterior muscles, and glucose transport was measured in vivo.RESULTSExpression of the obesity-associated R125W mutant significantly decreased insulin-stimulated glucose transport in the absence of changes in TBC1D1 PAS phosphorylation. Simultaneous expression of an inactive Rab-GTPase (GAP) domain of TBC1D1 in the R125W mutant reversed this decrease in glucose transport caused by the R125W mutant. Surprisingly, expression of TBC1D1 mutated to Ala on four conserved Akt and/or AMP-activated protein kinase predicted phosphorylation sites (4P) had no effect on insulin-stimulated glucose transport. In contrast, expression of the TBC1D1 4P mutant decreased contraction-stimulated glucose transport, an effect prevented by concomitant disruption of TBC1D1 Rab-GAP activity. There was no effect of the R125W mutation on contraction-stimulated glucose transport.CONCLUSIONSTBC1D1 regulates both insulin- and contraction-stimulated glucose transport, and this occurs via distinct mechanisms. The R125W mutation of TBC1D1 impairs skeletal muscle glucose transport, which could be a mechanism for the obesity associated with this mutation.

BackgroundSiberian apricot (Prunus sibirica L.) has emerged as a novel potential source of biodiesel in China, but the molecular regulatory mechanism of oil accumulation in Siberian apricot seed kernels (SASK) is still unknown at present. To better develop SASK oil as woody biodiesel, it is essential to profile transcriptome and to identify the full repertoire of potential unigenes involved in the formation and accumulation of oil SASK during the different developing stages.ResultsWe firstly detected the temporal patterns for oil content and fatty acid (FA) compositions of SASK in 7 different developing stages. The best time for obtaining the high quality and quantity of SASK oil was characterized at 60 days after flowering (DAF), and the representative periods (10, 30, 50, 60, and 70 DAF) were selected for transcriptomic analysis. By Illumina/Solexa sequencings, approximately 65 million short reads (average length = 96 bp) were obtained, and then assembled into 124,070 unigenes by Trinity strategy (mean size = 829.62 bp). A total of 3,000, 2,781, 2,620, and 2,675 differentially expressed unigenes were identified at 30, 50, 60, and 70 DAF (10 DAF as the control) by DESeq method, respectively. The relationship between the unigene transcriptional profiles and the oil dynamic patterns in developing SASK was comparatively analyzed, and the specific unigenes encoding some known enzymes and transcription factors involved in acetyl-coenzyme A (acetyl-CoA) formation and oil accumulation were determined. Additionally, 5 key metabolic genes implicated in SASK oil accumulation were experimentally validated by quantitative real-time PCR (qRT-PCR). Our findings could help to construction of oil accumulated pathway and to elucidate the molecular regulatory mechanism of increased oil production in developing SASK.ConclusionsThis is the first study of oil temporal patterns, transcriptome sequencings, and differential profiles in developing SASK. All our results will serve as the important foundation to further deeply explore the regulatory mechanism of SASK high-quality oil accumulation, and may also provide some reference for researching the woody biodiesel plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0213-3) contains supplementary material, which is available to authorized users.

This review surveys the chemical and biological literature dealing with the isolation, structural elucidation and bioactivity of hericenones and erinacines from the fruiting body and mycelium of Hericium erinaceus, concentrating on work that has appeared in the literature up to December 2009.