Translational Control Of Gene Expression Research Articles

Abstract 3332: Radiation-induced translational control of gene expression in glioblastoma stem-like cells

Abstract Radiation-induced gene expression has long been assumed to comprise a protective response against radiation-induced cell death. However, whereas radiation-induced changes in total cellular mRNA (i.e., the transcriptome) have been studied extensively, the vast majority of mRNA changes detected do not correlate with changes in the corresponding protein, a situation that has made it difficult to assign functional significance to radiation-induced gene expression. In contrast, analysis of polysome-bound RNA in established cell lines indicates that radiation selectively regulates gene translation, a process that operates independently from changes in the transcriptome. Moreover, characteristics of the radiation-induced translatome include a correlation between the genes whose translational activity is affected by radiation and the expression of their corresponding protein. To investigate the role of the translational control of gene expression in the radioresponse of glioblastoma (GBM), we have focused on a series of human glioblastoma stem-like cells (GSCs). Radiation-induced translatomes and transcriptomes were defined using microarray analysis of polysome-bound RNA and total RNA, respectively, for 4 GSCs and 2 normal cell types (astrocytes and pericytes). For each cell type, there was little overlap between the radiation-induced translatomes and transcriptomes. Subsequent analyses focused on those genes whose expression was only modified in the translatome, i.e., those subject to radiation-induced translational control. Hierarchical clustering of radiation-induced changes in KEGG Pathways showed that while the radiation-induced changes in the GSC lines were more similar to each other than to the normal CNS cells, there were also cell line-dependent changes. Rather than screen individual proteins, Ingenuity Pathway Analysis (IPA) was used to identify potential functional consequences of radiation-induced translational control. A significant network activated in all GSC translatomes was Response to DNA Damage. Consistent with activation of this pathway, post- irradiation addition of the mTOR inhibitor INK128, which inhibits translational control, slowed the dispersal of γH2AX foci. Among the radiation-induced translatome networks activated in a cell line specific manner was Mitochondrial Function. This was validated by the detection of radiation-induced changes in mitochondrial DNA and mass, which were prevented by addition of INK128 after irradiation. These results suggest that analysis of radiation-induced translatomes may provide novel insight into the general as well as cell specific mechanisms mediating radioresponse. In addition, they suggest that the translational control of gene expression may provide a source of molecular targets for GBM radiosensitization. Citation Format: Amy Wahba, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. Radiation-induced translational control of gene expression in glioblastoma stem-like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3332. doi:10.1158/1538-7445.AM2015-3332

Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression.

Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a cis-repressor at the 5' end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A trans-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability. We describe here a cis-repressor that can completely shut off translation of antibiotic-resistance reporters and a trans-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the trans-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control.

Translational regulation shapes the molecular landscape of complex disease phenotypes.

The extent of translational control of gene expression in mammalian tissues remains largely unknown. Here we perform genome-wide RNA sequencing and ribosome profiling in heart and liver tissues to investigate strain-specific translational regulation in the spontaneously hypertensive rat (SHR/Ola). For the most part, transcriptional variation is equally apparent at the translational level and there is limited evidence of translational buffering. Remarkably, we observe hundreds of strain-specific differences in translation, almost doubling the number of differentially expressed genes. The integration of genetic, transcriptional and translational data sets reveals distinct signatures in 3′UTR variation, RNA-binding protein motifs and miRNA expression associated with translational regulation of gene expression. We show that a large number of genes associated with heart and liver traits in human genome-wide association studies are primarily translationally regulated. Capturing interindividual differences in the translated genome will lead to new insights into the genes and regulatory pathways underlying disease phenotypes.

Meta-regulation of Arabidopsis auxin responses depends on tRNA maturation.

SummaryPolar transport of the phytohormone auxin throughout plants shapes morphogenesis and is subject to stringent and specific control. Here, we identify basic cellular activities connected to translational control of gene expression as sufficient to specify auxin-mediated development. Mutants in subunits of Arabidopsis Elongator, a protein complex modulating translational efficiency via maturation of tRNAs, exhibit defects in auxin-controlled developmental processes, associated with reduced abundance of PIN-formed (PIN) auxin transport proteins. Similar anomalies are observed upon interference with tRNA splicing by downregulation of RNA ligase (AtRNL), pointing to a general role of tRNA maturation in auxin signaling. Elongator Protein 6 (ELP6) and AtRNL expression patterns underline an involvement in adjusting PIN protein levels, whereas rescue of mutant defects by auxin indicates rate-limiting activities in auxin-controlled organogenesis. This emphasizes mechanisms in which auxin serves as a bottleneck for plant morphogenesis, translating common cellular activities into defined developmental readouts.

Ribosome Profiling Uncovers the Role of uORFs in Translational Control of Gene Expression during Erythroblast Differentiation

The erythroid progenitor compartment possesses a large expansion capacity, both in vivo and in vitro, which enables a rapid restoration of peripheral erythrocytes following severe blood loss. This expansion is tightly regulated to maintain erythrocyte numbers between narrow boundaries, and to balance expansion of the erythroid compartment against the availability of iron for heme and haemoglobin production. We previously observed that control of mRNA translation is crucial for expansion of the erythroid compartment. We also showed that translation of specific transcripts is impaired in Diamond Blackfan Anemia (DBA), a severe congenital anemia due to defective ribosome biosynthesis. Transcripts can be subject to translational control through domains in the 5’- or 3’UTR, including secondary structures, protein binding sequences and upstream open reading frames (uORFs). The presence of uORFs, including those starting at non-AUG codons in the 5’UTR, may alter the level of mRNA translation, but may also result in the expression of alternative protein isoforms because translation initiation may be redirected to more downstream start codons.The aim of our current studies is to provide a genome wide map of mRNA translation efficiency during erythropoiesis that can be used to investigate defective mRNA translation in, for instance, DBA. Ribosome profiling is a genome wide high-throughput sequencing technology for global mapping of translation initiation sites that allows translation analysis with codon resolution at the genome wide level. We first investigated translational changes occurring during differentiation of mouse erythroblasts. We used p53-deficient, growth factor dependent and differentiation competent immortalized erythroblast cultures that were expanded in presence of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids as T0, and subsequently differentiated the cells in presence of Epo for 17 and 46 hours (T17, and T46 samples). To obtain ribosome footprints, the cells were treated for 7 minutes with harringtonin or solvent, and subsequently for 5 minutes with cycloheximide, which arrests translation by stabilizing the ribosomes at translation initiation codons, or on all codons, respectively. We used optimized protocols for ribosome footprinting and data analysis, and focused the analysis on transcripts containing uORFs.First we performed a qualitative analysis of start codon usage. The ribosome footprint data proved to be superior to previously used polyribosome recruitment. In some cases polysome recruitment appeared to represent translation of an uORFs while the protein coding ORF is hardly translated (e.g. Csf2rb2, Puma). In another set of transcripts, we found uORFs that are differentially translated during differentiation, and thereby regulate differential translation from a downstream start codon (e.g. Klf3, Use1, CD47, Kell). Finally, comparison of ribosome footprints determined in erythroblasts and in myoblasts/myotubes revealed tissue specific translation regulation of otherwise ubiquitously expressed transcripts among which transcripts encoding ribosomal proteins. Second, we will perform quantitative analysis of mRNA translation in erythropoiesis through the comparison of ribosome footprint reads in an ORF with total mRNA reads obtained from total mRNA sequencing of the same sample.The obtained insight in transcript specific translation at codon resolution is of great value to understand many cellular processes during erythropoiesis, and will be of particular interest to understand responses to iron availability and reactive oxygen species that particularly affect translation of transcripts harboring uORFs. DisclosuresNo relevant conflicts of interest to declare.

Abstract SY42-01: Translational control and tumor stem cell radioresponse

Abstract SY42-01: Translational control and tumor stem cell radioresponse

Conserved regions of the DMD 3′ UTR regulate translation and mRNA abundance in cultured myotubes

Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease, is caused by mutations in the DMD gene, which encodes for the protein dystrophin. Its regulation is of therapeutic interest as even small changes in expression of functional dystrophin can significantly impact the severity of DMD. While tissue-specific distribution and transcriptional regulation of several DMD mRNA isoforms has been well characterized, the post-transcriptional regulation of dystrophin synthesis is not well understood. Here, we utilize qRTPCR and a quantitative dual-luciferase reporter assay to examine the effects of isoform specific DMD 5′ UTRs and the highly conserved DMD 3′ UTR on mRNA abundance and translational control of gene expression in C2C12 cells. The 5′ UTRs were shown to initiate translation with low efficiency in both myoblasts and myotubes. Whereas, two large highly conserved elements in the 3′ UTR, which overlap the previously described Lemaire A and D regions, increase mRNA levels and enhance translation upon differentiation of myoblasts into myotubes. The results presented here implicate an important role for DMD UTRs in dystrophin expression and delineate the cis-acting elements required for the myotube-specific regulation of steady-state mRNA levels and translational enhancer activity found in the DMD 3′ UTR.

Translational control of immune responses: from transcripts to translatomes.

Selective translational control of gene expression is emerging as a principal mechanism for the regulation of protein abundance that determines a variety of functions in both the adaptive immune system and the innate immune system. The translation-initiation factor eIF4E acts as a node for such regulation, but non-eIF4E mechanisms are also prevalent. Studies of 'translatomes' (genome-wide pools of translated mRNA) have facilitated mechanistic discoveries by identifying key regulatory components, including transcription factors, that are under translational control. Here we review the current knowledge on mechanisms that regulate translation and thereby modulate immunological function. We further describe approaches for measuring and analyzing translatomes and how such powerful tools can facilitate future insights on the role of translational control in the immune system.

Translational control of memory CD8 T cell differentiation (LYM4P.752)

Abstract CD8 T cells play an essential role in controlling viral as well as intracellular bacterial and parasitic infections. Memory CD8 T cells provide protective immunity upon re-encounter with the same pathogens. A better understanding of memory CD8 T cell development is critical for the rational design of vaccines. Transcriptome analyses in antigen-specific CD8 T cells have made a significant contribution to characterize global re-programming of gene expression during memory CD8 T cell differentiation. However, there has been minimal emphasis on understanding translational control of gene expression in antigen specific CD8 T cells. To examine mRNA translation during memory CD8 T cell differentiation, we performed longitudinal analyses of polysome profiles in antigen-specific CD8 T cells after acute LCMV infection. We found that mRNA translation was dynamically regulated during CD8 T cell responses; translation activity was significantly enhanced during clonal expansion phase, and it immediately returned to basal level at the peak of CD8 T cell responses. Genome-wide analyses of mRNA translation showed that a significant number of mRNAs were translationally regulated during CD8 T cell responses. Further experiments revealed that translational control of gene expression plays an important role in memory CD8 T cell formation. Thus, our studies provide a framework for understanding translational regulation that occurs during memory T cell differentiation.

Alternative translation initiation in immunity: MAVS learns new tricks

Translational control of gene expression contributes to various aspects of immune function [1]. Recent results by Brubaker et al. [2] show how alternative translation initiation produces distinct isoforms of Mitochondrial Antiviral Signaling (MAVS), an adaptor protein associated with RIG-I and MDA5 that possess unique immunomodulatory properties.

1H, 13C and 15N resonance assignment of WHEP domains of human glutamyl-prolyl tRNA synthetase

Human bifunctional glutamyl-prolyl tRNA synthetase (EPRS) contains three WHEP domains (R123) linking two catalytic domains. These three WHEP domains have been shown to be involved in protein-protein and protein-nucleic acid interactions. In translational control of gene expression, R12 repeats is known to interact with 3'UTR element in mRNAs of inflammatory gene for translational control mechanisms. While, R23 repeats interacts with NSAP1, which inhibits mRNA binding. Here we present the NMR chemical shift assignments for R12 (128 amino acids) as a 14 kDa recombinant protein and whole WHEP domains R123 (208 amino acids) as a 21 kDa recombinant protein. 97% of backbone and 98% of side-chain assignments have been completed in R12 analysis and 93 and 92% of backbone and side-chain, respectively, assignments have been completed in R123 analysis based upon triple-resonance experiments.

A Cross-Platform Comparison of Affymetrix and Agilent Microarrays Reveals Discordant miRNA Expression in Lung Tumors of c-Raf Transgenic Mice

Non-coding RNAs play major roles in the translational control of gene expression. In order to identify disease-associated miRNAs in precursor lesions of lung cancer, RNA extracts from lungs of either c-Raf transgenic or wild-type (WT) mice were hybridized to the Agilent and Affymetrix miRNA microarray platforms, respectively. This resulted in the detection of a range of miRNAs varying between 111 and 267, depending on the presence or absence of the transgene, on the gender, and on the platform used. Importantly, when the two platforms were compared, only 11–16% of the 586 overlapping genes were commonly detected. With the Agilent microarray, seven miRNAs were identified as significantly regulated, of which three were selectively up-regulated in male transgenic mice. Much to our surprise, when the same samples were analyzed with the Affymetrix platform, only two miRNAs were identified as significantly regulated. Quantitative PCR performed with lung RNA extracts from WT and transgenic mice confirmed only partially the differential expression of significant regulated miRNAs and established that the Agilent platform failed to detect miR-433. Finally, bioinformatic analyses predicted a total of 152 mouse genes as targets of the regulated miRNAs of which 4 and 11 genes were significantly regulated at the mRNA level, respectively in laser micro-dissected lung dysplasia and lung adenocarcinomas of c-Raf transgenic mice. Furthermore, for many of the predicted mouse target genes expression of the coded protein was also repressed in human lung cancer when the publically available database of the Human Protein Atlas was analyzed, thus supporting the clinical significance of our findings. In conclusion, a significant difference in a cross-platform comparison was observed that will have important implications for research into miRNAs.

GCN2 and TOR converge on aging

Coordinated responses in all tissues upon nutrient stress ensure the survival of an organism in periods of food unavailability. Two evolutionarily conserved, nutrient-sensing signaling pathways that promote stress adaptation following starvation are the general amino acid control pathway that activates GCN2 kinase and the target of rapamycin (TOR) kinase pathway [1]. In response to amino acid deprivation GCN2 is activated, upon binding of uncharged tRNAs, whereas TOR is inhibited through regulation of its localization. In both cases the outcome is reduction of global protein synthesis, albeit different mechanisms are involved. GCN2 directly phosphorylates the eukaryotic initiation factor 2α (eIF2α), whereas inactivation of TOR dephosphorylates the ribosomal protein S6 kinase (S6K) and the eukaryotic translation initiation factor 4E (eIF4)-binding protein (4E-BP). The result is repression of translation initiation of most mRNAs, accompanied by favored translation of specific mRNAs. Translational control of gene expression in response to nutrient and other environmental stresses contributes to stress management via energy saving and selective synthesis of stress-responsive proteins [2]. These effects might be also responsible for lifespan extension through down-regulation of mRNA translation. Inhibition of TOR signaling, by genetic or pharmacological means, has been associated with treatment of several diseases and increased lifespan in many organisms, including humans [3]. Similarly, a robust nutritional intervention that slows aging and its related pathology in diverse species is dietary restriction (DR), the effects of which, at the molecular level, are attributed largely to TOR inhibition. In consistence, deficiency of downstream targets of TOR such as S6K or translation factors/regulators extends lifespan in model systems [4]. Also, under conditions of impaired TOR, the activation of autophagy, a catabolic process that enhances degradation and recycling of damaged cellular components during ageing, contributes to longevity. Interestingly, regulation of autophagy in response to nutrient starvation involves GCN2 signaling, in yeast and mammalian cells [5]. Despite that both GCN2 and TOR can sense nutrient deprivation and regulate protein synthesis and autophagy, the impact of GCN2 signaling in longevity and its connection to TOR is not clear. Evidence in mice indicates that reduced amino acid levels activate GCN-2 and can suppress TOR activity on its targets S6K and 4E-BP. In our recent work [6], we showed that GCN-2 in Caenorhabditis elegans can influence the lifespan of nutrient-sensitized worms, through regulation of TOR signaling. Deletion of gcn-2 had no effect on growth and lifespan of wild-type animals under normal conditions but reduced their lifespan under amino acid limitation, as this was recapitulated through RNAi-mediated silencing of aminoacyl-tRNA synthetases genes. Moreover, disruption of gcn-2 decreased the long-life of eat-2 mutants (a genetic model of DR) and of TOR-defective worms. Longevity conferred by inactivation of the downstream target of TOR rsks-1/S6K, but not of ife-2/eIF4E, was also affected by GCN-2 loss. In C. elegans, the TOR-S6K signaling antagonizes a forkhead transcription factor, PHA-4/FoxA, with roles in early development, survival of first stage (L1) larvae under starvation and DR-mediated longevity of adults [7, 8]. We revealed that GCN-2 was required for the induction of pha-4 under conditions of amino acid deprivation or TOR inactivation. Consequently, the expression of known PHA-4 target genes, involved in stress defense and autophagy, was induced in a GCN-2-dependent manner thus contributing to stress survival and longevity of worms under nutrient or other stress (Figure ​(Figure11). Figure 1 Model depicting the function of GCN-2 in response to nutrient or environmental stress and its impact on survival and longevity of C. elegans. Although the ways in which PHA-4 regulates lifespan are not well-defined they may be relevant to the mechanisms used by L1 larvae to survive starvation [8]. PHA-4 has a broad role in regulation of gene expression but can preferentially bind to specific set of genes under distinct developmental or environmental conditions. Among the candidate PHA-4 targets in starved larvae are stress-responsive and autophagic genes, some of which are PHA-4-regulated in dietary restricted adults, suggesting common mechanisms. Intriguingly, genes involved in metabolic processes such as many nuclear hormone receptors (NHRs) and multiple regulators of sterol and fatty-acid metabolism constitute candidate targets of PHA-4 in starved larvae. Lipid metabolism and fat storage occurs mostly in the intestine of worms, where PHA-4 is induced upon DR, and has been linked to lifespan regulation. Thus, alterations in metabolic pathways in specific tissues, beyond cellular protection, could contribute to longevity induced by TOR inactivation during nutrient stress, but their dependence on GCN-2 remains to be addressed. Also, important questions remain with respect to the interplay between the GCN-2 and other stress signaling pathways that modulate lifespan under such conditions. Elucidation of the above is of great interest, as certain amino acid-restricted diets in rodents extend lifespan, improve their metabolic traits and confer protection from various age-related diseases.

Translational sensitivity of the Escherichia coli genome to fluctuating tRNA availability

The synthesis of protein from messenger RNA during translation is a highly dynamic process that plays a key role in controlling the efficiency and fidelity of genome-wide protein expression. The availability of aminoacylated transfer RNA (tRNA) is a major factor influencing the speed of ribosomal movement, which depending on codon choices, varies considerably along a transcript. Furthermore, it has been shown experimentally that tRNA availability can vary significantly under different growth and stress conditions, offering the cell a way to adapt translational dynamics across the genome. Existing models of translation have neglected fluctuations of tRNA pools, instead assuming fixed tRNA availabilities over time. This has lead to an incomplete understanding of this process. Here, we show for the entire Escherichia coli genome how and to what extent translational speed profiles, which capture local aspects of translational elongation, respond to measured shifts in tRNA availability. We find that translational profiles across the genome are affected to differing degrees, with genes that are essential or related to fundamental processes such as translation, being more robust than those linked to regulation. Furthermore, we reveal how fluctuating tRNA availability influences profiles of specific sequences known to play a significant role in translational control of gene expression.

The Akt/mTor signaling cascade is modified during placentation in the porcine uterine tissue

Recently we showed that essential components for the initiation of protein synthesis, namely the eukaryotic initiation factor 4E (eIF4E, mRNA-cap-binding protein) and its repressors 4E-BP1 as well as 4E-BP2, are proteolytically processed in the porcine endometrium during implantation. Here, the situation during placentation was compared with ovariectomized (OVX) animals and animals on pregnancy day 1 (PD1). Furthermore, the research was extended to factors which phosphorylate eIF4E and 4E-BPs and regulate their activities. These are the protein kinase B/mammalian target of rapamycin kinase (Akt/mTor) with the regulators Raptor and Rictor as well as the mitogen activated protein kinases (MAPKs): extra cellular-signal regulated kinase 1 and 2 (ERK1 and ERK2). Striking differences in the placentation site (PS) and the areas aside from PS (peri-PS) were observed. EIF4E and 4E-BP2 truncation as well as 4E-BP1 degradation took place in the endometrium of the peri-PS on PD24. Accompanied by a fragmentation of Akt/mTor, no expression of Rictor was observed, whereas the abundance of Raptor was not altered. On the contrary, MAPKs expression and phosphorylation remained almost stable in the peri-PS. In conclusion, the results indicated that on PD24 the translational regulation was shifted to 4E-BP2 control. Furthermore, the Akt/mTor signaling cascade seemed to be down regulated which suggest reduced phosphorylation of 4E-BP2. Whereas Akt was proteolyzed, the observed mTor fragments represented most likely splicing variants. The results indicate that translational control of gene expression is an important feature in the porcine endometrium during early pregnancy.

Hyperglycemia Mediates a Shift From Cap-Dependent to Cap-Independent Translation Via a 4E-BP1–Dependent Mechanism

Diabetes and its associated hyperglycemia induce multiple changes in liver function, yet we know little about the role played by translational control of gene expression in mediating the responses to these conditions. Here, we evaluate the hypothesis that hyperglycemia-induced O-GlcNAcylation of the translational regulatory protein 4E-BP1 alters hepatic gene expression through a process involving the selection of mRNA for translation. In both streptozotocin (STZ)-treated mice and cells in culture exposed to hyperglycemic conditions, expression of 4E-BP1 and its interaction with the mRNA cap-binding protein eIF4E were enhanced in conjunction with downregulation of cap-dependent and concomitant upregulation of cap-independent mRNA translation, as assessed by a bicistronic luciferase reporter assay. Phlorizin treatment of STZ-treated mice lowered blood glucose concentrations and reduced activity of the cap-independent reporter. Notably, the glucose-induced shift from cap-dependent to cap-independent mRNA translation did not occur in cells lacking 4E-BP1. The extensive nature of this shift in translational control of gene expression was revealed using pulsed stable isotope labeling by amino acids in cell culture to identify proteins that undergo altered rates of synthesis in response to hyperglycemia. Taken together, these data provide evidence for a novel mechanism whereby O-GlcNAcylation of 4E-BP1 mediates translational control of hepatic gene expression.

Puf Mediates Translation Repression of Transmission-Blocking Vaccine Candidates in Malaria Parasites

Translational control of gene expression plays an essential role in development. In malaria parasites, translational regulation is critical during the development of specialized transition stages between the vertebrate host and mosquito vector. Here we show that a Pumilio/FBF (Puf) family RNA-binding protein, PfPuf2, is required for the translation repression of a number of transcripts in gametocytes including two genes encoding the transmission-blocking vaccine candidates Pfs25 and Pfs28. Whereas studies to date support a paradigm of Puf-mediated translation regulation through 3′ untranslated regions (UTRs) of target mRNAs, this study, for the first time, identifies a functional Puf-binding element (PBE) in the 5′UTR of pfs25. We provide both in vitro and in vivo evidence to demonstrate that PfPuf2 binds to the PBEs in pfs25 and pfs28 to mediate translation repression. This finding provides a renewed view of Pufs as versatile translation regulators and sheds light on their functions in the development of lower branches of eukaryotes.

Abstract 69: In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128.

Abstract Radiation-induced translational control of gene expression has been shown to be a component of the cellular radioresponse. Furthermore, recent data suggest strategies targeting eIF4E may be effective for tumor cell radiosensitization. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of eIF4E and mRNA translation. The newly developed ATP-competitive mTOR inhibitor, INK128, inhibits both mTORC1 and mTORC2 activity, and is in currently undergoing evaluation in Phase 1 clinical trials. In the study described here we evaluated the effects of INK128 treatment on tumor cell radiosensitivity in vitro using a clonogenic survival assay and in vivo according to tumor growth delay. For tumor cell lines initiated from different histologic origins in vitro exposure to INK128 resulted in the complete inhibition of both mTORC1 and 2. Treatment with the same concentration of INK128 immediately after irradiation increased tumor cell radiosensitivity with DEFs greater than 1.3. To determine whether the INK128 induced radiosensitization could be extended to an in vivo model we used subcutaneous xenografts initiated from U251 or PSN1 tumor cells. INK128 treatment (oral gavage) inhibited mTOR activity in tumors established from both cell lines as defined by loss of 4E-BP1 phosphorylation. Furthermore, m7-GTP batch chromatography showed that INK128 treatment decreased cap-binding activity in xenografts. To evaluate the effects of INK128 on in vivo tumor radiosensitivity a tumor growth delay assay was performed using mice bearing U251 tumor xenografts. When tumors reached approximately 210mm3 mice were randomized into 4 groups: vehicle, radiation, INK128, and INK128 plus radiation. INK128 was delivered once a day (3 mg/kg, oral gavage) for four days with the tumor locally irradiated (2 Gy) 2h after each of the four drug treatments. Absolute growth delays were calculated as the time in days for tumors in treated mice to grow from 210 to 1000mm3 minus the time in days for tumors to reach the same size in vehicle treated mice. The absolute growth delays for INK128 alone and radiation alone groups were 1.7 and 12.7 days respectively. The growth delay in mice treated with the combination of INK128 and radiation was 22.2 days, which is greater than the sum of the growth delays caused by INK128 and radiation alone. The DEF, obtained by dividing the normalized tumor growth delay in mice treated with the radiation/PP242 combination (20.5) by the absolute growth delay in mice treated with radiation only (12.7), was 1.6. These results suggest that mTOR inhibition by the clinically relevant ATP-competitive mTOR inhibitor, INK128, enhances tumor cell radiosensitivity both in vitro and in vivo. Citation Format: Thomas J. Hayman, Tamalee Kramp, Kevin Camphausen, Philip J. Tofilon. In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 69. doi:10.1158/1538-7445.AM2013-69

Toxoplasma gondii Alba Proteins Are Involved in Translational Control of Gene Expression

Molecular mechanisms controlling gene expression in apicomplexan parasites remain poorly understood. Here, we report the characterization of two Toxoplasma gondii homologs of the ancient archeal Alba proteins named TgAlba1 and TgAlba2. The targeted disruption of TgAlba1 and TgAlba2 genes in both virulent type I and avirulent type II strains of T. gondii reveals that TgAlba proteins may have an important role in regulating stress response. We found that although the steady-state level of the Tgalba2 transcript is increased in the ΔTgalba1 null mutant parasites, the cognate TgAlba2 protein is undetectable, suggesting that TgAlba1 is required for translation of TgAlba2. Using a tandem affinity purification tag strategy combined with proteomic analyses, we provide evidence that many factors known to be involved in the translation machinery are co-purified with TgAlba1 and TgAlba2. We further performed RNA pull-down and microarray analyses to show that TgAlba1 and TgAlba2 bind to more than 30 RNAs including their own transcripts. Moreover, we demonstrate that the tight translational regulation of the TgAlba2 endogenous transcript relies on the presence of both its 3′ untranslated region and that of the TgAlba1 protein. Thus, our findings on TgAlba1 and TgAlba2 are consistent with a role in gene-specific translation.

Translational Homeostasis via the mRNA Cap-Binding Protein, eIF4E

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.