Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target.

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the Plasmodium falciparum protein kinase PfCLK3, which we used in combination with chemogenetics to validate PfCLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for PfCLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of PfCLK3 mediated rapid killing of asexual liver- and blood-stage P. falciparum and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against P. berghei and P. knowlesi Hence, our data establish PfCLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria.

In situ growth in early lung adenocarcinoma may represent precursor growth or invasive clone outgrowth-a clinically relevant distinction.

The switch from in situ to invasive tumor growth represents a crucial stage in the evolution of lung adenocarcinoma. However, the biological understanding of this shift is limited, and 'Noguchi Type C' tumors, being early lung adenocarcinomas with mixed in situ and invasive growth, represent those that are highly valuable in advancing our understanding of this process. All Noguchi Type C adenocarcinomas (n = 110) from the LATTICE-A cohort were reviewed and two patterns of in situ tumor growth were identified: those deemed likely to represent a true shift from precursor in situ to invasive disease ('Noguchi C1') and those in which the lepidic component appeared to represent outgrowth of the invasive tumor along existing airspaces ('Noguchi C2'). Overall Ki67 fraction was greater in C2 tumors and only C1 tumors showed significant increasing Ki67 from in situ to invasive disease. P53 positivity was acquired from in situ to invasive disease in C1 tumors but both components were positive in C2 tumors. Likewise, vimentin expression was increased from in situ to invasive tumor in C1 tumors only. Targeted next generation sequencing of 18 C1 tumors identified four mutations private to the invasive regions, including two in TP53, while 6 C2 tumors showed no private mutations. In the full LATTICe-A cohort, Ki67 fraction classified as either less than or greater than 10% within the in situ component of lung adenocarcinoma was identified as a strong predictor of patient outcome. This supports the proposition that tumors of all stages that have 'high grade' in situ components represent those with aggressive lepidic growth of the invasive clone. Overall these data support that the combined growth of Noguchi C tumors can represent two differing biological states and that 'Noguchi C1' tumors represent the genuine biological shift from in situ to invasive disease.

Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas.

RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.

p53 mutants cooperate with HIF-1 in transcriptional regulation of extracellular matrix components to promote tumor progression.

Mutations in the TP53 gene and microenvironmentally driven activation of hypoxia-inducible factor-1 (HIF-1) typically occur in later stages of tumorigenesis. An ongoing challenge is the identification of molecular determinants of advanced cancer pathogenesis to design alternative last-line therapeutic options. Here, we report that p53 mutants influence the tumor microenvironment by cooperating with HIF-1 to promote cancer progression. We demonstrate that in non-small cell lung cancer (NSCLC), p53 mutants exert a gain-of-function (GOF) effect on HIF-1, thus regulating a selective gene expression signature involved in protumorigenic functions. Hypoxia-mediated activation of HIF-1 leads to the formation of a p53 mutant/HIF-1 complex that physically binds the SWI/SNF chromatin remodeling complex, promoting expression of a selective subset of hypoxia-responsive genes. Depletion of p53 mutants impairs the HIF-mediated up-regulation of extracellular matrix (ECM) components, including type VIIa1 collagen and laminin-γ2, thus affecting tumorigenic potential of NSCLC cells in vitro and in mouse models in vivo. Analysis of surgically resected human NSCLC revealed that expression of this ECM gene signature was highly correlated with hypoxic tumors exclusively in patients carrying p53 mutations and was associated with poor prognosis. Our data reveal a GOF effect of p53 mutants in hypoxic tumors and suggest synergistic activities of p53 and HIF-1. These findings have important implications for cancer progression and might provide innovative last-line treatment options for advanced NSCLC.

CD40L/IL-4-stimulated CLL demonstrates variation in translational regulation of DNA damage response genes including ATM.

CD40L/interleukin-4 (IL-4) stimulation occurs in vivo in the tumor microenvironment and induces global translation to varying degrees in individuals with chronic lymphocytic leukemia (CLL) in vitro. However, the implications of CD40L/IL-4 for the translation of specific genes is not known. To determine the most highly translationally regulated genes in response to CD40L/IL-4, we carried out ribosome profiling, a next-generation sequencing method. Significant differences in the translational efficiency of DNA damage response genes, specifically ataxia-telangiectasia-mutated kinase (ATM) and the MRE11/RAD50/NBN (MRN) complex, were observed between patients, suggesting different patterns of translational regulation. We confirmed associations between CD40L/IL-4 response and baseline ATM levels, induction of ATM, and phosphorylation of the ATM targets, p53 and H2AX. X-irradiation was used to demonstrate that CD40L/IL-4 stimulation tended to improve DNA damage repair. Baseline ATM levels, independent of the presence of 11q deletion, correlated with overall survival (OS). Overall, we suggest that there are individual differences in translation of specific genes, including ATM, in response to CD40L/IL-4 and that these interpatient differences might be clinically important.

TAp73 contributes to the oxidative stress response by regulating protein synthesis.

TAp73 is a transcription factor that plays key roles in brain development, aging, and cancer. At the cellular level, TAp73 is a critical homeostasis-maintaining factor, particularly following oxidative stress. Although major studies focused on TAp73 transcriptional activities have indicated a contribution of TAp73 to cellular metabolism, the mechanisms underlying its role in redox homeostasis have not been completely elucidated. Here we show that TAp73 contributes to the oxidative stress response by participating in the control of protein synthesis. Regulation of mRNA translation occupies a central position in cellular homeostasis during the stress response, often by reducing global rates of protein synthesis and promoting translation of specific mRNAs. TAp73 depletion results in aberrant ribosomal RNA (rRNA) processing and impaired protein synthesis. In particular, polysomal profiles show that TAp73 promotes the integration of mRNAs that encode rRNA-processing factors in polysomes, supporting their translation. Concurrently, TAp73 depletion causes increased sensitivity to oxidative stress that correlates with reduced ATP levels, hyperactivation of AMPK, and translational defects. TAp73 is important for maintaining active translation of mitochondrial transcripts in response to oxidative stress, thus promoting mitochondrial activity. Our results indicate that TAp73 contributes to redox homeostasis by affecting the translational machinery, facilitating the translation of specific mitochondrial transcripts. This study identifies a mechanism by which TAp73 contributes to the oxidative stress response and describes a completely unexpected role for TAp73 in regulating protein synthesis.

Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a (Ink4a/Arf).

Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard.

An improved analysis methodology for translational profiling by microarray.

Translational regulation plays a central role in the global gene expression of a cell, and detection of such regulation has allowed deciphering of critical biological mechanisms. Genome-wide studies of the regulation of translation (translatome) performed on microarrays represent a substantial proportion of studies, alongside with recent advances in deep-sequencing methods. However, there has been a lack of development in specific processing methodologies that deal with the distinct nature of translatome array data. In this study, we confirm that polysome profiling yields skewed data and thus violates the conventional transcriptome analysis assumptions. Using a comprehensive simulation of translatome array data varying the percentage and symmetry of deregulation, we show that conventional analysis methods (Quantile and LOESS normalizations) and statistical tests failed, respectively, to correctly normalize the data and to identify correctly deregulated genes (DEGs). We thus propose a novel analysis methodology available as a CRAN package; Internal Control Analysis of Translatome (INCATome) based on a normalization tied to a group of invariant controls. We confirm that INCATome outperforms the other normalization methods and allows a stringent identification of DEGs. More importantly, INCATome implementation on a biological translatome data set (cells silenced for splicing factor PSF) resulted in the best normalization performance and an improved validation concordance for identification of true positive DEGs. Finally, we provide evidence that INCATome is able to infer novel biological pathways with superior discovery potential, thus confirming the benefits for researchers of implementing INCATome for future translatome studies as well as for existing data sets to generate novel avenues for research.

Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation.

Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5'-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N'-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5'-deoxy-5'-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5'-deoxy-5'-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5'-deoxy-5'-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents.

mTORC1-mediated translational elongation limits intestinal tumour initiation and growth.

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.

Decreased translation of Dio3 mRNA is associated with drug-induced hepatotoxicity.

Recent work has demonstrated the importance of post-transcriptional gene regulation in toxic responses. In the present study, we used two rat models to investigate mRNA translation in the liver following xenobiotic-induced toxicity. By combining polysome profiling with genomic methodologies, we were able to assess global changes in hepatic mRNA translation. Dio3 (iodothyronine deiodinase type III) was identified as a gene that exhibited specific translational repression and had a functional role in a number of relevant canonical pathways. Western blot analysis indicated that this repression led to reduced D3 (the protein expressed by Dio3) levels, enhanced over time and with increased dose. Using Northern blotting techniques and qRT-PCR (quantitative reverse transcription-PCR), we confirmed further that there was no reduction in Dio3 mRNA, suggesting that translational repression of Dio3 is an important determinant of the reduced D3 protein expression following liver damage. Finally, we show that drug-induced hepatotoxicity appears to cause localized disruptions in thyroid hormone levels in the liver and plasma. We suggest that this leads to reduced translation of Dio3 mRNA, which results in decreased D3 production. It may therefore be possible that this is an important mechanism by which the liver can, upon early signs of damage, act rapidly to maintain its own energy equilibrium, thereby avoiding global disruption of the hypothalamic-pituitary-thyroid axis.

Failure of translation of human adenovirus mRNA in murine cancer cells can be partially overcome by L4-100K expression in vitro and in vivo.

Adaptive immune responses may be vital in the overall efficacy of oncolytic viruses in human malignancies. However, immune responses to oncolytic adenoviruses are poorly understood because these viruses lack activity in murine cells, which precludes evaluation in immunocompetent murine cancer models. We have evaluated human adenovirus activity in murine cells. We show that a panel of murine carcinoma cells, including CMT64, MOVCAR7, and MOSEC/ID8, can readily be infected with human adenovirus. These cells also support viral gene transcription, messenger RNA (mRNA) processing, and genome replication. However, there is a profound failure of adenovirus protein synthesis, especially late structural proteins, both in vitro and in vivo, with reduced loading of late mRNA onto ribosomes. Our data also show that in trans expression of the nonstructural late protein L4-100K increases both the amount of viral mRNA on ribosomes and the synthesis of late proteins, accompanied by reduced phosphorylation of eIF2α and improved anticancer efficacy. These results suggest that murine models that support human adenovirus replication could be generated, thus allowing evaluation of human adenoviruses in immunocompetent mice.