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The pentacyclic acridinium methosulfate salt RHPS4 induces the 3 0 single-stranded guanine-rich telomeric overhang to fold into a G-quadruplex structure. Stabilisation of the latter is incompatible with an attachment of telomerase to the telomere and thus G-quadruplex ligands can effectively inhibit both the catalytic and capping functions of telomerase. In this study, we examined mechanisms underlying telomere uncapping by RHPS4 in uterus carcinoma cells (UXF1138L) with short telomeres and compared the susceptibility of bulk and clonogenic cancer cells to the G-quadruplex ligand. We show that treatment of UXF1138L cells with RHPS4 leads to the displacement of the telomerase catalytic subunit (hTERT) from the nucleus, induction of telomere-initiated DNAdamage signalling and chromosome fusions. We further report that RHPS4 is more potent against cancer cells that grow as colonies in soft agar than cells growing as monolayers. Human cord blood and HEK293T embryonic kidney cell colony forming units, however, were more resistant to RHPS4. RHPS4-treated UXF1138L xenografts had a decreased clonogenicity, showed loss of nuclear hTERT expression and an induction of mitotic abnormalities compared with controls. Although single-agent RHPS4 had limited in vivo efficacy, a combination of RHPS4 with the mitotic spindle poison Taxol caused tumour remissions and further enhancement of telomere dysfunction.

Although human T-cell lymphotropic virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), the role of viral gene expression in the progression to and maintenance of the leukemic state in vivo is unclear because of the inability of most previous studies to readily detect HTLV-I RNA in infected individuals. By using the reverse transcriptase-polymerase chain reaction, we detected spliced messages for the HTLV-I pX regulatory genes in primary uncultured cells from ATL patients and healthy asymptomatic carriers. In addition to the expected doubly spliced pX message, three alternatively spliced mRNAs were demonstrated (pXA17, pX-p21rex, and pX-orflI mRNAs, where orf = open reading frame). The same splice sites were shown in the messages from uncultured ATL cells and from the HTLV-I-producing C1O/MJ cell line. Alternatively spliced pX mRNAs have the potential to code for known and putative pX gene products. Among the transcripts is a monocistronic mRNA likely to code for p2l'rx (pX-p2lrx mRNA). Since alternative splicing of HTLV

In a breast tumor xenograft model, the MCT-1 oncogene increases the in vivo tumorgenicity of MCF7 cells by promoting angiogenesis and inhibiting apoptosis. Increases in the tumor microvascular density are accompanied by a strong reduction in the levels of the angiogenesis inhibitor thrombospondin-1 (TSP1), but the mechanisms underlying this process are unknown. We show that TSP1 expression is controlled, at least in part, by post-transcriptional events. Using RNA interference to knock down the expression of the RNA-binding protein HuR in MCF7 cells as well as HuR overexpression, we demonstrate that HuR plays an important role in translation of the TSP1 mRNA. Furthermore, employing the RIP-Chip assay yielded 595 transcripts with significantly altered binding to HuR in the more tumorigenic breast cancer clones compared with the weakly tumorigenic clones. These mRNAs clustered in several pathways implicated in the transformed phenotype, such as the RAS pathway (involved in mitogenesis), the PI3K pathway (evasion of apoptosis) and pathways mediating angiogenesis and the cellular response to hypoxia. These findings demonstrate for the first time that global changes in HuR-bound mRNAs are implicated in the evolution to a more tumorigenic phenotype in an in vivo tumor model and underscore the role of global mRNA-protein interactions toward tumor progression.

HIV necessitates host factors for successful completion of its life cycle. Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that forms two complexes, mTORC1 and mTORC2. Rapamycin is an allosteric inhibitor of mTOR that selectively inhibits mTORC1. Rapamycin interferes with viral entry of CCR5 (R5)-tropic HIV and with basal transcription of the HIV LTR, potently inhibiting replication of R5 HIV but not CXCR4 (X4)-tropic HIV in primary cells. The recently developed ATP-competitive mTOR kinase inhibitors (TOR-KIs) inhibit both mTORC1 and mTORC2. Using INK128 as a prototype TOR-KI, we demonstrate potent inhibition of both R5 and X4 HIV in primary lymphocytes (EC50 < 50 nM), in the absence of toxicity. INK128 inhibited R5 HIV entry by reducing CCR5 levels. INK128 also inhibited both basal and induced transcription of HIV genes, consistent with inhibition of mTORC2, whose activity is critical for phosphorylation of PKC isoforms and, in turn, induction of NF-κB. INK128 enhanced the antiviral potency of the CCR5 antagonist maraviroc, and had favorable antiviral interactions with HIV inhibitors of reverse transcriptase, integrase and protease. In humanized mice, INK128 decreased plasma HIV RNA by >2 log10 units and partially restored CD4/CD8 cell ratios. Targeting of cellular mTOR with INK128 (and perhaps others TOR-KIs) provides a potential strategy to inhibit HIV, especially in patients with drug resistant HIV strains.

The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL.

IntroductionEukaryotic cells have various mechanisms and levels by which gene expression can be regulated including: transcription, export of mRNA messages, mRNA stability, and posttranslational modifications. Protein synthesis is essential for cell viability, and controlling mRNA translation is a critical step in regulation of gene expression. Translation can be divided between 3 stages: initiation, elongation, and termination. Translational control is principally exerted by regulating the formation of the cap-dependent translation initiation complex. Translation initiation comprises a mechanism in which the eIF4F ternary complex (eIF4E, eIF4A, and eIF4G) recruits a 43S preinitiation complex containing a Met-tRNAi and multiple initiation factors (eIFs 1, 1A, 2, 3, and 5) to the 5Ј methyl-7-GTP cap complex on mRNA (reviewed in Sonenberg and Hinnebusch). 1 Once the preinitiation complex scans the 5Ј untranslated region of the mRNA and reaches the AUG start codon, the large (60S) ribosomal subunit joins the small (40S) ribosomal subunit and begins to synthesize the protein. 1 Deregulated protein synthesis plays an important role in human cancer and deregulated translational control has been recognized as an integral part of the malignant state. 2,3 In the past several years it has become clear that the efficiency of expression of key proteins involved in cell-growth regulation, proliferation, and apoptosis may be controlled at the translational level by changes in the activity of components of the protein synthesis machinery. 4,5 Various classes of mRNAs differ considerably in their translational efficiency. Typically, mRNAs coding for proteins positively involved in regulating cell growth and survival have a high degree of secondary structure in the 5Ј untranslated region (UTR). The translation of such messages is particularly sensitive to the activity of the cap-dependent translation-initiation machinery. 6 In view of the fact that translation factors are closely regulated by conditions that affect cell growth, it is not surprising that, experimentally, aberrant expression of some of these factors has been shown to induce malignant transformation of cells.Translation initiation has recently been shown to be a common downstream target of signal transduction pathways deregulated in cancer and initiated by mutated/overexpressed oncogenes and tumor suppressors. 7 Several previous publications indicate that aberrant control of protein synthesis contributes to lymphomagenesis 3,8 opening up possibilities for innovative therapeutics, that is, targeting the translational machinery. Below, we present an overview of potentially targetable translational machinery components and regulatory signaling pathways that represent a novel approach for the treatment of hematologic malignancies. eIF4FA major regulatory step in control of protein synthesis is translation initiation. Translation initiation is modulated by the association of a ternary complex of proteins, eukaryotic translation initiation factor F (eIF4F), composed of e...

Altered lipid metabolism and aberrant protein translation are strongly associated with cancerous outgrowth; however, the inter-regulation of these key processes is still underexplored in diffuse large B-cell lymphoma (DLBCL). Although fatty acid synthase (FASN) activity is reported to positively correlate with PI3K-Akt-mTOR pathway that can modulate protein synthesis, the precise impact of FASN inhibition on this process is still unknown. Herein, we demonstrate that attenuating FASN expression or its activity significantly reduces eIF4B (eukaryotic initiation factor 4B) levels and consequently overall protein translation. Through biochemical studies, we identified eIF4B as a bonafide substrate of USP11, which stabilizes and enhances eIF4B activity. Employing both pharmacological and genetic approaches, we establish that FASN-induced PI3K-S6Kinase signaling phosphorylates USP11 enhancing its interaction with eIF4B and thereby promoting oncogenic translation.