Mev Pathway Research Articles

Transcriptomic responses to grazing reveal the metabolic pathway leading to the biosynthesis of domoic acid and highlight different defense strategies in diatoms

BackgroundA major cause of phytoplankton mortality is predation by zooplankton. Strategies to avoid grazers have probably played a major role in the evolution of phytoplankton and impacted bloom dynamics and trophic energy transport. Certain species of the genus Pseudo-nitzschia produce the neurotoxin, domoic acid (DA), as a response to the presence of copepod grazers, suggesting that DA is a defense compound. The biosynthesis of DA comprises fusion of two precursors, a C10 isoprenoid geranyl pyrophosphate and l-glutamate. Geranyl pyrophosphate (GPP) may derive from the mevalonate isoprenoid (MEV) pathway in the cytosol or from the methyl-erythritol phosphate (MEP) pathway in the plastid. l-glutamate is suggested to derive from the citric acid cycle. Fragilariopsis, a phylogenetically related but nontoxic genus of diatoms, does not appear to possess a similar defense mechanism. We acquired information on genes involved in biosynthesis, precursor pathways and regulatory functions for DA production in the toxigenic Pseudo-nitzschia seriata, as well as genes involved in responses to grazers to resolve common responses for defense strategies in diatoms.ResultsSeveral genes are expressed in cells of Pseudo-nitzschia when these are exposed to predator cues. No genes are expressed in Fragilariopsis when treated similarly, indicating that the two taxa have evolved different strategies to avoid predation. Genes involved in signal transduction indicate that Pseudo-nitzschia cells receive signals from copepods that transduce cascading molecular precursors leading to the formation of DA. Five out of seven genes in the MEP pathway for synthesis of GPP are upregulated, but none in the conventional MEV pathway. Five genes with known or suggested functions in later steps of DA formation are upregulated. We conclude that no gene regulation supports that l-glutamate derives from the citric acid cycle, and we suggest the proline metabolism to be a downstream precursor.ConclusionsPseudo-nitzschia cells, but not Fragilariopsis, receive and respond to copepod cues. The cellular route for the C10 isoprenoid product for biosynthesis of DA arises from the MEP metabolic pathway and we suggest proline metabolism to be a downstream precursor for l-glutamate. We suggest 13 genes with unknown function to be involved in diatom responses to grazers.

ABCA1, apoA-I, and BTN3A1: A Legitimate Ménage à Trois in Dendritic Cells.

Human Vγ9Vδ2 T cells have the capacity to detect supra-physiological concentrations of phosphoantigens (pAgs) generated by the mevalonate (Mev) pathway of mammalian cells under specific circumstances. Isopentenyl pyrophosphate (IPP) is the prototypic pAg recognized by Vγ9Vδ2 T cells. B-cell derived tumor cells (i.e., lymphoma and myeloma cells) and dendritic cells (DCs) are privileged targets of Vγ9Vδ2 T cells because they generate significant amounts of IPP which can be boosted with zoledronic acid (ZA). ZA is the most potent aminobisphosphonate (NBP) clinically available to inhibit osteoclast activation and a very potent inhibitor of farnesyl pyrophosphate synthase in the Mev pathway. ZA-treated DCs generate and release in the supernatants picomolar IPP concentrations which are sufficient to induce the activation of Vγ9Vδ2 T cells. We have recently shown that the ATP-binding cassette transporter A1 (ABCA1) plays a major role in the extracellular release of IPP from ZA-treated DCs. This novel ABCA1 function is fine-tuned by physical interactions with IPP, apolipoprotein A-I (apoA-I), and butyrophilin-3A1 (BTN3A1). The mechanisms by which soluble IPP induces Vγ9Vδ2 T-cell activation remain to be elucidated. It is possible that soluble IPP binds to BTN3A1, apoA-I, or other unknown molecules on the cell surface of bystander cells like monocytes, NK cells, Vγ9Vδ2 T cells, or any other cell locally present. Investigating this scenario may represent a unique opportunity to further characterize the role of BTN3A1 and other molecules in the recognition of soluble IPP by Vγ9Vδ2 T cells.

The Hypoxia-Inducible Factor-1alpha Is Constitutively Upregulated in TP53 Disrupted CLL Cells: A Potential Target to Overcome Fludarabine Resistance

BACKGROUNDTreatment of patients with fludarabine-resistant chronic lymphocytic leukemia (CLL) is an unmet clinical need. Fludarabine resistance in CLL can be ascribed to intrinsic genetic features of the tumor cells, mainly consisting in TP53 disruption, and to the interactions of CLL cells with stromal cells (SC) of the tumor microenvironment. One of the main players of SC-induced fludarabine resistance is the CXCL12/CXCR4 axis, which activates in CLL cells the Ras/ERK1-2 and RhoA-dependent signaling pathways. To be active transducers Ras and RhoA need to undergo isoprenylation by means of small molecules produced by the mevalonate (Mev) pathway. We reported that the Mev pathway-regulated Ras and RhoA signaling cascades and the downstream hypoxia inducible factor (HIF)-1α/P-glycoprotein axis are more active in IGHV unmutated than in mutated CLL cells, leading to a constitutive protection from doxorubicin-induced cytotoxicity. HIF-1α is a transcription factor constitutively expressed in CLL cells and controls the expression of several genes implicated in survival and cell metabolism. The Mev pathway manipulation with simvastatin (Sim), as well as the targeted inhibition of ERK1-2, RhoA kinase and HIF-1α, restore the sensitivity of CLL cells to doxorubicin. The role of the Mev pathway, the Ras and RhoA signaling, and the transcription factor HIF-1α in regulating the SC-induced fludarabine resistance of TP53 disrupted (TP53dis) CLL cells is currently unknown. AIMThe aim of this study was to evaluate the activity of the Mev pathway-regulated Ras and RhoA signaling cascades and the transcription factor HIF-1α in TP53dis CLL cells, in order to identify biochemical and molecular targets potentially amenable to therapeutic intervention. METHODSPurified CLL cells were cultured alone or with the M2-10B4 SC line. Cell cultures were exposed to fludarabine (10 μM), Sim (1 μM) or the HIF-1α inhibitor YC-1 (10 μM). The activity of the Mev pathway was measured by quantification of [3H]acetate-labeled cholesterol and farnesyl pyrophosphate produced by CLL cells. Ras and RhoA activities were evaluated by pull-down assay and ELISA, respectively. ERK1-2 phosphorylation was evaluated by Western Blot. RhoA kinase, Akt and HIF-1α activities were measured with specific immunoassay. The glucose flux through glycolysis and tricarboxylic acid cycle was measured by radiolabeling cells with [6-14C]-glucose and quantifying the amount of glucose transformed into CO2. Cell viability was analysed by Annexin-V/propidium Iodide immunostaining and flow cytometry. Patients with TP53 mutation or 17p deletion were considered TP53dis. RESULTSCo-culture with SC upregulated the Mev pathway activity of CLL cells, activated the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling, and increased the transcriptional activity of HIF-1α. Blocking the Mev pathway by Sim significantly reduced the SC-induced upregulation of Ras and RhoA signalling pathways, and counteracted the SC-mediated protection of CLL cells from fludarabine-induced cytotoxicity. Based on these results we evaluated the activity of the Mev pathway, and the Ras and RhoA signalling in TP53dis and TP53 wild-type (TP53wt) CLL cells. There was no difference between the two groups in terms of Mev pathway activity, Ras/ERK1-2 and RhoA/RhoA kinase signal transduction, and Akt activity. By contrast, we found that TP53dis CLL cells had a significantly higher transcriptional activity of HIF-1α than TP53wt CLL cells (p=0.02). CLL patients carrying a percentage of 17p deletion >60% in the tumor clone had the higher values of HIF-1α activity. As a result, TP53dis CLL cells overexpressed the glycolytic HIF-1α target gene α-enolase and had a more active glycolytic activity than TP53wt CLL cells (p=0.003). In TP53dis CLL cells, Sim significantly inhibited the Mev pathway, and the Ras and RhoA signalling, but had no effect on the upregulated transcriptional activity of HIF-1α and on the protective effect exerted by SC toward fludarabine-induced cytotoxicity. Conversely, the HIF-1α inhibitor YC-1 partially restored the fludarabine sensitivity of TP53dis CLL cells, as shown by its ability to counteract the protective effect exerted by SC toward fludarabine-induced cell death. CONCLUSIONSOur data demonstrate that the targeted inhibition of HIF-1α is a promising strategy to circumvent the SC-induced fludarabine resistance of TP53dis CLL cells. DisclosuresMarchetti:GILEAD: Consultancy, Research Funding; JANSSEN: Other: tavel, accomodation, expenses; SANOFI: Membership on an entity's Board of Directors or advisory committees; GILEAD: Other: teaching, Research Funding; NOVARTIS: Research Funding. Gaidano:MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding. Boccadoro:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees. Massaia:Gilead: Research Funding; Janssen: Honoraria; Roche: Honoraria. Coscia:Roche: Honoraria, Other: Advisory board; Mundipharma: Honoraria.

The Mevalonate Metabolic Pathway and the CXCL12/CXCR4 Axis Reciprocally Interact and Are Implicated in Fludarabine Resistance of Chronic Lymphocytic Leukemia Cells

BACKGROUNDTreatment of fludarabine-resistant chronic lymphocytic leukemia (CLL) patients is an unmet clinical need. Fludarabine resistance in CLL depends on intrinsic molecular features of the tumor cells, and on bidirectional interactions occurring between CLL cells and stromal cells (SC) of the tumor microenvironment. One of the main players of SC-induced fludarabine resistance is the CXCL12/CXCR4 axis. CXCR4 is a G protein-coupled receptor constitutively expressed on CLL cells. The binding of CXCR4 with CXCL12 activates the Ras/ERK1-2/Akt and the RhoA-dependent signalling pathways. To be active transducers Ras and RhoA need to undergo a post-translational modification (i.e. isoprenylation) by means of small molecules produced by the mevalonate (Mev) pathway. We have recently demonstrated that the Mev pathway is more active in IGHV unmutated than in mutated CLL cells, and is amenable to pharmacological manipulation by statins (i.e. simvastatin [Sim]). It is currently unknown whether the Mev pathway and its pharmacological targeting are implicated in the modulation of the CXCL12/CXCR4 axis and in the SC-induced fludarabine resistance of CLL cells. AIMThe aim of this study was to investigate the reciprocal interactions between the Mev pathway and the CXCL12/CXCR4 axis, in order to identify potential targets to counteract the constitutive and SC-induced fludarabine resistance of CLL cells. METHODSImmuno-magnetically purified patient-derived CLL cells were cultured alone or with murine SC (M2-10B4 cell line). In selected experiments, cell cultures were exposed to human recombinant CXCL12 (100 μg/ml), CXCR4 inhibitor AMD3100 (5 μg/ml), fludarabine (F-ara-A, 10 μM), Sim (1 μM), ERK1-2 kinase inhibitor PD98059 (10 μM), RhoA kinase inhibitor Y276 (10 μM), HIF-1α inhibitor YC-1 (10 μM). The activity of the Mev pathway was measured by the quantification of metabolites [i.e. cholesterol and farnesyl pyrophosphate (FPP)] produced by CLL cells after 24 h incubation with 1 μCi of [3H]acetate. Ras and RhoA activities were evaluated measuring their GTP-bound fraction, taken as an index of the G-protein activation, respectively by pull-down assay and by an ELISA based assay. ERK1-2 and HIF-1α phosphorylation were evaluated by Western Blot. RhoA kinase, Akt and HIF-1α activities were measured with specific immunoassay kit. The amount of CXCL12 in culture supernatants was assessed by ELISA assay. Cell viability was determined by Annexin-V/propidium Iodide immunostaining and flow cytometry analysis. RESULTSCo-culture with SC upregulated the Mev pathway activity of CLL cells, as shown by the increased production of cholesterol and FPP. This SC-induced increase in the Mev pathway activity was followed by the activation of the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling, the upregulation of the pro-survival factor Akt, and an increase in the transcriptional activity of HIF-1α. These biological and molecular effects were identically observed when CLL cells were exposed to recombinant CXCL12, and were completely abrogated by the CXCR4 antagonist AMD3100, thus showing the key role of the CXCL12/CXCR4 axis in the SC-induced modulation of the Mev pathway and the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling. On the other hand, blocking the Mev pathway by Sim and targeting ERK1-2 kinases, RhoA kinase and HIF-1α by specific small-molecule inhibitors significantly reduced the constitutive activity and the SC-induced upregulation of the Ras/ERK1-2 and RhoA/RhoA kinase signal transduction. A regulatory role of the Mev pathway on the CXCL12/CXCR4 axis was observed not only in CLL cells but also in SC, as shown by the significant reduction in CXCL12 secretion by SC exposed to Sim. In the last set of experiments, we found that the inhibition of the Mev pathway by Sim potentiated the direct cytotoxic effect of fludarabine against CLL cells. Even more importantly, both Sim and the downstream HIF-1α inhibitor YC-1 were capable of counteracting the SC-mediated protection of CLL cells from fludarabine-induced cytotoxicity. CONCLUSIONSOur data demonstrate that the Mev pathway has a regulatory role on the CXCL12/CXCR4 axis and on the SC-mediated protective effects toward spontaneous and fludarabine-induced CLL cell death. The upstream inhibition of the Mev pathway and the downstream targeting of HIF-1α are promising strategies to circumvent fludarabine resistance in CLL. DisclosuresBoccadoro:Janssen-Cilag: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

The Mevalonate Pathway and Downstream Signal Transducers As Therapeutic Targets to Overcome Multidrug Resistance in Chronic Lymphocytic Leukemia (CLL)

AbstractAbstract 3881 Background:The mutational status of tumor immunoglobulin heavy chain variable region (IGHV) is a reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. The tumor microenvironment actively supports the survival of CLL cells and confers a multidrug resistance (MDR) phenotype to CLL cells. MDR is due to the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. Pgp is under the positive control of the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α) which is activated by isoprenylated Ras/Rho-dependent downstream signaling pathways. Ras and Rho isoprenylation are regulated by the mevalonate (Mev) pathway activity suggesting that this pathway can be exploited as a metabolic checkpoint to regulate chemresistance. Aim:The aim of this study was twofold: 1) to investigate the correlation between chemoresistance and the activity of the Mev pathway and Ras/Rho-A downstream signaling pathways in purified M and UM CLL cells under basal conditions and after incubation with stromal cells; 2) to evaluate the chemosensitizing effects of agents specifically targeting the Mev pathway and downstream signaling pathways under the same culture conditions. Methods:M and UM CLL cells were cultured in the presence and in the absence of murine stromal cells (M210B4) and exposed to Zoledronic acid (ZA) (1 μmol/L), Simvastatine (Sim) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L), HIF-1α inhibitor YC-1 (10 μmol/L) and Doxorubicine (Doxo) (1 μmol/L). The Mev pathway activity was measured by cells radiolabelling with [14C]-mevalonic acid and thin layer chromatography. Ras, ERK1/2 and Akt activity were detected by Western blot. Rho, Rho Kinase and HIF-1α activity were assessed by ELISA. Mdr1 expression was measured by Real Time-PCR. PgP activity was evaluated by measuring Doxo intracellular accumulation. Doxo cytotoxicity was assessed by annexin V and propidium iodide staining. Results:The Mev pathway is significantly more active in UM than in M CLL cells. This hypermetabolic activity translates into a higher activation of Ras/Akt and Rho/Rho kinase signaling pathways and higher expression of the phosphorylated active form of HIF-1α. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to a more effective Doxo extrusion and therefore better survival upon Doxo exposure. M210B4 stromal cells further protect UM CLL cells from Doxo induced cell death by upregulating Mev pathway activity, HIF-1α/mdr1/PgP axis activation, and Doxo extrusion. Targeting the Mev pathway of UM cells with ZA and Mev reduces the basal activity of HIF-1α/mdr1/PgP axis and significantly increases Doxo retention and cytotoxicity. Similar effects are obtained with PD85 and YC1–10 which are specific inhibitors of the downstream molecules ERK-1/2 and HIF-1α, respectively. All these agents are able to overcome the protective effect exerted by stromal cells by significantly increasing PgP activity and Doxo-induced cell death. Conclusions:Our data demonstrate that the Ras- and Rho-dependent HIF-1α/mdr1/PgP axis is more active and associated with higher levels of MDR in UM compared with M CLL cells. Targeting the Mev pathway and/or downstream signalling pathways is a promising strategy to circumvent basal and stroma-mediated chemoresistance especially in UM CLL cells. Disclosures:Massaia:Novartis Farma S.p.A: Honoraria, Research Funding.

Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts

Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl- d-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants.

The Mevalonate Pathway As a Metabolic Target to Circumvent Multidrug-Resistance in Chronic Lymphocytic Leukemia Cells

Abstract 735The mutational status of the tumor immunoglobulin heavy chain variable region (IGHV) is a very reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. We have recently shown that UM CLL cells are more dependent on microenvironment-mediated signals for survival (Coscia M. et al, Leukemia, 2011), but it is currently unknown whether this also translates into an enhanced chemotherapy resistance. Multi drug resistance (MDR) is mediated by the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. The Pgp protein is the product of the mdr1 gene whose regulation is under the positive control of the mevalonate (Mev) metabolic pathway, the downstream Ras/RhoA dependent signaling pathways and the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α). The aim of this study was to investigate the MDR phenotype and signaling cascade in M and UM CLL cells under basal conditions and after exposure to prosurvival microenvironment-mediated signals.Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from untreated CLL patients. More than 90% pure CLL cells were co-cultured with the murine stromal cell line M2-10B4. Culture conditions included: medium alone, Zoledronic acid (ZA) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L) and the HIF-1α inhibitor YC-1 (10 μmol/L). The level of activity of the Mev pathway was evaluated by measuring the intermediate metabolite farnesyl pyrophosphate (FPP) and the final product cholesterol (Chol) as previously described (Coscia M. et al, J Cell Mol Med, 2010). The Ras and RhoA activity, was evaluated by measuring the GTP binding. Rho kinase activity was measured using the CycLex Rho Kinase Assay Kit (CycLex Co., Nagano). The following antibodies were used for Western Blot (WB) analyses: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2; anti-ERK 1/2; anti-HIF-1α; anti-Pgp; anti-GAPDH. The activity of HIF-1 in the nuclear extracts was assessed by the TransAM™ HIF-1 Transcription Factor Assay Kit (Active Motif). Mdr1 gene expression was evaluated by RT-PCR. Intracellular Doxorubicine (Doxo) accumulation was evaluated by cytofluorimetric analyses.We have found that the Mev pathway is significantly accelerated in freshly isolated UM vs M CLL cells. This hypermetabolic activity translates into a higher activation of the Ras/ERK-1/2 and RhoA/Rho kinase signaling pathways, and to a significantly more active HIF-1α in UM compared to M CLL cells. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to Pgp expression and more effective Doxo extrusion in these cells. The co-culture of CLL cells with the murine stromal cell line M2-10B4 further accelerated the Mev pathway and activated the HIF-1α/mdr1/PgP axis leading to even lower intracellular Doxo retention. This stroma-mediated effect was particularly evident in UM CLL cells. Targeting of the Mev pathway with ZA and statins, and targeting of ERK-1/2 and HIF-1α with specific inhibitors (PD85 and YC1-10, respectively) significantly reduced basal and stroma-induced activity of HIF-1α/mdr1/PgP axis, and significantly increased the intracellular concentrations of Doxo in UM CLL cells. These data indicate that the pharmacological inhibition of the Mev pathway and of the downstream HIF-1α/mdr1/PgP axis can be regarded as a novel potential strategy to circumvent basal and environment-mediated chemoresistance of CLL cells. Disclosures:Massaia:Novartis Farma S.p.A: Honoraria, Research Funding, scientific adviser board.

The Defective Proliferation of Vgamma9Vdelta2 T Cells to Zoledronic Acid In Chronic Lymphocytic Leukemia (CLL) Is a Powerful Time to First Treatment (TFT) Predictor Associated with the IGHV Mutational Status

AbstractAbstract 3602The clinical course of chronic lymphocytic leukemia (CLL) depends on the intrinsic tumor cell features but also on the interactions between tumor cells, local microenvironment and host immunity. The interplay between CLL cells and conventional αβ T cells has already been investigated in details, whereas very little is known about the role of Vgamma9Vdelta2 T cells. These cells have intrinsic antitumor properties which can be further enhanced by stimulation with aminobisphosphonates such as zoledronic acid (ZA) via monocytes or other antigen-presenting cells. ZA targets the mevalonate (Mev) pathway and induces the intracellular accumulation and release of intermediate metabolites, like isopentenyl pyrophosphate (IPP), which are very similar to the natural ligands of Vgamma9Vdelta2 T cells.In this study we have performed a phenotypic and functional analysis of Vgamma9Vdelta2 T cells in 93 untreated CLL patients and correlated the results with intrinsic CLL cell features and clinical outcome. Stimulation of peripheral blood mononuclear cells with ZA induced Vgamma9Vdelta2 T cell proliferation in 47/93 patients (responders, R), but not in 46/93 patients (non-responders, NR). Vgamma9Vdelta2 T-cell subset distribution of R CLL was similar to healthy donors, whereas effector memory (EM) and terminally differentiated effector memory (TEMRA) cells predominated in NR CLL. A significant association was found between the R/NR status of Vgamma9Vdelta2 T cells and the mutational status of the tumor IGHV genes: 77% of R patients were M, whereas 70% of UM patients were NR. To test the hypothesis that this difference reflected a different activity of the Mev pathway in M and UM CLL cells, we performed a bioinformatic elaboration of data obtained from gene expression profiling of CLL cells and a biochemical quantification of the Mev pathway in CLL cells including the intermediate metabolites farnesyl pyrophosphate (FPP) and IPP, and the final product cholesterol. The Mev pathway was significantly more active in UM than in M CLL cells, suggesting that the IPP overproduction by UM CLL cells is responsible for a chronic stimulation of Vgamma9Vdelta2 T cells leading to their differentiation into EM and TEMRA cells. This biochemical-driven immunoediting process has clinical implications. After a median follow-up of 46 months from diagnosis, univariate analysis identified R status as a predictor of reduced TFT (NR: 59 months vs R: not reached; p=0.01). The R/NR status also allows to further identify two subsets in UM CLL patients (R-UM and NR-UM) with significantly different TFT (NR-UM: 32 months vs R-UM: not reached; p=0.02). In multivariate analysis, Binet stage (P=0.027), IGHV mutational status (p=0.016), R/NR status (p=0.007), high and low-risk cytogenetic abnormalities (p<0.001) and lymphocytosis at the time of diagnosis (p<0.001) were independent TFT predictors.In conclusion, we have identified a novel TFT predictor based on a putative interaction between the Mev pathway of CLL cells and Vgamma9Vdelta2 T cells. These results further strengthen the importance of tumor-host immune interactions in CLL evolution. Disclosures:Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia:Novartis: Honoraria, Research Funding.

Probing Ligand-binding Pockets of the Mevalonate Pathway Enzymes from Streptococcus pneumoniae

Diphosphomevalonate (Mev.pp) is the founding member of a new class of potential antibiotics targeting the Streptococcus pneumoniae mevalonate (Mev) pathway. We have synthesized a series of Mev.pp analogues designed to simultaneously block two steps in this pathway, through allosteric inhibition of mevalonate kinase (MK) and, for five of the analogues, by mechanism-based inactivation of diphosphomevalonate decarboxylase (DPM-DC). The analogue series expands the C(3)-methyl group of Mev.pp with hydrocarbons of varying size, shape, and chemical and physical properties. Previously, we established the feasibility of a prodrug strategy in which unphosphorylated Mev analogues could be enzymatically converted to the active Mev.pp forms by the endogenous MK and phosphomevalonate kinase. We now report the kinetic parameters for the turnover of non-, mono-, and diphosphorylated analogues as substrates and inhibitors of the three mevalonate pathway enzymes. The inhibition of MK by Mev.pp analogues revealed that the allosteric site is selective for compact, electron-rich C(3)-subsitutents. The lack of reactivity of analogues with DPM-DC provided evidence, counter to the existing model, for a decarboxylation transition state that is concerted rather than dissociative. The Mev pathway is composed of three structurally and functionally conserved enzymes that catalyze consecutive steps in a metabolic pathway. The current work reveals that these enzymes exhibit significant differences in specificity toward R-group substitution at C(3) and that these patterns are explained well by changes in the volume of the C(3) R-group-binding pockets of the enzymes.

Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-009-2219-x) contains supplementary material, which is available to authorized users.

Dendritic Cells Can Be Primed to Concurrently Activate Both Unconventional Gamma/delta and Conventional alpha/beta T Cells.

Vgamma9/Vdelta2 (gamma/delta) T cells represent the major subset of unconventional T cells circulating in the peripheral blood. Gamma/delta T cells play a major role in immune defenses against microbes, stressed cells and tumor cells. This property is based on their capability to naturally recognize phosphoantigens (pAgs), which are produced via the mevalonate (Mev) or the DOXP pathway in mammalian and nonmammalian cells, and induced self-ligands, which are de novo expressed or upregulated on the surface of stressed or tumor cells. Interestingly, gamma/delta T cells can also be activated by aminobisphosphonates (ABP)-treated monocytes. We have previously shown that ABP specifically target the Mev pathway of monocytes and induce the accumulation of phosphorylated Mev metabolites naturally recognized by gamma/delta T cells. The aim of this work was to determine whether ABP-treated dendritic cells (DC) can also activate gamma/delta T cells and whether this activation, if any, is detrimental or beneficial to the generation of antigen (Ag)-specific MHC-restricted immune responses mediated by conventional alpha/beta T cells. To this end, we have generated highly purified immature (iDC) and mature DC (mDC) from peripheral blood monocytes of healthy donors and incubated with zoledronic acid (Zol) for 24 hours. Zol is the most potent ABP currently available for clinical use. Zol treatment did not affect the phenotype and immunostimulatory properties of iDC and mDC. Zol-treated iDC and mDC induced a rapid and vigorous expansion of central memory and effector memory gamma/delta T cells. Zol-treated iDC were more potent inducers of gamma/delta T-cell activation than mDC and monocytes. Activated gamma/delta T cells displayed antitumor activity and expressed on the cell surface the appropriate antigen repertoire to target secondary lymphoid organs and exert costimulatory activity on conventional alpha/beta T cells. Indeed, an in vitro model showed that antigen-specific MHC-restricted immune responses againt the influenza matrix peptide were significantly improved by the concurrent activation of gamma/delta T cells. This is the first report showing that: 1) DC can simultaneously be primed to activate both gamma/delta and alpha/beta T cells; 2) the former act as cellular adjuvants for the development of adaptive immune responses. In conclusion, large numbers of gamma/delta T cells with effector and costimulatory activities can rapidly be generated by Zol-treated iDC/mDC. This strategy is worth of further investigation to improve adoptive cell therapy and vaccine interventions against tumors and infections.