Rationale for Combining Bispecific T Cell Activating Antibodies With Checkpoint Blockade for Cancer Therapy

T cells have been established as core effectors for cancer therapy;this has moved the focus of therapeutic endeavors to effectively enhance or restore T cell tumoricidal activity rather than directly target cancer cells. Both antibodies targeting the checkpoint inhibitory molecules programmed death receptor 1 (PD1), PD-ligand 1 (PD-L1) and cytotoxic lymphocyte activated antigen 4 (CTLA4), as well as bispecific antibodies targeting CD3 and CD19 are now part of the standard of care. In particular, antibodies to checkpoint molecules have gained broad approval in a number of solid tumor indications, such as melanoma or non-small cell lung cancer based on their unparalleled efficacy. In contrast, the efficacy of bispecific antibody-derivatives is much more limited and evidence is emerging that their activity is regulated through diverse checkpoint molecules. In either case, both types of compounds have their limitations and most patients will not benefit from them in the long run. A major aspect under investigation is the lack of baseline antigen-specific T cells in certain patient groups, which is thought to render responses to checkpoint inhibition less likely. On the other hand, bispecific antibodies are also restricted by induced T cell anergy. Based on these considerations, combination of bispecific antibody mediated on-target T cell activation and reversal of anergy bears high promise. Here, we will review current evidence for such combinatorial approaches, as well as ongoing clinical investigations in this area. We will also discuss potential evidence-driven future avenues for testing

PD1-CD28 Fusion Protein Enables CD4+ T Cell Help for Adoptive T Cell Therapy in Models of Pancreatic Cancer and Non-hodgkin Lymphoma

Background: Interaction of the programmed death receptor 1 (PD-1) and its ligand, PD-L1, suppresses T cell activity and permits tumors to evade T cell-mediated immune surveillance. We have recently demonstrated that antigen-specific CD8+ T cells transduced with a PD1-CD28 fusion protein are protected from D-1-mediated inhibition. We have now investigated the potential of PD1-CD28 fusion protein-transduced CD4+ T cells alone or in combination with CD8+ T cells for immunotherapy of pancreatic cancer and non-Hodgkin lymphoma. Methods: OVA-specific CD4+ and CD8+ were retrovirally transduced with the PD1-CD28 fusion protein. Cytokine release, proliferation, cytotoxic activity, and phenotype of transduced T cells were assessed in the context of Panc02-OVA (murine pancreatic cancer model) and E.G7-PD-L1 (murine T cell lymphoma model) cells. Results: Stimulation of PD1-CD28 fusion protein-transduced CD4+ T cells with anti-CD3 and recombinant PD-L1 induced specific T cell activation, as measured by IFN-y release and T cell proliferation. Coculture with Panc02-OVA or E.G7-PD-L1 tumor cells also led to specific activation of CD4+ T cells. Cytokine release and T cell proliferation was most effective when tumor cells simultaneously encountered genetically engineered CD4+ and CD8+ T cells. Synergy between both cell populations was also observed for specific tumor cell lysis. T cell cytotoxicity was mediated via granzyme B release and mediated enhanced tumor control in vivo. Transduced CD4+ and CD8+ T cells in co-culture with tumor cells developed a predominant central memory phenotype over time. Different ratios of CD4+ and CD8+ transduced T cells led to a significant increase of IFN-y and IL-2 secretion positively correlating with CD4+ T cell numbers used. Mechanistically, IL-2 and MHC-I were central to the synergistic activity of CD4+ and CD8+ T cells, since neutralization of IL-2 prevented the crosstalk between these cell populations. Conclusion: PD1-CD28 fusion protein-transduced CD4+ T cells significantly improved anti-tumoral effect of fusion protein-transduced CD8+ T cells. Thus, our results indicate that PD1-CD28 fusion protein-transduced CD4+ T cells have the potential to overcome the PD-1-PD-L1 immunosuppressive axis in pancreatic cancer and non-Hodgkin lymphoma

Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia

A single-cell screening approach identifies targets for CAR-T cells in acute myeloid leukemia. Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development

Nouvelle thérapie anti-tumorale multi-cibles basée sur la dégradation des ARNms à demi-vie courte

One of the innovative aspects of anti-cancer therapies is the possibility of preventing tumor growth by blocking blood supply. Cancer cells induce the formation of their own blood vessels from pre-existing vasculature, a process called angiogenesis. One of the most important proangiogenic factors is vascular endothelial growth factor (VEGF). The success of bevacizumab (a humanized anti-VEGF monoclonal antibody) combined to chemotherapy for the treatment of human metastatic cancers has validated VEGF as an efficient target. However, despite the initial enthusiasm, resistance to these anti-angiogenic treatments resulting from compensatory mechanisms occurs upon time. For this reason, there is a real need for new anti-angiogenic drugs that will target the angiogenic process through distinct mechanisms. In 2010, our laboratory has successfully developed an anti-angiogenic and anti-tumoral therapy based on destabilization of short-lived mRNAs by the zinc finger protein TIS11b. However, the therapeutic protein was highly unstable, thus making it difficult to further characterize the experimental therapy. In this context, the main task of my thesis was the optimization of TIS11b stability and activity followed by the evaluation of the multi-target action of our novel protein on tumor development. In a first part of this work, biochemical and molecular approaches allowed us to demonstrate that phosphorylation of the C-terminal serine S334 in TIS11b protein markedly increases its stability. In addition, deletion of the N-terminal domain of TIS11b highly increases its protein stability without affecting its activity. Therefore, we integrated N-terminal truncation (ZnC) and C-terminal substitution of S334 by an aspartate to mimic a permanent phosphorylation at S334 (ZnCS334D) as a novel TIS11b engineering strategy. Both proteins were fused subsequently to a cell-penetrating peptide polyarginine (R9). In vitro studies revealed that R9-ZnC and R9-ZnCS334D inhibit VEGF expression in the murine breast cancer cells 4T1. In addition, R9-ZnCS334D impaired proliferation, migration, invasion and anchorage-independent growth of 4T1 cells. In vivo, intra-tumoral injection of either protein significantly reduced VEGF expression and tumor vascularization. Strikingly, antibody array analyses of tumor extracts demonstrated a reduced expression of several chemokines such as Fractalkine, MCP-1, NOV, SDF-1 and Pentraxin upon R9-ZnC or R9-ZnCS334D treatment. These factors, which are produced by several cell types within tumor tissue, are key drivers of tumor angiogenesis, tumor-promoting inflammation and invasion. Furthermore, the expression of markers of the epithelial-to-mesenchymal transition was also significantly reduced, suggesting an anti-metastatic effect of R9-ZnC and R9-ZnCS334D. Thus, we provide R9-ZnC and R9-ZnCS334D as potential novel multi-target agents which inhibit key hallmarks of cancer progression. This work supports the emerging link between mRNA stability and cancer and proposes novel concepts for the development of innovative anti-cancer therapies.La formation de nouveaux vaisseaux sanguins ou angiogenèse soutient la croissance tumorale en fournissant l'oxygène et les nutriments qui lui sont nécessaires. Le rôle clé du facteur de croissance de l'endothélium vasculaire VEGF dans ce processus a suscité le développement de stratégies anti-angiogéniques pour le traitement du cancer. Cependant, des travaux précliniques et des données cliniques suggèrent l'émergence de résistances aux anti-angiogéniques, en raison notamment de la redondance des facteurs de croissance pro-angiogéniques. Il est donc nécessaire de développer des stratégies alternatives plus efficaces. En 2010, notre laboratoire a apporté la preuve de concept d'une thérapie anti-tumorale et anti-angiogénique innovante basée la dégradation des ARNm à demi-vie courte par la protéine à doigts de zinc TIS11b. Néanmoins, l'instabilité de la protéine thérapeutique a entravé la caractérisation plus détaillée de cette stratégie. Dans ce contexte, l'objectif majeur de ma thèse était l'optimisation de la stabilité et de l'activité de TIS11b et l'évaluation de son efficacité thérapeutique. Pour cela, nous avons généré une nouvelle protéine TIS11b génétiquement modifiée sur la base d'études biochimiques et moléculaires. Notamment, nous avons observé que la phosphorylation de la sérine 334 située dans le domaine C-terminal de TIS11b augmente de façon très significative la stabilité de la protéine et potentialise son activité déstabilisatrice de l'ARNm du VEGF. De plus, la délétion du domaine N-terminal augmente également la stabilité de TIS11b sans altérer son activité. Nous avons alors généré deux nouvelles protéines thérapeutiques, la protéine ZnC et la protéine ZnC334D pour laquelle la troncation du domaine N-terminal et la substitution de la sérine S334 par un aspartate mimant une phosphorylation ont été combinées. Les nouvelles protéines ont été fusionnées à une étiquette polyarginine R9 leur permettant de traverser les membranes cellulaires (R9-ZnC et R9-ZnCS334D). Nous avons montré que R9-ZnC et R9-ZnCS334D inhibent l'expression de VEGF in vitro dans la lignée de cancer du sein murin 4T1. De plus, R9-ZnCS334D exerce une activité anti-proliférative, anti-migratoire et anti-invasive dans ces cellules. In vivo, l'injection intra-tumorale de R9-ZnCS334D dans des tumeurs 4T1 préétablies inhibe significativement l'expression du VEGF, la croissance et la vascularisation tumorales. De façon remarquable, l'analyse des extraits tumoraux indique que le traitement diminue l'expression de chimiokines clés dans les processus d'angiogenèse, d'inflammation et d'invasion (Fractalkine, MCP-1, NOV, SDF-1, Pentraxin…). Enfin, R9-ZnC et R9-ZnCS334D inhibent l'expression de marqueurs de la transition épithélio-mésenchymateuse, un processus impliqué dans la dissémination métastatique. L'ensemble de ces travaux indique que R9-ZnC et R9-ZnCS334D sont des molécules anti-tumorales multi-cibles, qui inhibent plusieurs étapes clés de la progression tumorale. Cette étude confirme que le ciblage de la stabilité des ARNm est une stratégie prometteuse et novatrice pour le développement de nouvelles thérapies anti-cancéreuses

Nouvelle thérapie anti-tumorale multi-cibles basée sur la dégradation des ARNms à demi-vie courte

One of the innovative aspects of anti-cancer therapies is the possibility of preventing tumor growth by blocking blood supply. Cancer cells induce the formation of their own blood vessels from pre-existing vasculature, a process called angiogenesis. One of the most important proangiogenic factors is vascular endothelial growth factor (VEGF). The success of bevacizumab (a humanized anti-VEGF monoclonal antibody) combined to chemotherapy for the treatment of human metastatic cancers has validated VEGF as an efficient target. However, despite the initial enthusiasm, resistance to these anti-angiogenic treatments resulting from compensatory mechanisms occurs upon time. For this reason, there is a real need for new anti-angiogenic drugs that will target the angiogenic process through distinct mechanisms. In 2010, our laboratory has successfully developed an anti-angiogenic and anti-tumoral therapy based on destabilization of short-lived mRNAs by the zinc finger protein TIS11b. However, the therapeutic protein was highly unstable, thus making it difficult to further characterize the experimental therapy. In this context, the main task of my thesis was the optimization of TIS11b stability and activity followed by the evaluation of the multi-target action of our novel protein on tumor development. In a first part of this work, biochemical and molecular approaches allowed us to demonstrate that phosphorylation of the C-terminal serine S334 in TIS11b protein markedly increases its stability. In addition, deletion of the N-terminal domain of TIS11b highly increases its protein stability without affecting its activity. Therefore, we integrated N-terminal truncation (ZnC) and C-terminal substitution of S334 by an aspartate to mimic a permanent phosphorylation at S334 (ZnCS334D) as a novel TIS11b engineering strategy. Both proteins were fused subsequently to a cell-penetrating peptide polyarginine (R9). In vitro studies revealed that R9-ZnC and R9-ZnCS334D inhibit VEGF expression in the murine breast cancer cells 4T1. In addition, R9-ZnCS334D impaired proliferation, migration, invasion and anchorage-independent growth of 4T1 cells. In vivo, intra-tumoral injection of either protein significantly reduced VEGF expression and tumor vascularization. Strikingly, antibody array analyses of tumor extracts demonstrated a reduced expression of several chemokines such as Fractalkine, MCP-1, NOV, SDF-1 and Pentraxin upon R9-ZnC or R9-ZnCS334D treatment. These factors, which are produced by several cell types within tumor tissue, are key drivers of tumor angiogenesis, tumor-promoting inflammation and invasion. Furthermore, the expression of markers of the epithelial-to-mesenchymal transition was also significantly reduced, suggesting an anti-metastatic effect of R9-ZnC and R9-ZnCS334D. Thus, we provide R9-ZnC and R9-ZnCS334D as potential novel multi-target agents which inhibit key hallmarks of cancer progression. This work supports the emerging link between mRNA stability and cancer and proposes novel concepts for the development of innovative anti-cancer therapies.La formation de nouveaux vaisseaux sanguins ou angiogenèse soutient la croissance tumorale en fournissant l'oxygène et les nutriments qui lui sont nécessaires. Le rôle clé du facteur de croissance de l'endothélium vasculaire VEGF dans ce processus a suscité le développement de stratégies anti-angiogéniques pour le traitement du cancer. Cependant, des travaux précliniques et des données cliniques suggèrent l'émergence de résistances aux anti-angiogéniques, en raison notamment de la redondance des facteurs de croissance pro-angiogéniques. Il est donc nécessaire de développer des stratégies alternatives plus efficaces. En 2010, notre laboratoire a apporté la preuve de concept d'une thérapie anti-tumorale et anti-angiogénique innovante basée la dégradation des ARNm à demi-vie courte par la protéine à doigts de zinc TIS11b. Néanmoins, l'instabilité de la protéine thérapeutique a entravé la caractérisation plus détaillée de cette stratégie. Dans ce contexte, l'objectif majeur de ma thèse était l'optimisation de la stabilité et de l'activité de TIS11b et l'évaluation de son efficacité thérapeutique. Pour cela, nous avons généré une nouvelle protéine TIS11b génétiquement modifiée sur la base d'études biochimiques et moléculaires. Notamment, nous avons observé que la phosphorylation de la sérine 334 située dans le domaine C-terminal de TIS11b augmente de façon très significative la stabilité de la protéine et potentialise son activité déstabilisatrice de l'ARNm du VEGF. De plus, la délétion du domaine N-terminal augmente également la stabilité de TIS11b sans altérer son activité. Nous avons alors généré deux nouvelles protéines thérapeutiques, la protéine ZnC et la protéine ZnC334D pour laquelle la troncation du domaine N-terminal et la substitution de la sérine S334 par un aspartate mimant une phosphorylation ont été combinées. Les nouvelles protéines ont été fusionnées à une étiquette polyarginine R9 leur permettant de traverser les membranes cellulaires (R9-ZnC et R9-ZnCS334D). Nous avons montré que R9-ZnC et R9-ZnCS334D inhibent l'expression de VEGF in vitro dans la lignée de cancer du sein murin 4T1. De plus, R9-ZnCS334D exerce une activité anti-proliférative, anti-migratoire et anti-invasive dans ces cellules. In vivo, l'injection intra-tumorale de R9-ZnCS334D dans des tumeurs 4T1 préétablies inhibe significativement l'expression du VEGF, la croissance et la vascularisation tumorales. De façon remarquable, l'analyse des extraits tumoraux indique que le traitement diminue l'expression de chimiokines clés dans les processus d'angiogenèse, d'inflammation et d'invasion (Fractalkine, MCP-1, NOV, SDF-1, Pentraxin…). Enfin, R9-ZnC et R9-ZnCS334D inhibent l'expression de marqueurs de la transition épithélio-mésenchymateuse, un processus impliqué dans la dissémination métastatique. L'ensemble de ces travaux indique que R9-ZnC et R9-ZnCS334D sont des molécules anti-tumorales multi-cibles, qui inhibent plusieurs étapes clés de la progression tumorale. Cette étude confirme que le ciblage de la stabilité des ARNm est une stratégie prometteuse et novatrice pour le développement de nouvelles thérapies anti-cancéreuses

A novel multi-target cancer therapy based on destabilization of short-lived mRNAs

La formation de nouveaux vaisseaux sanguins ou angiogenèse soutient la croissance tumorale en fournissant l'oxygène et les nutriments qui lui sont nécessaires. Le rôle clé du facteur de croissance de l'endothélium vasculaire VEGF dans ce processus a suscité le développement de stratégies anti-angiogéniques pour le traitement du cancer. Cependant, des travaux précliniques et des données cliniques suggèrent l'émergence de résistances aux anti-angiogéniques, en raison notamment de la redondance des facteurs de croissance pro-angiogéniques. Il est donc nécessaire de développer des stratégies alternatives plus efficaces. En 2010, notre laboratoire a apporté la preuve de concept d'une thérapie anti-tumorale et anti-angiogénique innovante basée la dégradation des ARNm à demi-vie courte par la protéine à doigts de zinc TIS11b. Néanmoins, l'instabilité de la protéine thérapeutique a entravé la caractérisation plus détaillée de cette stratégie. Dans ce contexte, l'objectif majeur de ma thèse était l'optimisation de la stabilité et de l'activité de TIS11b et l'évaluation de son efficacité thérapeutique. Pour cela, nous avons généré une nouvelle protéine TIS11b génétiquement modifiée sur la base d'études biochimiques et moléculaires. Notamment, nous avons observé que la phosphorylation de la sérine 334 située dans le domaine C-terminal de TIS11b augmente de façon très significative la stabilité de la protéine et potentialise son activité déstabilisatrice de l'ARNm du VEGF. De plus, la délétion du domaine N-terminal augmente également la stabilité de TIS11b sans altérer son activité. Nous avons alors généré deux nouvelles protéines thérapeutiques, la protéine ZnC et la protéine ZnC334D pour laquelle la troncation du domaine N-terminal et la substitution de la sérine S334 par un aspartate mimant une phosphorylation ont été combinées. Les nouvelles protéines ont été fusionnées à une étiquette polyarginine R9 leur permettant de traverser les membranes cellulaires (R9-ZnC et R9-ZnCS334D). Nous avons montré que R9-ZnC et R9-ZnCS334D inhibent l'expression de VEGF in vitro dans la lignée de cancer du sein murin 4T1. De plus, R9-ZnCS334D exerce une activité anti-proliférative, anti-migratoire et anti-invasive dans ces cellules. In vivo, l'injection intra-tumorale de R9-ZnCS334D dans des tumeurs 4T1 préétablies inhibe significativement l'expression du VEGF, la croissance et la vascularisation tumorales. De façon remarquable, l'analyse des extraits tumoraux indique que le traitement diminue l'expression de chimiokines clés dans les processus d'angiogenèse, d'inflammation et d'invasion (Fractalkine, MCP-1, NOV, SDF-1, Pentraxin…). Enfin, R9-ZnC et R9-ZnCS334D inhibent l'expression de marqueurs de la transition épithélio-mésenchymateuse, un processus impliqué dans la dissémination métastatique. L'ensemble de ces travaux indique que R9-ZnC et R9-ZnCS334D sont des molécules anti-tumorales multi-cibles, qui inhibent plusieurs étapes clés de la progression tumorale. Cette étude confirme que le ciblage de la stabilité des ARNm est une stratégie prometteuse et novatrice pour le développement de nouvelles thérapies anti-cancéreuses.One of the innovative aspects of anti-cancer therapies is the possibility of preventing tumor growth by blocking blood supply. Cancer cells induce the formation of their own blood vessels from pre-existing vasculature, a process called angiogenesis. One of the most important proangiogenic factors is vascular endothelial growth factor (VEGF). The success of bevacizumab (a humanized anti-VEGF monoclonal antibody) combined to chemotherapy for the treatment of human metastatic cancers has validated VEGF as an efficient target. However, despite the initial enthusiasm, resistance to these anti-angiogenic treatments resulting from compensatory mechanisms occurs upon time. For this reason, there is a real need for new anti-angiogenic drugs that will target the angiogenic process through distinct mechanisms. In 2010, our laboratory has successfully developed an anti-angiogenic and anti-tumoral therapy based on destabilization of short-lived mRNAs by the zinc finger protein TIS11b. However, the therapeutic protein was highly unstable, thus making it difficult to further characterize the experimental therapy. In this context, the main task of my thesis was the optimization of TIS11b stability and activity followed by the evaluation of the multi-target action of our novel protein on tumor development. In a first part of this work, biochemical and molecular approaches allowed us to demonstrate that phosphorylation of the C-terminal serine S334 in TIS11b protein markedly increases its stability. In addition, deletion of the N-terminal domain of TIS11b highly increases its protein stability without affecting its activity. Therefore, we integrated N-terminal truncation (ZnC) and C-terminal substitution of S334 by an aspartate to mimic a permanent phosphorylation at S334 (ZnCS334D) as a novel TIS11b engineering strategy. Both proteins were fused subsequently to a cell-penetrating peptide polyarginine (R9). In vitro studies revealed that R9-ZnC and R9-ZnCS334D inhibit VEGF expression in the murine breast cancer cells 4T1. In addition, R9-ZnCS334D impaired proliferation, migration, invasion and anchorage-independent growth of 4T1 cells. In vivo, intra-tumoral injection of either protein significantly reduced VEGF expression and tumor vascularization. Strikingly, antibody array analyses of tumor extracts demonstrated a reduced expression of several chemokines such as Fractalkine, MCP-1, NOV, SDF-1 and Pentraxin upon R9-ZnC or R9-ZnCS334D treatment. These factors, which are produced by several cell types within tumor tissue, are key drivers of tumor angiogenesis, tumor-promoting inflammation and invasion. Furthermore, the expression of markers of the epithelial-to-mesenchymal transition was also significantly reduced, suggesting an anti-metastatic effect of R9-ZnC and R9-ZnCS334D. Thus, we provide R9-ZnC and R9-ZnCS334D as potential novel multi-target agents which inhibit key hallmarks of cancer progression. This work supports the emerging link between mRNA stability and cancer and proposes novel concepts for the development of innovative anti-cancer therapies

Cibler la dégradation des ARNms médiée par les éléments riches en A et U à l'aide d'une forme active tronquée de la protéine à doigts de zincs TIS11b/BRF1 affecte les principales propriétés de la tumorigenèse mammaire.

International audienceAltered expression of regulatory RNA-binding proteins (RBPs) in cancer leads to abnormal expression of mRNAs encoding many factors involved in cancer hallmarks. While conventional anticancer therapies usually target one pathway at a time, targeting key RBP would affect multiple genes and thus overcome drug resistance. Among the Tristetraprolin family of RBP, TIS11b/BRF1/ZFP36L1 mediates mRNA decay through binding to Adenylate/Uridylate (AU-rich elements) in mRNA 3'-untranslated region and recruitment of mRNA degradation enzymes. Here, we show that TIS11b is markedly underexpressed in three breast cancer cell lines, as well as in breast tumor samples. We hypothesized that restoring intracellular TIS11b levels could impair cancer cell phenotypic traits. We thus generated a derivative of TIS11b called R9-ZnCS334D, by combining N-terminal domain deletion, serine-to-aspartate substitution at position 334 to enhance the function of the protein and fusion to the cell-penetrating peptide polyarginine R9. R9-ZnCS334D not only blunted secretion of vascular endothelial growth factor (VEGF) but also inhibited proliferation, migration, invasion, and anchorage-independent growth of murine 4T1 or human MDA-MB-231 breast cancer cells. Moreover, R9-ZnCS334D prevented endothelial cell organization into vessel-like structures, suggesting that it could potentially target various cell types within the tumor microenvironment. In vivo, injection of R9-ZnCS334D in 4T1 tumors impaired tumor growth, decreased tumor hypoxia, and expression of the epithelial-to-mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin. R9-ZnCS334D also hindered the expression of chemokines and proteins involved in cancer-related inflammation and invasion including Fractalkine (CX3CL1), SDF-1 (CXCL12), MCP-1 (CCL2), NOV (CCN3), and Pentraxin-3 (PTX3). Collectively, our data indicate that R9-ZnCS334D counteracts multiple traits of breast cancer cell aggressiveness and suggest that this novel protein could serve as the basis for innovative multi-target therapies in cancer

A novel combined approach to detect androgenic activities with yeast based assays in Schizosaccharomyces pombe and Saccharomyces cerevisiae

We describe the construction and validation of novel test systems for detecting androgenic activities using a combination of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By applying the reporter enhanced Green Fluorescent Protein (EGFP) the incubation time could be reduced to only 24 h if compared to the classical p-galactosidase reporter (48 h). Both yeast systems were validated by analyzing the effects of seven androgens as well as five anti-androgens. One androgen (stanozolol) could be detected ten times more sensitive in S. cerevisiae than in S. pombe. Three of the five anti-androgenic substances showed no or only a slight effect in both yeast assays. The other two anti-androgens could be detected much better in S. pombe. Additionally, we could show that both yeast assays tolerated 10% urine within the media and still were capable to detect dihydrotestosterone at a concentration of 10(-8) M suggesting the use of the assays for applied doping pre-screening. In summary, the novel androgen-sensitive yeast assays have a large potential for various applications, e.g. as pre-screening in doping analysis or cattle feeding. A combination of both assays, exploiting these two phylogenetic very different yeasts, allows detection of the activity of a wide range of androgenic substances. (C) 2010 Elsevier Ireland Ltd. All rights reserved

The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

International audienceTPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function

Using Antigen-Specific B Cells to Combine Antibody and T Cell-Based Cancer Immunotherapy

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumordirected T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigenbiotin tetramers, resulted in the development of an antigenpresenting phenotype and the induction of a tumor antigenspecific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigenspecific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NYESO- 1-specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. (C) 2017 AACR