Frédéric Pont

Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties

We previously synthesized dendrogenin A and hypothesized that it could be a natural metabolite occurring in mammals. Here we explore this hypothesis and report the discovery of dendrogenin A in mammalian tissues and normal cells as an enzymatic product of the conjugation of 5,6α-epoxy-cholesterol and histamine. Dendrogenin A was not detected in cancer cell lines and was fivefold lower in human breast tumours compared with normal tissues, suggesting a deregulation of dendrogenin A metabolism during carcinogenesis. We established that dendrogenin A is a selective inhibitor of cholesterol epoxide hydrolase and it triggered tumour re-differentiation and growth control in mice and improved animal survival. The properties of dendrogenin A and its decreased level in tumours suggest a physiological function in maintaining cell integrity and differentiation. The discovery of dendrogenin A reveals a new metabolic pathway at the crossroads of cholesterol and histamine metabolism and the existence of steroidal alkaloids in mammals.

First-in-man Phase 1 Clinical Trial of Gene Therapy for Advanced Pancreatic Cancer: Safety, Biodistribution, and Preliminary Clinical Findings

This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.

The gene expression profile of phosphoantigen-specific human γδ T lymphocytes is a blend of αβ T-cell and NK-cell signatures

Global transcriptional technologies have revolutionised the study of lymphoid cell populations, but human γδ T lymphocytes specific for phosphoantigens remain far less deeply characterised by these methods despite the great therapeutic potential of these cells. Here we analyse the transcriptome of circulating TCRVγ+ γδ T cells isolated from healthy individuals, and their relation with those from other lymphoid cell subsets. We report that the gene signature of phosphoantigen‐specific TCRVγ+ γδ T cells is a hybrid of those from αβ T and NK cells, with more ‘NK‐cell’ genes than αβ T cells have and more ‘T‐cell’ genes than NK cells. The expression profile of TCRVγ+ γδ T cells stimulated with phosphoantigen recapitulates their immediate physiological functions: Th1 cytokine, chemokine and cytotoxic activities reflect their high mitotic activity at later time points and do not indicate antigen‐presenting functions. Finally, such hallmarks make the transcriptome of γδ T cells, whether resting or clonally expanding, clearly distinctive from that of NK/T or peripheral T‐cell lymphomas of the γδ subtype.

Characterization of the arabinogalactan protein 31 (AGP31) of Arabidopsis thaliana: new advances on the Hyp-O-glycosylation of the Pro-rich domain.

Background: AGP31 is a multidomain plant cell wall hydroxyproline-rich glycoprotein. The position of Hyp and the distribution of carbohydrates is unknown. Results: Most Hyp of the Pro-rich domain are isolated within repeated motifs and carry glycans of various sizes. Conclusion: AGP31 glycoforms are very heterogeneous. Significance: This might be the first evidence for new Hyp-O-glycans. Proteins are important actors in plant cell walls because they contribute to their architecture and their dynamics. Among them, hydroxyproline (Hyp)-rich glycoproteins constitute a complex family of O-glycoproteins with various structures and functions. In this study, we characterized an atypical Hyp-rich glycoprotein, AGP31 (arabinogalactan protein 31), which displays a multidomain organization unique in Arabidopsis thaliana, consisting of a short arabinogalactan protein (AGP) motif, a His stretch, a Pro-rich domain, and a C-terminal PAC (PRP-AGP containing Cys) domain. The use of various mass spectrometry strategies was innovative and powerful: it permitted us to locate Hyp residues, to demonstrate the presence of carbohydrates, and to refine their distribution over the Pro-rich domain. Most Hyp were isolated within repeated motifs such as KAOV, KSOV, K(PO/OP)T, K(PO/OP)V, T(PO/OP)V, and Y(PO/OP)T. A few extensin-like motifs with contiguous Hyp (SOOA and SOOT) were also found. The Pro-rich domain was shown to carry Gal residues on isolated Hyp but also Ara residues. The existence of new type Hyp-O-Gal/Ara-rich motifs not recognized by the β-glucosyl Yariv reagent but interacting with the peanut agglutinin lectin was proposed. In addition, the N-terminal short AGP motif was assumed to be substituted by arabinogalactans. Altogether, AGP31 was found to be highly heterogeneous in cell walls because arabinogalactans could be absent, Hyp-O-Gal/Ara-rich motifs of different sizes were observed, and truncated forms missing the C-terminal PAC domain were found, suggesting degradation in muro and/or partial glycosylation prior to secretion.

Phosphoantigens Overcome Human TCRVγ9+ γδ Cell Immunosuppression by TGF-β: Relevance for Cancer Immunotherapy

Human γδ cells expressing TCRVγ9 are HLA-unrestricted CTLs with high relevance for cancer immunotherapy. Many tumor cell types produce TGF-β, however, a cytokine strongly immunosuppressive for conventional T CD4, CD8, and NK cells. Whether TGF-β also inhibits TCRVγ9+ lymphocytes was unknown. Because phosphoantigens (PAgs), such as bromohydrin pyrophosphate, selectively activate the antitumor functions of TCRVγ9+ T cells, in this study, we investigated whether TGF-β modulates these functions. We report that TGF-β does not block activation of TCRVγ9+ T cells but inhibits their PAg/IL-2–induced proliferation and maturation into effector cells and finally reduces the cytotoxic activity of these γδ T cells when exposed to lymphoma target cells. TGF-β did not bias their differentiation pattern toward γδ Th17 or γδ regulatory T cells. Nevertheless, increasing doses of PAg stimulus countered TGF-β inhibition. So, although TGF-β impairs TCRVγ9+ γδ cells like other cytolytic lymphocytes, PAg alone or combined to therapeutic mAb has the ability to bypass its immunosuppressive activity.

Pancreatic preneoplastic lesions plasma signatures and biomarkers based on proteome profiling of mouse models.

Background:Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a mortality that is almost identical to incidence. Because early detected PDAC is potentially curable, blood-based biomarkers that could detect currently developing neoplasia would improve patient survival and management. PDAC develops from pancreatic intraepithelial neoplasia (PanIN) lesions, graded from low grade (PanIN1) to high grade (PanIN3). We made the hypothesis that specific proteomic signatures from each precancerous stage exist and are detectable in plasma.Methods:We explored the peptide profiles of microdissected PanIN cells and of plasma samples corresponding to the different PanIN grade from genetically engineered mouse models of PDAC using capillary electrophoresis coupled to mass spectrometry (CE-MS) and Chip-MS/MS.Results:We successfully characterised differential peptides profiles from PanIN microdissected cells. We found that plasma from tumor-bearing mice and age-matched controls exhibit discriminative peptide signatures. We also determined plasma peptide signatures corresponding to low- and high-grade precancerous step present in the mice pancreas using the two mass spectrometry technologies. Importantly, we identified biomarkers specific of PanIN3.Conclusions:We demonstrate that benign and advanced PanIN lesions display distinct plasma peptide patterns. This strongly supports the perspectives of developing a non-invasive screening test for prediction and early detection of PDAC.

Human Monocyte Recognition of Adenosine-Based Cyclic Dinucleotides Unveils the A2a Gαs Protein-Coupled Receptor Tonic Inhibition of Mitochondrially Induced Cell Death

ABSTRACT Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cells in vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a Gαs-coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic. In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34+ cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a Gαs protein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death.

Large-scale microarray profiling reveals four stages of immune escape in non-Hodgkin lymphomas

ABSTRACT Non-Hodgkin B-cell lymphoma (B-NHL) are aggressive lymphoid malignancies that develop in patients due to oncogenic activation, chemo-resistance, and immune evasion. Tumor biopsies show that B-NHL frequently uses several immune escape strategies, which has hindered the development of checkpoint blockade immunotherapies in these diseases. To gain a better understanding of B-NHL immune editing, we hypothesized that the transcriptional hallmarks of immune escape associated with these diseases could be identified from the meta-analysis of large series of microarrays from B-NHL biopsies. Thus, 1446 transcriptome microarrays from seven types of B-NHL were downloaded and assembled from 33 public Gene Expression Omnibus (GEO) datasets, and a method for scoring the transcriptional hallmarks in single samples was developed. This approach was validated by matching scores to phenotypic hallmarks of B-NHL such as proliferation, signaling, metabolic activity, and leucocyte infiltration. Through this method, we observed a significant enrichment of 33 immune escape genes in most diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) samples, with fewer in mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL) samples. Comparing these gene expression patterns with overall survival data evidenced four stages of cancer immune editing in B-NHL: non-immunogenic tumors (stage 1), immunogenic tumors without immune escape (stage 2), immunogenic tumors with immune escape (stage 3), and fully immuno-edited tumors (stage 4). This model complements the standard international prognostic indices for B-NHL and proposes that immune escape stages 3 and 4 (76% of the FL and DLBCL samples in this data set) identify patients relevant for checkpoint blockade immunotherapies.

Cyclic dinucleotides modulate human T-cell response through monocyte cell death

Cyclic dinucleotides, a class of microbial messengers, have been recently identified in bacteria, but their activity in humans remains largely unknown. Here, we have studied the function of cyclic dinucleotides in humans. We found that c‐di‐AMP and cGAMP, two adenosine‐based cyclic dinucleotides, activated T lymphocytes in an unusual manner through monocyte cell death. c‐di‐AMP and cGAMP induced the selective apoptosis of human monocytes, and T lymphocytes were activated by the direct contact with these dying monocytes. The ensuing T‐cell response comprised cell‐cycle exit, phenotypic maturation into effector memory cells and proliferation arrest, but not cell death. This quiescence was transient since T cells remained fully responsive to further restimulation. Together, our results depict a novel activation pattern for human T lymphocytes: a transient quiescence induced by c‐di‐AMP‐ or cGAMP‐primed apoptotic monocytes.

nwCompare and AutoCompare Softwares for Proteomics and Transcriptomics Data Mining – Application to the Exploration of Gene Expression Profiles of Aggressive Lymphomas

The global protein and gene expression profiling technologies have revolutionized the study of normal and malignant cells. Transcriptomes permitted to delineate subtypes of B-cell lymphomas which were otherwise histologically and clinically undistinguishable. Although the data mining of proteomes or transcriptomes from these malignant cells can unveil new aspects of their biology, tools to simultaneously compare several samples are scarce. Here we depict nwCompare and Autocompare, two new freewares we developed with this aim, and examplify their use for the comparative data mining of transcriptomes from normal human B cells and B cell lymphomas such as follicular lymphomas (FL) and diffuse large Bcell lymphomas (DLBCL).