Widespread use of microarrays has generated large amounts of data, the interrogation of the publi... more Widespread use of microarrays has generated large amounts of data, the interrogation of the public microarray repositories, identifying similarities between microarray experiments is now one of the major challenges. Approaches using defined group of genes, such as pathways and cellular networks (pathway analysis), have been proposed to improve the interpretation of microarray experiments. We propose a novel method to compare microarray experiments at the pathway level, this method consists of two steps: first, generate pathway signatures, a set of descriptors recapitulating the biologically meaningful pathways related to some clinical/biological variable of interest, second, use these signatures to interrogate microarray databases. We demonstrate that our approach provides more reliable results than with gene-based approaches. While gene-based approaches tend to suffer from bias generated by the analytical procedures employed, our pathway based method successfully groups together si...
Human Th17 cells have a limited proliferative capacity compared to other T-cell subsets. We have ... more Human Th17 cells have a limited proliferative capacity compared to other T-cell subsets. We have shown that human Th17 cells display impaired IL-2 production due to IL-4-induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B-cell traslocation gene) antiproliferative protein family, which prevents cell-cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR-activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)-related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR-mediated expansion not only by blocking the molecular pathway involved in the activation of the IL-2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.
Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target a... more Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.
We studied whether pharmacological blockade of the IL-1β-mediated signaling, rapidly activated in... more We studied whether pharmacological blockade of the IL-1β-mediated signaling, rapidly activated in forebrain by epileptogenic injuries, affords neuroprotection in two different rat models of status epilepticus (SE). As secondary outcome, we measured treatment's effect on SE-induced epileptogenesis. IL-1β signaling was blocked by systemic administration of two antiinflammatory drugs, namely human recombinant IL-1 receptor antagonist (anakinra), the naturally occurring and clinically used competitive IL-1 receptor type 1 antagonist, and VX-765 a specific non-peptide inhibitor of IL-1β cleavage and release. Antiinflammatory drugs were given 60min after antiepileptic (AED) drug-controlled SE induced by pilocarpine, or 180min after unrestrained electrical SE, for 7days using a protocol yielding therapeutic drug levels in brain. This drug combination significantly decreased both IL-1β expression in astrocytes and cell loss in rat forebrain. Neuroprotection and the antiinflammatory effect were more pronounced in the electrical SE model. Onset of epilepsy, and frequency and duration of seizures 3months after electrical SE were not significantly modified. Transcriptomic analysis in the hippocampus showed that the combined treatment did not affect the broad inflammatory response induced by SE during epileptogenesis. In particular, the treatment did not prevent the induction of the complement system and Toll-like receptors, both contributing to cell loss and seizure generation. We conclude that the IL-1β signaling represents an important target for reducing cell loss after SE. The data highlight a new class of clinically tested agents affording neuroprotection after a delayed post-injury intervention. Earlier blockade of this rapid onset inflammatory pathway during SE, or concomitant treatment with antiinflammatory drugs targeting additional components of the broad inflammatory response to SE, or co-treatment with AEDs, is likely to be required for optimizing beneficial outcomes.
To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) t... more To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.
There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstra... more There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies.
The constant improvement of high-throughput technologies has led to a great increase in generated... more The constant improvement of high-throughput technologies has led to a great increase in generated data per single experiment. Pathway analysis is a widespread method to understand experimental results at the system level. Pathway Processor 2.0 is an upgrade over the original Pathway Processor program developed in 2002, extended to support more species, analysis methods, and RNAseq data in addition to microarrays through a simple Web-based interface. The tool can perform two different types of analysis: the first covers the traditional Fisher's Test used by Pathway Processor and topology-aware analyses, which take into account the propagation of changes over the whole structure of a pathway, and the second is a new pathway-based method to investigate differences between phenotypes of interest. Common problems and troubleshooting are also discussed.
Widespread use of microarrays has generated large amounts of data, the interrogation of the publi... more Widespread use of microarrays has generated large amounts of data, the interrogation of the public microarray repositories, identifying similarities between microarray experiments is now one of the major challenges. Approaches using defined group of genes, such as pathways and cellular networks (pathway analysis), have been proposed to improve the interpretation of microarray experiments. We propose a novel method to compare microarray experiments at the pathway level, this method consists of two steps: first, generate pathway signatures, a set of descriptors recapitulating the biologically meaningful pathways related to some clinical/biological variable of interest, second, use these signatures to interrogate microarray databases. We demonstrate that our approach provides more reliable results than with gene-based approaches. While gene-based approaches tend to suffer from bias generated by the analytical procedures employed, our pathway based method successfully groups together si...
Human Th17 cells have a limited proliferative capacity compared to other T-cell subsets. We have ... more Human Th17 cells have a limited proliferative capacity compared to other T-cell subsets. We have shown that human Th17 cells display impaired IL-2 production due to IL-4-induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B-cell traslocation gene) antiproliferative protein family, which prevents cell-cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR-activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)-related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR-mediated expansion not only by blocking the molecular pathway involved in the activation of the IL-2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.
Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target a... more Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.
We studied whether pharmacological blockade of the IL-1β-mediated signaling, rapidly activated in... more We studied whether pharmacological blockade of the IL-1β-mediated signaling, rapidly activated in forebrain by epileptogenic injuries, affords neuroprotection in two different rat models of status epilepticus (SE). As secondary outcome, we measured treatment's effect on SE-induced epileptogenesis. IL-1β signaling was blocked by systemic administration of two antiinflammatory drugs, namely human recombinant IL-1 receptor antagonist (anakinra), the naturally occurring and clinically used competitive IL-1 receptor type 1 antagonist, and VX-765 a specific non-peptide inhibitor of IL-1β cleavage and release. Antiinflammatory drugs were given 60min after antiepileptic (AED) drug-controlled SE induced by pilocarpine, or 180min after unrestrained electrical SE, for 7days using a protocol yielding therapeutic drug levels in brain. This drug combination significantly decreased both IL-1β expression in astrocytes and cell loss in rat forebrain. Neuroprotection and the antiinflammatory effect were more pronounced in the electrical SE model. Onset of epilepsy, and frequency and duration of seizures 3months after electrical SE were not significantly modified. Transcriptomic analysis in the hippocampus showed that the combined treatment did not affect the broad inflammatory response induced by SE during epileptogenesis. In particular, the treatment did not prevent the induction of the complement system and Toll-like receptors, both contributing to cell loss and seizure generation. We conclude that the IL-1β signaling represents an important target for reducing cell loss after SE. The data highlight a new class of clinically tested agents affording neuroprotection after a delayed post-injury intervention. Earlier blockade of this rapid onset inflammatory pathway during SE, or concomitant treatment with antiinflammatory drugs targeting additional components of the broad inflammatory response to SE, or co-treatment with AEDs, is likely to be required for optimizing beneficial outcomes.
To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) t... more To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.
There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstra... more There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies.
The constant improvement of high-throughput technologies has led to a great increase in generated... more The constant improvement of high-throughput technologies has led to a great increase in generated data per single experiment. Pathway analysis is a widespread method to understand experimental results at the system level. Pathway Processor 2.0 is an upgrade over the original Pathway Processor program developed in 2002, extended to support more species, analysis methods, and RNAseq data in addition to microarrays through a simple Web-based interface. The tool can perform two different types of analysis: the first covers the traditional Fisher's Test used by Pathway Processor and topology-aware analyses, which take into account the propagation of changes over the whole structure of a pathway, and the second is a new pathway-based method to investigate differences between phenotypes of interest. Common problems and troubleshooting are also discussed.
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