Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of... more Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent fla...
Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- a... more Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- and HER2-negative phenotype and in most cases, a relatively aggressive clinical behaviour. The lack of specific targeted therapies and low efficiency of currently available chemotherapies spurred several clinical trials in the last few years. Despite encouraging results, TNBC still remains a major unmet medical need that prompted us to explore the role of 863 epigenetic modulators in TNBC cell survival.Methods: A comprehensive siRNA library was screened to explore the role of known epigenetic modulators in TNBC cell viability and growth. The knock-down effect was evaluated for 863 epigenetic genes using 4 siRNAs/gene in two TNBC and a non-TNBC cell lines using ATP-based luminescence and nuclei count image-based assays. Considering siRNA off-target effects, four analysis methods including a classical threshold-based analysis and three ranking methods were applied to determine on-target hit...
Efficient cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of a... more Efficient cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumour immune responses. Yet, several steps of antigen intracellular traffic during cross-presentation are incompletely understood: in particular, the molecular mechanisms and the relative importance of antigen import from endocytic compartments into the cytosol. Here, we asked whether antigen import into the cytosol is rate-limiting for cross-presentation and anti-tumour immunity. By screening 700 FDA-approved drugs, we identified 37 import enhancers. We focused on prazosin and tamoxifen, and generated proteomic organellar maps of drug-treated DCs, covering the subcellular localisations of over 2000 proteins. By combining organellar mapping, quantitative proteomics, microscopy, and bioinformatics, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release into the cytosol. Enhancing antigen import facilitates cross-presentation of...
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupl... more Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.
Motivation High Content Screening (HCS) is an important tool in drug discovery and characterisati... more Motivation High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, High Content drug screens are performed on one single cell line. Yet, a single cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterise drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process. Results The contribution of this paper is twofold. First, we investigate whether we can predict drug mechanism of action (MOA) at the molecular level without optimisation of the MOA classes to the screen specificities. To this end, we benchmark a set of algorithms within a conventional pipeline, and evaluate their MOA prediction performance according to a statistically rigorous framework. Second...
We aim at the identification of myosin motor proteins that control trafficking at the Golgi appar... more We aim at the identification of myosin motor proteins that control trafficking at the Golgi apparatus. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identify MYO1C as a novel player at the Golgi. We demonstrate that depletion of MYO1C induces Golgi apparatus fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi apparatus that colocalize with Golgi-associated actin dots. Interestingly, MYO1C depletion leads to loss of cellular F-actin, and Golgi apparatus decompaction is also observed after the inhibition or loss of the Arp2/3 complex. We show that the functional consequences of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes branched actin at the Golgi apparatus that facilitates the arrival of incoming transport at the Golgi.
Proteins destined to the cell surface are conveyed through membrane-bound compartments using the ... more Proteins destined to the cell surface are conveyed through membrane-bound compartments using the secretory pathway. Multiple secretory routes exist in cells, which paves the way to the development of inhibitory molecules able to specifically perturb the transport of a chosen cargo. We used differential high-content screening of chemical libraries to identify molecules reducing the secretion of CCR5, the major co-receptor for HIV-1 entry. Three molecules strongly affected the anterograde transport of CCR5, without inhibiting the transport of the related G protein-coupled receptors CCR1 and CXCR4. These three molecules perturb the transport of endogenous CCR5 and decrease the entry of HIV in human primary target cells. Two molecules were found to share the same mode of action, inhibiting palmitoylation of CCR5. Our results demonstrate that secretory routes can be specifically targeted which allows to envisage novel strategies to provoke the intracellular retention or rerouting of secr...
High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, ... more High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, drug screens are performed in one single cell line. Yet, a single cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterise drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process. The contribution of this paper is twofold. First, we investigate whether we can predict drug mechanism of action (MOA) at the molecular level without optimisation of the MOA classes to the screen specificities. To this end, we benchmark a set of algorithms within a conventional pipeline, and evaluate their MOA prediction performance according to a statistically rigorous framework. Second, we extend this conventional pi...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 15, 2018
Targeted therapies that use the signaling pathways involved in prostate cancer are required to ov... more Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance. We established four chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by and silencing, and mass spectrometry analysis was used to identify its downstream effectors. We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulate...
Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeuti... more Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeutic drugs mainly because they allow for scanning a wide spectrum of possible targets at once. However, despite compelling methodological advances, posterior identification of a compound's mechanism of action (MOA) has remained difficult and highly refractory to automated analyses. Methods such as the cell painting assay and multiplexing fluorescent dyes to reveal broadly relevant cellular components were recently suggested for MOA prediction. We demonstrated that adding fluorescent dyes to a single assay has limited impact on MOA prediction accuracy, as monitoring only the nuclei stain could reach compelling levels of accuracy. This observation suggested that multiplexed measurements are highly correlated and nuclei stain could possibly reflect the general state of the cell. We then hypothesized that combining unrelated and possibly simple cell-based assays could bring a solution that ...
Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivat... more Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These result...
Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of... more Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent fla...
Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- a... more Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- and HER2-negative phenotype and in most cases, a relatively aggressive clinical behaviour. The lack of specific targeted therapies and low efficiency of currently available chemotherapies spurred several clinical trials in the last few years. Despite encouraging results, TNBC still remains a major unmet medical need that prompted us to explore the role of 863 epigenetic modulators in TNBC cell survival.Methods: A comprehensive siRNA library was screened to explore the role of known epigenetic modulators in TNBC cell viability and growth. The knock-down effect was evaluated for 863 epigenetic genes using 4 siRNAs/gene in two TNBC and a non-TNBC cell lines using ATP-based luminescence and nuclei count image-based assays. Considering siRNA off-target effects, four analysis methods including a classical threshold-based analysis and three ranking methods were applied to determine on-target hit...
Efficient cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of a... more Efficient cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumour immune responses. Yet, several steps of antigen intracellular traffic during cross-presentation are incompletely understood: in particular, the molecular mechanisms and the relative importance of antigen import from endocytic compartments into the cytosol. Here, we asked whether antigen import into the cytosol is rate-limiting for cross-presentation and anti-tumour immunity. By screening 700 FDA-approved drugs, we identified 37 import enhancers. We focused on prazosin and tamoxifen, and generated proteomic organellar maps of drug-treated DCs, covering the subcellular localisations of over 2000 proteins. By combining organellar mapping, quantitative proteomics, microscopy, and bioinformatics, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release into the cytosol. Enhancing antigen import facilitates cross-presentation of...
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupl... more Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.
Motivation High Content Screening (HCS) is an important tool in drug discovery and characterisati... more Motivation High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, High Content drug screens are performed on one single cell line. Yet, a single cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterise drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process. Results The contribution of this paper is twofold. First, we investigate whether we can predict drug mechanism of action (MOA) at the molecular level without optimisation of the MOA classes to the screen specificities. To this end, we benchmark a set of algorithms within a conventional pipeline, and evaluate their MOA prediction performance according to a statistically rigorous framework. Second...
We aim at the identification of myosin motor proteins that control trafficking at the Golgi appar... more We aim at the identification of myosin motor proteins that control trafficking at the Golgi apparatus. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identify MYO1C as a novel player at the Golgi. We demonstrate that depletion of MYO1C induces Golgi apparatus fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi apparatus that colocalize with Golgi-associated actin dots. Interestingly, MYO1C depletion leads to loss of cellular F-actin, and Golgi apparatus decompaction is also observed after the inhibition or loss of the Arp2/3 complex. We show that the functional consequences of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes branched actin at the Golgi apparatus that facilitates the arrival of incoming transport at the Golgi.
Proteins destined to the cell surface are conveyed through membrane-bound compartments using the ... more Proteins destined to the cell surface are conveyed through membrane-bound compartments using the secretory pathway. Multiple secretory routes exist in cells, which paves the way to the development of inhibitory molecules able to specifically perturb the transport of a chosen cargo. We used differential high-content screening of chemical libraries to identify molecules reducing the secretion of CCR5, the major co-receptor for HIV-1 entry. Three molecules strongly affected the anterograde transport of CCR5, without inhibiting the transport of the related G protein-coupled receptors CCR1 and CXCR4. These three molecules perturb the transport of endogenous CCR5 and decrease the entry of HIV in human primary target cells. Two molecules were found to share the same mode of action, inhibiting palmitoylation of CCR5. Our results demonstrate that secretory routes can be specifically targeted which allows to envisage novel strategies to provoke the intracellular retention or rerouting of secr...
High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, ... more High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, drug screens are performed in one single cell line. Yet, a single cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterise drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process. The contribution of this paper is twofold. First, we investigate whether we can predict drug mechanism of action (MOA) at the molecular level without optimisation of the MOA classes to the screen specificities. To this end, we benchmark a set of algorithms within a conventional pipeline, and evaluate their MOA prediction performance according to a statistically rigorous framework. Second, we extend this conventional pi...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 15, 2018
Targeted therapies that use the signaling pathways involved in prostate cancer are required to ov... more Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance. We established four chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by and silencing, and mass spectrometry analysis was used to identify its downstream effectors. We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulate...
Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeuti... more Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeutic drugs mainly because they allow for scanning a wide spectrum of possible targets at once. However, despite compelling methodological advances, posterior identification of a compound's mechanism of action (MOA) has remained difficult and highly refractory to automated analyses. Methods such as the cell painting assay and multiplexing fluorescent dyes to reveal broadly relevant cellular components were recently suggested for MOA prediction. We demonstrated that adding fluorescent dyes to a single assay has limited impact on MOA prediction accuracy, as monitoring only the nuclei stain could reach compelling levels of accuracy. This observation suggested that multiplexed measurements are highly correlated and nuclei stain could possibly reflect the general state of the cell. We then hypothesized that combining unrelated and possibly simple cell-based assays could bring a solution that ...
Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivat... more Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These result...
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Papers by Elaine Del Nery