Periodic Reporting for period 1 - MONET (Merkel cell polyomavirus Oncogenic Network)
Reporting period: 2019-09-02 to 2021-09-01
The aim of the MONET project is to finely decipher the Merkel Cell polyomavirus (MCPyV) oncogenic mechanisms in Merkel cell carcinoma (MCC). Viral pathogens are estimated to be responsible of ~12% of cancers worldwide, thus deserving extensive investigations and representing useful models for the study of oncogenesis mechanisms. The MCC is an aggressive neuroendocrine skin cancer detected in ~2500 patients per year in Europe. It has recently been linked with a clonal integration of MCPyV in more than 80% of cases. MCPyV is a 6-protein encoding virus, expressing only two proteins with reported oncogenic functions. This minimal transformation system thus allows system-wide study of its associated oncogenic mechanisms. Using cutting-edge mass spectrometry-based techniques in human cancer cell systems, these studies overall objective is to identify new protein-protein interactions essential for tumorigenesis. It aims at underpinning future translational researches, and hence appears as a fundamental step for therapeutic development. It has however been impacted by the COVID-19, as explain further in the next section.
Preliminary interactomics results unveil a novel axis potentially involved in MCPyV tumor antigens (TAs)-driven oncogenesis. Mining these proteomic and functional approaches allowed for a better understanding of MCPyV-associated carcinogenesis. In a broader view, it defines a new framework for identifying druggable targets in pathogen-driven cancers. This project will underpin future translational researches, and hence appears as a fundamental step for therapeutic development.
Preliminary interactomics results unveil a novel axis potentially involved in MCPyV tumor antigens (TAs)-driven oncogenesis. Mining these proteomic and functional approaches allowed for a better understanding of MCPyV-associated carcinogenesis. In a broader view, it defines a new framework for identifying druggable targets in pathogen-driven cancers. This project will underpin future translational researches, and hence appears as a fundamental step for therapeutic development.
The Researcher first did implement the proximal interactomics technologies (BioID and TurboID) in the host laboratory and then built stable inducible cell lines for the MONET project. During the COVID-19 lockdown, there was no mean to keep working on other subject than the SARS-CoV-2. The MONET project was then redirected using the molecular tools developed. This deviation was agreed upon with PO. Given the restriction, switching topic to SARS-CoV-2 was a mean to keep working in laboratory.
SARS-CoV-2 proximal interactome. We applied BioID to SARS-CoV-2 to delineate the first SARS-CoV-2 proximal interactome in human cells. The functional significance of our dataset is supported by the CRISPR-Cas screens identifying SARS-CoV-2 essential host factors. 432 protein-coding genes have been identified as essential in three or more independent studies. Our works identified 120 factors of them and provides molecular ties to at least one viral protein, more than any other study. We have applied our BioID pipeline to the 27 SARS-CoV-2 proteins, and performed the BioID experiments in un-stimulated cells or under poly(I:C) treatment to mimic viral infection. We thus have determined the first and most complete proteome proximal interactome of SARS-CoV-2, as well as the only virus-host proximal interactome upon immune stimulation.
Given that our interactome describes 5,626/10,185 PPIs not detected in any other study, we implemented N2H as an orthogonal approach to characterize these novel PPIs. We thus sampled 289 PPIs to identify the direct partners of viral proteins. We explored the binary, direct interactions between 156 high-confidence-proximal host factors and their cognate viral proteins. Binary virus-host interactions were measured using the N2H method, a Split-Nanoluciferase Complementation assay performed in human cells. A total of 85/289 BioID identified proximity interactions were detected at high confidence (p<0.01) of one or several viral proteins in the N2H assay (p<0.05). Thus, over 30% of the tested interactions are direct. We typically obtain 15-20% cross-validation when testing interactions identified by IP-MS technique, supposedly the gold standard to detect PPIs.
Our SARS-CoV-2 study identified NSP13-USP13 interaction by BioID, which was characterized as direct by N2H. When tested in an infectious context, a recently characterized UPS13 inhibitory compound, the Spautin-1, remarkably impaired SARS-CoV-2 replication, with an IC50 ~1µM, lower than gc376 (reference compound for in vitro SARS-CoV-2 life cycle inhibition), while no toxicity has been observed on cells treated at these doses. This molecule is under further characterization at our collaborator laboratory at Institut Pasteur, Paris.
MONET (initial plan). After lockdown, the initial MONET project resumed. TAs Interactomes assembly was achieved and analyzed to build the first functional hypothesis. Mass spectrometry-based experiments were extended to explore the changes induced in the proteome of cells expressing sT and LT at different times of induction. GST pull-down followed by mass spectrometry were done, using recombinant sT and LT fused to GST tag to identify which proximal interactors were able to directly bind the TAs. Given the epigenetic enrichment within the TAs proximal interactors, the TAs deregulate the cell transcriptome (thus proteome) through alterations the DNA binding proteins functions at the deregulated genes sites. Bioinformatic analysis are ongoing to identify which TAs proximal interactors are binding groups of genes encoding proteins identified as deregulated in our whole proteome analysis (ENCODE analysis). Results highlights are the finalization of the basal TAs proximal interactomes, the identification of EP400 as a new FBXW7 candidate target, the identification of PIN1 as the potential missing link between tLT-FBXW7 in oncogenesis, and the identification of a potential new addressing mechanism of PIN1 to FBXW7. Overall, although delayed and truncated, the MONET project has provided a wealth of data and opened several functional hypotheses which have been further explored.
SARS-CoV-2 proximal interactome. We applied BioID to SARS-CoV-2 to delineate the first SARS-CoV-2 proximal interactome in human cells. The functional significance of our dataset is supported by the CRISPR-Cas screens identifying SARS-CoV-2 essential host factors. 432 protein-coding genes have been identified as essential in three or more independent studies. Our works identified 120 factors of them and provides molecular ties to at least one viral protein, more than any other study. We have applied our BioID pipeline to the 27 SARS-CoV-2 proteins, and performed the BioID experiments in un-stimulated cells or under poly(I:C) treatment to mimic viral infection. We thus have determined the first and most complete proteome proximal interactome of SARS-CoV-2, as well as the only virus-host proximal interactome upon immune stimulation.
Given that our interactome describes 5,626/10,185 PPIs not detected in any other study, we implemented N2H as an orthogonal approach to characterize these novel PPIs. We thus sampled 289 PPIs to identify the direct partners of viral proteins. We explored the binary, direct interactions between 156 high-confidence-proximal host factors and their cognate viral proteins. Binary virus-host interactions were measured using the N2H method, a Split-Nanoluciferase Complementation assay performed in human cells. A total of 85/289 BioID identified proximity interactions were detected at high confidence (p<0.01) of one or several viral proteins in the N2H assay (p<0.05). Thus, over 30% of the tested interactions are direct. We typically obtain 15-20% cross-validation when testing interactions identified by IP-MS technique, supposedly the gold standard to detect PPIs.
Our SARS-CoV-2 study identified NSP13-USP13 interaction by BioID, which was characterized as direct by N2H. When tested in an infectious context, a recently characterized UPS13 inhibitory compound, the Spautin-1, remarkably impaired SARS-CoV-2 replication, with an IC50 ~1µM, lower than gc376 (reference compound for in vitro SARS-CoV-2 life cycle inhibition), while no toxicity has been observed on cells treated at these doses. This molecule is under further characterization at our collaborator laboratory at Institut Pasteur, Paris.
MONET (initial plan). After lockdown, the initial MONET project resumed. TAs Interactomes assembly was achieved and analyzed to build the first functional hypothesis. Mass spectrometry-based experiments were extended to explore the changes induced in the proteome of cells expressing sT and LT at different times of induction. GST pull-down followed by mass spectrometry were done, using recombinant sT and LT fused to GST tag to identify which proximal interactors were able to directly bind the TAs. Given the epigenetic enrichment within the TAs proximal interactors, the TAs deregulate the cell transcriptome (thus proteome) through alterations the DNA binding proteins functions at the deregulated genes sites. Bioinformatic analysis are ongoing to identify which TAs proximal interactors are binding groups of genes encoding proteins identified as deregulated in our whole proteome analysis (ENCODE analysis). Results highlights are the finalization of the basal TAs proximal interactomes, the identification of EP400 as a new FBXW7 candidate target, the identification of PIN1 as the potential missing link between tLT-FBXW7 in oncogenesis, and the identification of a potential new addressing mechanism of PIN1 to FBXW7. Overall, although delayed and truncated, the MONET project has provided a wealth of data and opened several functional hypotheses which have been further explored.
Alike all researches unfolding along the COVID-19 crisis, the MONET project has been greatly slowed down by the sanitary crisis and associated restrictions. However, it has allowed: (i) the identification of MCpyV TAs proximal interactors; (ii) the orthogonal validation of several of them; (iii) characterizing the impact of their expression on the cell proteome, including modifications which can be linked to transformation (normal to oncogenic state); and (iv), the formulation of several hypothesis, on oncogenesis but also on fundamental mechanisms which are currently investigated.
Given the scientific reorientation of the work towards SARS-CoV-2 virus-host interactions, MONET has also provided an unmatched wealth of data and thus contributes to the global knowledge needed to combat the COVID-19 pandemic. The latest developments of this work prove that proximal interactomics outperform classical IP-MS experiments in detecting direct interactors (which is counter-intuitive but clear based on our data). We have also identified a candidate therapeutic molecule which could be envision for clinical management of severe COVID-19. Further experiments are ongoing. The potential impact of such results could have an important impact on the sanitary crisis management. The MONET project, despite exceptional circumstances, has thus provided significant advances which will contribute to a better understanding of MCC mechanisms, fundamental cell biology, and SARS-CoV-2 infectious pathways. Together, we believe this project has and will have a significant impact on these subjects.
Given the scientific reorientation of the work towards SARS-CoV-2 virus-host interactions, MONET has also provided an unmatched wealth of data and thus contributes to the global knowledge needed to combat the COVID-19 pandemic. The latest developments of this work prove that proximal interactomics outperform classical IP-MS experiments in detecting direct interactors (which is counter-intuitive but clear based on our data). We have also identified a candidate therapeutic molecule which could be envision for clinical management of severe COVID-19. Further experiments are ongoing. The potential impact of such results could have an important impact on the sanitary crisis management. The MONET project, despite exceptional circumstances, has thus provided significant advances which will contribute to a better understanding of MCC mechanisms, fundamental cell biology, and SARS-CoV-2 infectious pathways. Together, we believe this project has and will have a significant impact on these subjects.