A model for network-based identification and pharmacological targeting of aberrant, replication-permissive transcriptional programs induced by viral infection

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1. Version published to 10.1038/s42003-022-03663-8

   Jul 19, 2022
2. 

   Version published to 10.21203/rs.3.rs-1287631/v1 on Research Square

   Feb 4, 2022
3. 

   ScreenIT

   Jul 6, 2021

   SciScore for 10.1101/2021.07.03.451001: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Secondary antibody goat antimouse IR 800 (Thermo) and DNA dye Draq5 (Thermo) were diluted 1/10,000 in blocking buffer and incubated for 1h at RT.	Secondary antibody goat antimouse suggested: None DNA dye Draq5 (Thermo) suggested: None Draq5 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The virus was amplified in Vero E6 cells and used at a passage 3 for all experiments as previously described(24, 34).	Vero E6 suggested: RRID:CVCL_XD71)
   Media containing drugs was removed and 106 Focus Forming Units (FFU) (as determined in Vero cells) of SARS-CoV-2 was added to each well for 1 hour at 37°C.	Vero s…

   SciScore for 10.1101/2021.07.03.451001: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Secondary antibody goat antimouse IR 800 (Thermo) and DNA dye Draq5 (Thermo) were diluted 1/10,000 in blocking buffer and incubated for 1h at RT.	Secondary antibody goat antimouse suggested: None DNA dye Draq5 (Thermo) suggested: None Draq5 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The virus was amplified in Vero E6 cells and used at a passage 3 for all experiments as previously described(24, 34).	Vero E6 suggested: RRID:CVCL_XD71)
   Media containing drugs was removed and 106 Focus Forming Units (FFU) (as determined in Vero cells) of SARS-CoV-2 was added to each well for 1 hour at 37°C.	Vero suggested: None
   RNA-Seq raw-counts data for Calu-3, H1299 and Caco-2 cell line models were obtained from Gene Expression Omnibus Database (Gene Expression Omnibus (GEO), GSE148729)(23).	Caco-2 suggested: None
   Specifically, protein activity signatures in response to SARS-CoV-2 infection of the lung adenocarcinoma cell lines (Calu-3, H1299 and A549), lung organoids and human lung tissue samples were inferred with the VIPER algorithm using the lung adenocarcinoma context-specific network.	Calu-3 suggested: None A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   For Calu-3 and H1299 cell lines, we considered as “SARS-CoV-2-infected” all the cells with at least 1 sequencing read mapping to the SARS-CoV-2 genome.	H1299 suggested: None
   Enrichment of Viral Checkpoint MRs on infection essential genes identified by CRISPR screens: CRISPR screen results (z-score) were downloaded from the supplementary data of Wei et.al(9) (Vero-E6 cells) and Schneider et.	Vero-E6 suggested: None
   (7) (Huh-7.5 cells).	Huh-7.5 suggested: RRID:CVCL_7927)
   Software and Algorithms
   Sentences	Resources
   Quantifications of infection was carried out by quantifying the number of infected cells (mKate positive cells) in infected and not infected samples using CellProfiler.	CellProfiler suggested: None
   RNA-Seq raw-counts data for Calu-3, H1299 and Caco-2 cell line models were obtained from Gene Expression Omnibus Database (Gene Expression Omnibus (GEO), GSE148729)(23).	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
   Raw-count data was normalized using the variance stabilization transformation (VST) procedure as implemented in the DESeq package from Bioconductor(49)	DESeq suggested: (DESeq, RRID:SCR_000154)
   Differential gene expression signatures for the Wyler’s dataset(23) (GSE148729) were computed by comparing the SARS-CoV-2 infected samples against the centroid—i.e. the average expression of each gene—of the closest matched non-infected (mock) samples as identified by unsupervised clustering.	Wyler’s suggested: None
   The optimal number of clusters was estimated by silhouette-score analysis as implemented in the “fviz_nbclust” function of the “factoextra” package (https://cran.r-project.org/web/packages/factoextra/index.html).	https://cran.r-project.org/web/packages/factoextra/index.html suggested: (factoextra, RRID:SCR_016692)
   Lung, colon and rectal adenocarcinoma context-specific models of transcriptional regulation were reverse-engineered, based on 517 lung, 459 colon and 167 rectal adenocarcinoma samples in The Cancer Genome Atlas (TCGA) with the ARACNe algorithm(13, 50), as discussed in(19).	ARACNe suggested: (ARACNE, RRID:SCR_002180)
   Briefly, gene expression profiles were transformed to differential gene expression signatures using the “scale” method—i.e. z-score transformation—as implemented in the VIPER package(12).	VIPER suggested: None
   Enrichment of the top 50 most activated, and the top 50 most inactivated proteins in response to SARS-CoV-2 infection, obtained after integrating (average) all 10 single-cell protein activity signatures, on each CRISPR experiment z-score signature, and on their Stouffer’s integration, were estimated by GSEA.	GSEA suggested: (SeqGSEA, RRID:SCR_005724)
   Based on the successful outcomes observed with OncoTreat when evaluated in the context of tumor suppression, we sought to develop a novel, analogous algorithm, ViroTreat, to identify small molecule compounds capable of suppressing viral infection by targeting the Viral Checkpoint module.	OncoTreat suggested: None
   The lung adenocarcinoma, colon and rectal context-specific interactomes are available as part of the “aracne.networks” R-system’s package from Bioconductor.	Bioconductor suggested: (Bioconductor, RRID:SCR_006442)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Such a holistic approach to matching disease dependencies to drug MoA overcomes the inherent limitations of drug repurposing efforts that focus on inhibitors of individual proteins or single pathways to thwart viral infectivity as part of a host cell-directed strategy. Elucidation of Viral Checkpoint MRs requires availability of gene expression signatures that accurately reflect virusmediated changes in the host cell transcriptome. Thus, to avoid confounding effects by model-idiosyncratic mechanisms and to ensure identification of more universal and reproducible MR proteins, we dissected the Viral Check-point from multiple, complementary models, including transformed cell lines and normal 3D-organoid cultures representing both airway and GI epithelium lineages. In addition, to avoid additional confounding effects arising from infection heterogeneity, we performed VIPER analysis at the single cell level, thereby mitigating the contribution of non-infected cells, which represent the majority of the tissue, based on reads mapping to the SARS-CoV-2 genome. Similarly, we avoided confounding effects arising from single cell transcriptional state heterogeneity by comparing each infected cell to a small pool of the closest non-infected cells, based on MR analysis, as controls. Finally, to achieve context-specific understanding of drug MoA, the analysis was performed in tissues reflective of the biology of infected cells based on conservation of their most differentially active MRs, a...

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT02066532	Unknown status	Ruxolitinib in Combination With Trastuzumab in Metastatic HE…
   NCT02632071	Completed	ACY-1215 + Nab-paclitaxel in Metastatic Breast Cancer
   NCT03211988	Recruiting	Entinostat Neuroendocrine (NE) Tumor
   NCT04492891	Recruiting	Cyclosporine For The Treatment Of COVID-19(+)
   NCT04351763	Recruiting	Amiodarone or Verapamil in COVID-19 Hospitalized Patients Wi…
   NCT04509999	Withdrawn	Bicalutamide to Block TMPRSS2 in Males With COVID-19 Infecti…

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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4. 

   Version published to 10.1101/2021.07.03.451001v1 on bioRxiv

   Jul 4, 2021