Lorenzo Tomassoni

The impact of SARS-CoV-2 in the lung has been extensively studied, yet the molecular regulators of host-cell programs hijacked by the virus in distinct human airway epithelial cell populations remain poorly understood. Some of the reasons include overreliance on transcriptomic profiling and use of nonprimary cell systems. Here we report a network-based analysis of single-cell transcriptomic profiles able to identify master regulator (MR) proteins controlling SARS-CoV-2–mediated reprogramming in pathophysiologically relevant human ciliated, secretory, and basal cells. This underscored chromatin remodeling, endosomal sorting, ubiquitin pathways, as well as proviral factors identified by CRISPR assays as components of the viral-host response in these cells. Large-scale drug perturbation screens revealed 11 candidate drugs able to invert the entire MR signature activated by SARS-CoV-2. Leveraging MR analysis and perturbational profiles of human primary cells represents an innovative approach to investigate pathogen-host interactions in multiple airway conditions for drug prioritization.

Although the impact of SARS-CoV-2 in the lung has been extensively studied, the molecular regulators and targets of the host-cell programs hijacked by the virus in distinct human airway epithelial cell populations remain poorly understood. This is in part ascribed to the use of nonprimary cell systems, overreliance on single-cell gene expression profiling that does not ultimately reflect protein activity, and bias toward the downstream effects rather than their mechanistic determinants. Here we address these issues by network-based analysis of single cell transcriptomic profiles of pathophysiologically relevant human adult basal, ciliated and secretory cells to identify master regulator (MR) protein modules controlling their SARS-CoV-2-mediated reprogramming. This uncovered chromatin remodeling, endosomal sorting, ubiquitin pathways, as well as proviral factors identified by CRISPR analyses as components of the host response collectively or selectively activated in these cells. Large-scale perturbation assays, using a clinically relevant drug library, identified 11 drugs able to invert the entire MR signature activated by SARS-CoV-2 in these cell types. Leveraging MR analysis and perturbational profiles of human primary cells represents a novel mechanism-based approach and resource that can be directly generalized to interrogate signatures of other airway conditions for drug prioritization.

IPI-926 treatment alters cellular composition in the tumor microenvironment.

Myeloid network.Regulatory network for PDAC-associated myeloid cells, listing the inferred transcriptional targets (Target) for each regulatory protein (Regulator). Association weight (AW) and association mode (AM) are scores to quantify strength/direction of interaction. Sign indicates directionality of the interaction (1 = transactivating, -1 = transrepressing).

Abrogation of SHH signaling fuels hypersprouting in human and murine PDAC explants.

Overview of single cell regulatory network analysis.

Explant cellular populations throughout culture.

Supplementary Table 10: Antibodies for IHC and IF.Overview of antibodies used for stainings in human PDAC or KPC-derived tissues.Supplementary Table 11: Primer sequences for qRT-PCR.Primers for qRT-PCR-based quantification of ChIP and mRNA samples.Supplementary Table 12: Freezer dryer settings for sponge production.Specific settings for optimal sponge production.Supplementary Table 13: Explant media composition.List of reagents used for human PDAC and murine explants.

Explant cellular populations throughout culture.