Multiplexed single-cell transcriptomics reveals diverse phenotypic outcomes for pathogenic SHP2 variants

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is an important regulator of Ras/MAPK signaling and a signaling hub downstream of receptor tyrosine kinases and other transmembrane receptors. Germline missense PTPN11 mutations cause developmental disorders such as Noonan Syndrome, whereas somatic mutations drive diverse cancers. While many pathogenic mutations enhance SHP2 catalytic activity, some are inactivating or alter protein-protein interactions, confounding our understanding of how SHP2 dysregulation causes diseases. Here, we perform multiplexed single-cell transcriptional profiling in cells expressing clinically diverse SHP2 variants, with and without receptor tyrosine kinase stimulation. We find that loss of catalytic activity does not phenocopy SHP2 knock-out at the gene expression level, and we show evidence for both convergent phenotypic effects from mechanistically distinct mutations, as well as the divergent effects from structurally similar mutations. These findings provide a framework for understanding how structural perturbations to SHP2 impact cellular outcomes. Single-cell RNA-seq libraries were generated using three-level single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) of basal or EGF-stimulated HEK 293 SHP2 variant cells. Cells transfected with different SHP2 variants and exposed to varying EGF concentrations were hashed at either 24hr or 96hr and pooled prior to sci-RNA-seq using the sci-Plex nuclear barcoding strategy. This nuclear barcoding strategy relies on fixation of barcode containing well-specific oligos that are specific to a given cell type, replicate, or treatment condition. Cells represent unique timepoint, SHP2 variant, EGF concentration, and replicate.