shp2 mutant screen  (Element Biosciences Inc)

Journal: Science Advances

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

doi: 10.1126/sciadv.aea9389

Figure Lengend Snippet: ( A ) SHP2 receives input from a variety of cell signaling pathways, and SHP2 activation by binding to phosphoproteins has a diverse array of potential signaling and transcriptional outcomes. ( B ) Pie charts showing disease-driving genes for various human diseases, as identified in DNA sequencing of patient cohorts ( , – ). PTPN11 mutations underlie both congenital disorders and cancers. ( C ) Positions and frequencies of missense mutations in SHP2 along its 593-residue sequence. Pathogenic mutations were obtained from the ClinVar dataset. Cancer-associated mutations were obtained from the COSMIC and TCGA databases.

Article Snippet: The resulting libraries were sequenced on the Element Biosciences AVITI for the initial SHP2 mutant screen according to the manufacturer’s instructions and on the Illumina NovaSeq XPlus (Novogene) for the follow-up SHP2 Q510 screen.

Techniques: Protein-Protein interactions, Activation Assay, Binding Assay, DNA Sequencing, Residue, Sequencing

Journal: Science Advances

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

doi: 10.1126/sciadv.aea9389

Figure Lengend Snippet: ( A ) Schematic overview of RNA sequencing experiment to probe effects of SHP2 on gene expression. ( B ) Percentage of PTPN11 -expressing cells in the SHP2 KO population, SHP2 WT -transfected cells, and mock-transfected cells, of the total number of cells sequenced for those respective samples. ( C ) Volcano plots showing SHP2-induced DEGs for SHP2 KO and SHP2 WT at 24 hours. Any significant (FDR < 0.05) transcript with a ꞵ coefficient of >0.25 or < −0.25 is colored. ( D ) The same as (C), but for mock-transfected versus SHP2 KO . ( E ) Volcano plots showing EGF-induced DEGs for SHP2 KO (top) and SHP2 WT (bottom). Any significant (FDR < 0.05) transcript with a normalized ꞵ coefficient of >0.25 or < −0.25 is colored. ( F ) GSEA shows pathways of DEGs for SHP2 WT and SHP2 KO . * denotes FDR < 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001. ( G ) Four gene modules were identified between SHP2 WT and SHP2 KO . SHP2 WT without EGF stimulation behaves most similar to SHP2 KO . Low EGF is defined as 12.5 to 50 ng/ml; high EGF is defined as 100 to 1000 ng/ml.

Article Snippet: The resulting libraries were sequenced on the Element Biosciences AVITI for the initial SHP2 mutant screen according to the manufacturer’s instructions and on the Illumina NovaSeq XPlus (Novogene) for the follow-up SHP2 Q510 screen.

Techniques: RNA Sequencing, Gene Expression, Expressing, Transfection

Journal: Science Advances

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

doi: 10.1126/sciadv.aea9389

Figure Lengend Snippet: ( A ) Overview of mutants studied in this screen and their position on the protein. Additional descriptions of the mutants are given in table S3. ( B ) Heatmap of ꞵ coefficient correlation (Pearson’s ρ) with unsupervised hierarchical clustering, comparing SHP2 WT and all SHP2 variants at 24 hours. ( C ) Pseudo-bulked log 2 fold-change expression of cells grouped by time point, SHP2 variant (or mock-transfected cells), and EGF dose, against unstimulated SHP2 KO cells. Genes were filtered to the union of DEGs across all mutants (5209 genes). UMAP dimensions for 24 hours (hrs., left) and 96 hours (right). Colors of each mutant represent DEG correlation cluster at 24 hours, as seen in (B). ( D ) Number of DEGs per SHP2 variant, compared to SHP2 KO , at 24 hours. ( E ) The same as (D), but each SHP2 mutant compared to SHP2 WT .

Article Snippet: The resulting libraries were sequenced on the Element Biosciences AVITI for the initial SHP2 mutant screen according to the manufacturer’s instructions and on the Illumina NovaSeq XPlus (Novogene) for the follow-up SHP2 Q510 screen.

Techniques: Expressing, Variant Assay, Transfection, Mutagenesis

Journal: Science Advances

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

doi: 10.1126/sciadv.aea9389

Figure Lengend Snippet: ( A ) Overview of MrVI. MrVI model was trained on our 24 hours dataset, in which each combination of SHP2 variant and EGF-dose is defined as the sample of origin (96 unique samples), and replicate defined as the technical factor (two unique replicates). ( B ) UMAP of MrVI Z -space for all single cells (light gray), excluding SHP2 KO cells for visualization. Full MrVI Z -space can be found in fig. S4A. Each respective EGF dose group is indicated per UMAP. ( C ) UMAP of MrVI Z -space for all single cells, excluding SHP2 KO cells. Colors indicate clusters as identified by Leiden community detection. ( D ) Bar plots for each SHP2 mutant and their distribution across clusters. ( E ) UMAPs of the MrVI Z -space for SHP2 WT and SHP2 R138Q . ( F ) Volcano plot showing DEGs between SHP2 R138Q and SHP2 WT across EGF-concentrations. Significant genes (normalized effect size < −0.15 or > 0.15, FDR < 0.05) are labeled in dark gray (SHP2 WT ) and red (SHP2 R138Q ). ( G ) Dose response curves show reduced EGF-response of SHP2 R138Q compared to SHP2 WT . Data points and error bars represent the means and SD from three independent transfections, stimulation, and blotting experiments.

Article Snippet: The resulting libraries were sequenced on the Element Biosciences AVITI for the initial SHP2 mutant screen according to the manufacturer’s instructions and on the Illumina NovaSeq XPlus (Novogene) for the follow-up SHP2 Q510 screen.

Techniques: Variant Assay, Mutagenesis, Labeling, Transfection

Journal: Science Advances

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

doi: 10.1126/sciadv.aea9389

Figure Lengend Snippet: ( A ) UMAP of the MrVI Z -space for SHP2 T507K and SHP2 Q510K shows absence of cells in cluster 1. ( B ) Schematic showing the structure and activity changes in SHP2 Q510K relative to SHP2 WT , SHP2 E76K , and SHP2 T507K . The Q510K mutation shifts the protein toward the open conformation, while also enhancing Sprouty1 dephosphorylation. ( C ) AlphaFold 3 models of SHP2 T507K (top) and SHP2 Q510K (bottom), bound to Y53-phosphorylated Sprouty1 in the active site, showing the proximity of K507 and K510 with E55 on Sprouty1. ( D ) Catalytic efficiencies of full-length SHP2 WT , SHP2 T507K , and SHP2 Q510K against DiFMUP. ( E ) The same as (D), but for the isolated PTP domains. ( F ) Melting temperatures for full-length SHP2 WT , SHP2 T507K , SHP2 Q510K , and SHP2 Q510E . SHP2 E76K , a known open conformation mutant, is shown for reference. ( G ) The same as (F), but for isolated PTP domains. ( H ) Dephosphorylation assay with PTP WT , PTP T507K , and PTP Q510K showing switch in substrate preferences. Full peptide sequences are indicated in Materials and Methods. ( I ) Pairwise MrVI counterfactual cell distances, showing the largest distances between SHP2 WT and any Q-loop mutant. SHP2 Q510K and SHP2 Q510R show the smallest distance observed. ( J ) Heatmap for DEGs for SHP2 Q510K/R versus SHP2 WT . Z -scored mean expression for SHP2 Q510K , SHP2 Q510R , and SHP2 Q510E are visualized. Gene names in bold represent chaperone and protein folding genes. Red gene names represent EGF response genes.

Article Snippet: The resulting libraries were sequenced on the Element Biosciences AVITI for the initial SHP2 mutant screen according to the manufacturer’s instructions and on the Illumina NovaSeq XPlus (Novogene) for the follow-up SHP2 Q510 screen.

Techniques: Activity Assay, Mutagenesis, De-Phosphorylation Assay, Isolation, Expressing