Journal: JCI Insight

Article Title: CoREST complex inhibition alters RNA splicing to promote neoantigen expression and enhance tumor immunity

doi: 10.1172/jci.insight.190287

Figure Lengend Snippet: ( A ) Overview of immunoprecipitation–mass spectrometry experiment completed in duplicate. ( B ) Gene ontology analysis for proteins found to have significant baseline interactions (Log 2 FC > 1, P < 0.05) with LSD1 (top) and RCOR1 (bottom). Enrichment analysis was performed using the hypergeometric test with multiple test correction by the Benjamini-Hochberg method. ( C ) Venn diagram of RNA splicing proteins found to significantly interact with LSD1 and RCOR1. ( D ) Volcano plots of LSD1 (left) and RCOR1 (right) baseline interactions lost with corin treatment (red). Labeled points are proteins from the overlap in C . Statistical analysis was performed using the heteroscedastic t test. ( E ) IP-WB analysis of CoREST complex–U2AF2 and CoREST complex–SRSF1 interactions with DMSO or corin treatment (24 hours, 2.5 μM). ( F ) SDS-PAGE and Coomassie Blue staining of purified proteins. ( G ) GST pull-down assay using purified GST-tagged U2AF2 (amino acids 85–471) or SRSF1, and purified CoREST complex (LHC) or LSD1 protein.

Article Snippet: Mice were randomly assigned treatment groups (vehicle control, anti–PD-1, corin, and corin + anti–PD-1) and treated with 200 μg/mice of corin (HY-111048, MedChemExpress) or 200 μL vehicle control (5% DMSO/PBS) by daily intraperitoneal (i.p.) injections starting from day 6 after the tumor injection.

Techniques: Immunoprecipitation, Mass Spectrometry, Labeling, SDS Page, Staining, Purification, Pull Down Assay

Journal: JCI Insight

Article Title: CoREST complex inhibition alters RNA splicing to promote neoantigen expression and enhance tumor immunity

doi: 10.1172/jci.insight.190287

Figure Lengend Snippet: ( A ) Heatmap of significant KEGG and Hallmark pathways ( P < 0.05) across 6 melanoma cell lines treated with corin (24 hours, 2.5 μM) in duplicate. Pathways are ranked by average normalized enrichment score (NES), and cell lines are grouped based on phenotype. ( B ) Gene Set Enrichment Analysis plots for each cell line showing a significant negative enrichment for “KEGG Spliceosome.” ( C – E ) Heatmap of splicing factor genes significantly downregulated by corin treatment ( q < 0.01, Log2FC < –0.5) across all 6 melanoma cell lines clustered using Euclidean distance ( C ). Representative Western blot of downregulated splicing factors across 6 melanoma cell lines treated with DMSO ( D ) or corin ( C ) and quantification of biological replicates ( n = 3) ( E ). Data are shown as mean ± SD. ( F ) Kaplan-Meier plot of TCGA-SKCM patient survival based on median U2AF2 expression. Significance was determined using a log-rank test. ( G ) IP-WB analysis of CoREST-U2AF2 interactions following DMSO ( D ) and corin ( C ) treatment (24 hours, 2.5 μM) in V5-tagged U2AF2 overexpression SKMEL5 cells.

Article Snippet: Mice were randomly assigned treatment groups (vehicle control, anti–PD-1, corin, and corin + anti–PD-1) and treated with 200 μg/mice of corin (HY-111048, MedChemExpress) or 200 μL vehicle control (5% DMSO/PBS) by daily intraperitoneal (i.p.) injections starting from day 6 after the tumor injection.

Techniques: Western Blot, Expressing, Over Expression

Journal: JCI Insight

Article Title: CoREST complex inhibition alters RNA splicing to promote neoantigen expression and enhance tumor immunity

doi: 10.1172/jci.insight.190287

Figure Lengend Snippet: ( A ) Summary of significant RNA splicing changes across 6 melanoma cell lines treated with corin (ΔPSI ≥ |0.1|, q < 0.05) in duplicate. ( B ) PSI levels for all significant SE events following DMSO and corin treatment. Statistical comparisons were performed using a 2-sample t test to assess differences in PSI value between treatment groups within each cell line. P values were adjusted for multiple comparisons using the Bonferroni correction (* P adj < 0.05, **** P adj < 0.0001). ( C ) UpSet plot of skipped exon (SE) events that are exclusive to the differentiated phenotype, dedifferentiated phenotype, or shared by all cell lines (blue). ( D ) Unsupervised hierarchical clustering heatmap based on Euclidian distance of shared skipped exon inclusion levels. Rows are melanoma cell lines clustered by treatment and columns are shared inclusion events. ( E ) Gene ontology dot plot of the top pathways affected by corin-induced differential exon inclusion across all cell lines (median P adj < 0.01). Enrichment analysis was performed using the hypergeometric test with multiple test correction by the Benjamini-Hochberg method.

Article Snippet: Mice were randomly assigned treatment groups (vehicle control, anti–PD-1, corin, and corin + anti–PD-1) and treated with 200 μg/mice of corin (HY-111048, MedChemExpress) or 200 μL vehicle control (5% DMSO/PBS) by daily intraperitoneal (i.p.) injections starting from day 6 after the tumor injection.

Techniques:

Journal: JCI Insight

Article Title: CoREST complex inhibition alters RNA splicing to promote neoantigen expression and enhance tumor immunity

doi: 10.1172/jci.insight.190287

Figure Lengend Snippet: ( A ) Overview of neopeptide discovery and MHC binding predictions ( B ) UpSet plot of neopeptides (8–11 mers) produced with corin treatment of melanoma cells that are exclusive to the differentiated phenotype, dedifferentiated phenotype, or shared by all cell lines (blue). ( C ) Jaccard similarity index comparing corin-induced neopeptide production across all melanoma cell lines. ( D ) Number of corin-induced neopeptides predicted to bind to SKMEL5 HLAs based on 2 prediction tools: HLAthena and NetMHCPan4.1 (%Rank < 2). ( E ) Overlap of SKMEL5 corin-induced neopeptide HLA binders for each allele predicted by both tools. ( F ) Heatmap showing binding scores, PSI values, and junction counts of predicted SKMEL5 corin-induced neopeptides identified by SNAF and Splicetools. The top 15 unique candidates are labeled. ( G ) Histogram plots of peptides recovered from MHC-IP/MS in SKMEL5 DMSO and corin-treated (72 hours, 1 μM) samples for each replicate ( n = 2). ( H ) Identification of SKMEL5 corin-induced neopeptides recovered by MHC IP-MS. Corin-exclusive peptides are those identified from the IP-MS that appear in at least 1 corin replicate but neither DMSO replicate. Predicted peptides are those identified by binding scores in F . ( I ) Immunogenicity score predictions for neopeptide candidates. Green peptides are those identified from IP-MS and red peptides are additional candidates selected for immunogenicity validation assays based on immunogenic prediction and scores from F . ( J ) Ex vivo IFN-γ ELISpot assay for each candidate neopeptide tested with CEF and PHA positive controls. HLA-matched PBMCs were prestimulated with synthesized peptides (10 μg/mL) for 14 days in IL-2/IL-7 media. APCs were isolated from CD4 and CD8 depleted PBMCs, loaded with peptides (10 μg/mL), and seeded at a ratio of 3:1 with the prestimulated T cells in a 96 well ELISpot plate and analyzed for IFN-γ + T cells. ( K ) Quantification of ex vivo IFN-γ ELISpot assay illustrated in J . Statistical analysis was performed using multiple 2-tailed unpaired t tests. Data are shown as mean ± SD.

Article Snippet: Mice were randomly assigned treatment groups (vehicle control, anti–PD-1, corin, and corin + anti–PD-1) and treated with 200 μg/mice of corin (HY-111048, MedChemExpress) or 200 μL vehicle control (5% DMSO/PBS) by daily intraperitoneal (i.p.) injections starting from day 6 after the tumor injection.

Techniques: Binding Assay, Produced, Labeling, Protein-Protein interactions, Immunopeptidomics, Biomarker Discovery, Ex Vivo, Enzyme-linked Immunospot, Synthesized, Isolation

Journal: JCI Insight

Article Title: CoREST complex inhibition alters RNA splicing to promote neoantigen expression and enhance tumor immunity

doi: 10.1172/jci.insight.190287

Figure Lengend Snippet: ( A ) Schematic for corin + immunotherapy combination treatment in a melanoma xenograft mouse model. Six- to 10-week-old female C57BL/6 mice were inoculated with 2.5 × 10 5 B16-F10 cells. Mice were treated with 200 μg/mouse of corin or 200 μL vehicle control (5% DMSO/PBS) by daily i.p. injection starting from day 6 after tumor initiation. For anti–PD-1 treatment, mice were treated with 150 μg/mice anti–PD-1 or isotype control antibody 3 times/week starting from day 7 after tumor grafting. Ten mice were included in each treatment group. Tumors were measured 3 times/week and tumor volume, tumor weight, body weight change, spleen weight were measured. ( B and C ) Line plot and quantification of tumor volumes from day 7 to day 15 comparing DMSO, αPD-1, corin, and αPD-1 + corin treatment. ( D ) Histogram of tumor volumes depicted in B . ( E ) Histogram of body weight change relative to day 0 in animals treated with DMSO, αPD-1, corin, and αPD-1 + corin. ( F ) Histogram of spleen weights in animals treated with DMSO, αPD-1, corin, and αPD-1 + corin. Statistical analyses for C – F were performed using an ordinary 1-way ANOVA with Holm-Šídák’s correction for multiple comparisons. Data are shown as mean ± SD. ( G ) scRNA-Seq UMAP of the immune population (CD45 + ) isolated from B16-F10 melanomas. ( H ) Heatmap of the marker genes used to define immune subpopulations in G . ( I ) Subset UMAP of the T cell compartment comparing αPD-1 treatment to the combination of αPD-1 + corin. ( J ) Stacked bar plot of the T cell compartments in I . ( K ) Violin plots of significant DEGs (Log 2 FC > |1|, P adj < 0.05) in T cell populations isolated from αPD-1 versus αPD-1 + corin-treated B16-F10 melanomas. ( L ) GSEA plots for T cell populations isolated from αPD-1 versus αPD-1 + corin-treated B16-F10 melanomas showing enrichment for cytokine activity, leukocyte migration in inflammation, antigen response, and immune response in the αPD-1 + corin–treated tumors.

Article Snippet: Mice were randomly assigned treatment groups (vehicle control, anti–PD-1, corin, and corin + anti–PD-1) and treated with 200 μg/mice of corin (HY-111048, MedChemExpress) or 200 μL vehicle control (5% DMSO/PBS) by daily intraperitoneal (i.p.) injections starting from day 6 after the tumor injection.

Techniques: Control, Injection, Isolation, Marker, Activity Assay, Migration