Journal: bioRxiv
Article Title: RESTRICT-seq enables time-gated CRISPR screens and uncovers novel epigenetic dependencies of SCC resistance
doi: 10.1101/2025.09.17.676440
Figure Lengend Snippet: (A) Comparative Robust Rank Aggregation (RRA) analysis of depleted genes from AZD4547-treated RESTRICT-seq vs constitutive wtCas9 CRISPR screens at P8, relative to vehicle-treated controls. (B) RRA analysis depicting normalized drug effect in wtCas9 and arCas9-ON CRISPR screens. (C) Comparison of the normalized drug effect obtained from RESTRICT-seq with results from a previously published AZD4547-focused constitutive wtCas9 kinome-wide screen. Colored points indicate significant hits. (D) Retrospective single-cell RNA sequencing (scRNA-seq) analysis of FGFR expression levels in KRT14-expressing cells from treatment-naïve cSCC tumors. n = 3 biological replicates. (E) Expression levels of top significantly depleted target genes from RESTRICT-seq AZD4547 screen in FGFR-positive and FGFR-negative K14+ cells from treatment-naïve cSCC tumors. (F) Schematic overview of the small molecule inhibitor screen analysis of synergistic lethality. (G) 10-point dose-response inhibition screen and IC50 determinations for the PAK1 inhibitor AZ13705339 in human SCC cell lines, as well as 392-1 and MR041718 tumorigenic mouse cSCC cell lines. n = 3 technical replicates. (H) Average IC50 values (µM) for selective inhibitors targeting the top significantly depleted genes identified by RESTRICT-seq (i.e., KAT2B/EP300, KAT6A, PAK1), in mouse (MR041718, 392-1) and human SCC lines (A431, JHU-029, SCC-25). Assay included GSK4028 as enantiomeric negative control and AZD4547 as positive control. n = 3 technical replicates from three separate experiments. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s test for multiple comparisons (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). (I) Synergistic lethality matrix showing cooperative effects of AZD4547 with AZ13705339 in metastatic tumor-derived mouse (MR041718) and (J) human (A431) cSCC cells. (K) Schematic overview of the SCC human survival meta-analysis encompassing retrospective sequencing data from over 5,000 SCC tumor patients. (L) Overall median months survival of SCC patients with wild-type (unaltered) or altered alleles encoding top significantly depleted target genes identified by RESTRICT-seq. Red arrows depict relative difference in overall survival months among patients with altered alleles.
Article Snippet: Drug synergy and dose–response assays were conducted using the following small-molecule inhibitors: PAK1 inhibitors AZ13705339 (Tocris, 6177) and NVS-PAK-1 (Tocris, 6132); FGFR inhibitor AZD4547 (Abcam, ab216311); KAT6A inhibitors WM-8014 (Tocris, 6693; ApexBio, A9779) and WM-1119 (MedChem Express, HY-102058); and CBP/P300 inhibitors SGC-CBP30 (Santa Cruz, 1613695-14-9), garcinol (Santa Cruz, 78824-30-3), GSK4027 (Sigma-Aldrich, SML2018), and its enantiomeric control GSK4028 (Sigma-Aldrich, SML2024).
Techniques: CRISPR, Comparison, RNA Sequencing, Expressing, Inhibition, Negative Control, Positive Control, Derivative Assay, Sequencing
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