Figure 4. A two tier loss of function screen for modulators of the response to WINi.

(A) Two-tier screen design. In the first tier, Cas9-expressing MV4;11 cells were transduced with a genome-wide sgRNA library and treated with 2 μM C6 until a resistant cell population emerged. sgRNA representation in the pre-treatment population was compared to the post-treatment population. In the second tier, cells were transduced with a custom library of distinct sgRNAs targeting non-pan-essential “hits” from the first tier, cultured in the presence of DMSO, C6, or C16, and sgRNA representation in C6/C16-treated cultures compared to that from DMSO-treated cultures. Created with BioRender.com. (B) Volcano plot, showing gene-level changes in sgRNA representation from the first tier (orange indicates FDR < 0.05). Datapoints corresponding to TP53, RPL22, and CDKN2A are indicated. See Figure 4—source data 1 for full output of the Tier 1 screen. (C) Comparison of gene-level changes in sgRNA representation in C6- and C16-treated populations in the second tier screen, each compared to DMSO-treated populations (red indicates FDR < 0.05; black indicates non-targeting control sgRNAs). See Figure 4—source data 2 for full output of the Tier 2 screen. (D) Top: Overlap of genes from the Tier 2 screen with enriched (left) or depleted (right) sgRNAs in C6- and C16-treated MV4;11 populations, compared to the DMSO control. Bottom: Overlap of genes with enriched (left) or depleted (right) sgRNAs in the first versus second tiers of the screen. “Tier 1” contains only those genes targeted in the Tier 2 screen. “Tier 2” contains the intersection of genes with altered sgRNAs in both the C6 and C16 treatments. (E) Ranked heatmap, representing the mean gene-level Log2 fold change (FC) of sgRNAs from the C6 and C16 treatments in the Tier 2 screen, as well as gene enrichment analysis outputs. Note that “Signal transduction by p53 class mediator” is a GO:BP term (orange); “p53” assignments (yellow) were added by manual curation.