Fig. 1: DCAF15 is an AML-biased E3 ubiquitin ligase dependency.
From: DCAF15 control of cohesin dynamics sustains acute myeloid leukemia
a Schematic describing the design of CULLIN-RING E3 ubiquitin ligase (CRL) domain-focused sgRNA library. If known, sgRNAs targeted the receptor-adaptor interacting domain of the E3 (indicated with arrow), otherwise targeted first exon of gene. N8 NEDD8, R RING, U Ubiquitin. b Schematic describing workflow for CRISPR dropout screen using CRL sgRNA library. Protein domain Essentiality Score (ES) calculated for each gene as average[log2(final sgRNA abundance +1/initial sgRNA abundance)]. c Heatmap results for selected CRL genes and controls. Genes ranked by AML-biased ES, defined by the difference of ES in AML versus non-AML cell lines. d Overall survival of AML patients using GEPIA platform83 with low (bottom 75%) or high (top 25%) DCAF15 mRNA expression (TCGA-LAML dataset [https://www.cancer.gov/tcga]). TPM Transcripts Per Million. n = 106. log-rank test, **p-value < 0.01 (p = 0.0069). e DCAF15 mRNA expression in n = 252 AML patient samples compared to n = 6 normal hematopoietic stem cells (HSC) samples according to microarray expression profiling from GSE4251984,85, probe 91952_at. Welch’s two-sided t-test, *p-value < 0.05 (p = 0.0193). Center line shows the median, box limits show 75th and 25th percentiles, whiskers show minimum-maximum values, with the outlier shown as independent dot (Tukey method). Generated using BloodSpot platform86. f Competition-based proliferation assay performed in Cas9 + MV4-11 cell line. sgRNA+ populations monitored over time for mCherry expression by flow cytometry. sgRNA+ proportions normalized to day 3 post lentiviral sgRNA infection monitored over 23 days. n = 3 biologically independent replicates. sgROSA26, negative control; sgPCNA, positive control. g MV4-11 Cas9+ cell lines stably expressing empty vector (EV) or HA-tagged CRISPR-resistant DCAF15[ntG207C] were infected with ROSA26-targeting (negative control) or DCAF15-targeting sgRNAs. Cells treated with DMSO or 3 µM indisulam for 6 h and lysates analyzed by Western blot for indicated proteins. Immunoblots representative of two independent experiments. h Competition-based proliferation assay performed in MV4-11 Cas9+ cell lines stably expressing empty vector (EV) or DCAF15[ntG207C] and infected with ROSA26-targeting (negative control) or DCAF15-targeting sgRNAs. sgRNA+ populations monitored over time for mCherry expression by flow cytometry. sgRNA+ proportions normalized to day 6 post lentiviral sgRNA infection monitored over 24 days. n = 3 biologically independent replicates. Two-way ANOVA (with Bonferroni’s multiple comparisons test), ****padj < 0.0001, *padj < 0.05 (padj = 0.021), ns not-significant. Source data are provided as a Source Data file.
