a, Drug sensitivity profiles of tepoxalin and seven
additional compounds where ABCB1 RNA expression was the top
predictive genomic feature of the PRISM activity profile (n = 489 cell lines).
Tepoxalin was the only compound tested where high ABCB1 expression was
associated with sensitivity rather than resistance to tepoxalin. Cell line
viability is depicted with ABCB1 gene expression from CCLE
RNAseq log2 RPKM data. Cell lines are ranked by mean viability with a ceiling at
100%. b, ABCB1 knockout is the top hit that
rescues tepoxalin activity in a genome-wide CRISPR/Cas9 gene knockout screen.
LS1034-Cas9 cells (pXPR_311) were infected with the Brunello sgRNA library,
selected with puromycin, and treated with 16 μM tepoxalin versus vehicle
control with two replicates in independent flasks. Cells were passaged every
3–4 days over a 30-day period. Two-sided p-values are computed using
MAGeCK-MLE and shown versus gene-level log fold-changes. c,
ABCB1 overexpression sensitizes to tepoxalin activity in a
genome-wide CRISPR/dCas9 gene activation screen. LS1034-dCas9 cells (pXPR_109)
were infected with the Calabrese sgRNA library, selected with puromycin, and
treated with 16 μM tepoxalin versus vehicle control with two replicates
in independent flasks. Cells were passaged every 3–4 days over a 14-day
period. Two-sided p-values are computed using MAGeCK-MLE and shown versus
gene-level log fold-changes. d, Tepoxalin cellular competition
assay following ABCB1 knockout. LS1034-Cas9 cells (pXPR_311)
were stably infected with Firefly luciferase and parental LS1034 cells (without
Cas9) were stably infected with Renilla luciferase. Cells were mixed in 1:1
ratio an infected with the indicated sgRNA construct against
ABCB1 or an intergenic region on chromosome 2 (negative
control). Following puromycin selection, cell mixtures were treated with 16
μM tepoxalin. Firefly to Renilla luminescence ratio is plotted as log
fold-change over time. Mean of three technical replicates is shown and results
are representative of three independent experiments.