Fig. 4: Transcriptional plasticity permits diverse mechanisms of resistance to ERK/MAPK inhibition.

a Schematic depicting CRISPR/Cas9 loss-of-function screen. b Replicate-to-replicate comparison of gene-level essentiality phenotypes in MIA PaCa-2 cells treated with DMSO (1:1000) alone. Essential controls are shown in red, non-essential controls in blue, and non-targeting controls in yellow. c Scatter plot of genes included in loss-of-function screen comparing gene expression changes in MIA PaCa-2 cells treated with SCH772984 (1 µM) compared to DMSO (1:1000) for either one week (early) or eight weeks (stable resistance), with loss-of-function gene score indicated by color gradient. Gene score is calculated as the log2 fold change of the fractional representations (time final/time zero) of resistant cells treated with SCH772984 (1 µM) compared to parental cells treated with DMSO (1:1000) at the screen midpoint (19 days). d Gene-level representation of essential phenotypes in evolved resistant MIA PaCa-2 cells treated with SCH772984 (1 µM) at the screen midpoint (19 days), ranked by their mean log2-transformed gene score across duplicates; the insert shows the effect of ABCG2 loss in parental cells treated with DMSO (1:1000, left), resistant cells treated with SCH772984 (1 µM, middle), and resistant cells treated with DMSO (1:1000, right) at all three screen time points (15, 19, and 23 days). e Immunoblot of treatment-naïve parental MIA PaCa-2 cells and three independently evolved resistant derivatives of this same cell line to SCH772984 (1 µM). f Crystal violet staining of MIA Paca-2 cells evolved resistant to SCH772984 (1 µM) with either no alteration, control knockout, or ABCG2 knockout, treated with either SCH772984 (1 µM) or DMSO (1:1000) for one week; representative photograph of all conditions performed in triplicate. g Eight-point growth inhibition assay of parental and evolved resistant MIA PaCA-2 cells treated with increasing concentrations of SCH772984, performed in triplicate; one triplicate set of evolved resistant cells is treated with the ABCG2 inhibitor Elacridar (1 µM) in the background. h GI50 values derived from eight-point dose-response curves of parental and evolved resistant MIA PaCA-2 cells treated with increasing concentrations of the indicated chemotherapies, with and without Elacridar (1 µM). i Genome browser view of ChIP-seq tracks across the ABCG2 gene coding locus depicting H3K27Ac density in MIA PaCa-2 cell treated with DMSO (1:1000, parental), SCH772984 (1 µM) for one week (Early), or SCH772984 (1 µM) for eight weeks (Resistant); known enhancer elements within the ABCG2 coding region are depicted with blue boxes. Error bars represent standard deviations.