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Fig. 3 | Molecular Cancer

Fig. 3

From: Uncovering cancer vulnerabilities by machine learning prediction of synthetic lethality

Fig. 3

Synthetic Lethal interaction between CDKN2A and TYMP with TYMS.a Violin plot showing TYMS dependency (0 lowest, 1 highest) with respect to mutation status of CDKN2A in DepMap data. Each point represents corresponding cell line and dependency value. P-value was calculated using Mann–Whitney U test. b A panel of cancer cell lines carrying wildtype CDKN2A: MDA-MB-157, HCC1937, missense mutant: DU-145, NCI-H1703, nonsense mutant: CAL27, deleted for CDKN2A locus: CAL62, HOP62, NCI-H292, KYSE-140, KYSE-70, KYSE-450, splice site mutant: M14, were tested with increasing concentrations of PMX (0, 0.001, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5 and 15 μM). Cell viability was measured with CellTiter Glo after incubation of the cells with PMX for 96 h. Drug response curves were generated and IC50 values shown in brackets (μM) next to each cell line were calculated from at least 3 biological and 9 technical repeats. c Heatmap showing CDKN2A mutation status (red box = nonsense mutation, green boxes = missense mutation, white boxes = WT); CDKN2A, TYMP, TYMS, GART, DHFR expression status for cell lines used in this study. Color scale corresponds to (log2(TPM) + 1) values based on RNA-Seq. d Cancer cell lines were treated with PBS or 5 μM PMX for 48 h. Western blot was performed for the proteins involved in Thymidine nucleotide metabolism (DHFR, GART, TYMS, TK1 and TYMP), DNA damage checkpoint marker (phospho-CHK1 (S345)) and apoptosis marker (cleaved-PARP1). VINCULIN served as a loading control. Cell lines labeled with red color are CDKN2A-deficient and show sensitivity to PMX in (b). Quantification of these blots are available in Additional file 2. e PMX-sensitive cancer cell lines were supplemented with PBS or 50 μM of thymidine to the media during PMX (50 nM or 5 μM) treatment. Cell viability was measured using live-cell protease (CellTiter Fluor) and % viability was calculated compared to the control treatment. Boxplots were generated from data from at least 3 biological and technical repeats. In the boxplots, centerlines mark the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend to 5th and 95th percentiles. P-values were calculated using Mann–Whitney U test. f CAL27 and CAL62 cells were transfected with gRNA targeting TYMP. Five days post transfection, control or TYMP KO cells were treated with increasing doses of PMX (0, 0,25, 0.5, 1, 5 and 15 μM) for 96 h. Drug response curves were generated using data from 8 and 2 biological replicates, respectively. (Right) Western blot analysis of the indicated proteins 5 days post gRNA transfection. g MDA-MB-157 cells were transfected with gRNA targeting TYMP, CDKN2A or both genes. Five days post transfection, control or KO cells were treated as described in (f). Drug response curves were generated from at least 3 biological replicates. (Right) Western blot analysis of the indicated proteins 5 days post gRNA transfection. h RPE1 TP53−/−; CMYC, RPE1 TP53−/−; CMYC;CDKN2A−/−, RPE1 TP53−/−; CMYC; TYMP, RPE1 TP53−/−; CMYC; TYMP;CDKN2A−/−,cells were transfected with two different gRNA against TYMS and viability were measured 7 days using CellTiter Glo. Values were normalized to scrambled gRNA transfection and were plotted from at least 9 biological replicates. In the boxplots, centerlines mark the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend to 5th and 95th percentiles. P-values were calculated using a Mann–Whitney U test. (Right) Western blot analysis of the generated RPE-1 cell lines. i Prediction of TYMS dependency by different genetic backgrounds. DepMap cancer cell lines grouped by their CDKN2A mutation and TYMP expression status. In each group the ratios of the percentage of TYMS dependent/TYMS independent cell lines were calculated and plotted. j TYMS dependency distribution is shown as boxplots. Cell lines are grouped by their CDKN2A and TYMP expression status. CDKN2A deficiency/proficiency is defined by the presence of a mutation or copy number loss and TYMP status is defined by tissue as high and low expressed using the median as a cut-off

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