A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs

Abstract

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.

Figure 1

a. Schematic view of the composition of the SLC-focused CRISPR/Cas9 library and experimental outline of the genetic screen. b. Schematic view of the compound sets used in this study and the sequential filtering steps applied to the selection of a final set of 60 drugs for screening. c. Treeplot view of the drug classes and subclasses included in the screening set.

Figure 2

a. sgRNA-level enrichment for samples treated with 10X IC₅₀ methotrexate, as determined by DESeq2. All six sgRNAs targeting the SLC19A1 gene showed significant enrichment (n=2). b. Gene-level enrichment for samples treated with 10X IC₅₀ methotrexate, as determined by GSEA. Average log2 fold change for the significant sgRNAs for each gene is shown in the x-axis. Circle size indicates the number of significant sgRNAs. (n=2). c. Overview of significantly enriched SLCs (FDR≤1%) identified upon treatment with different compounds. Significant enrichments for all different doses of the same compound are merged together (union), selecting the most significant value in the case of repeated hits. SLC genes are ordered by name, and treatments are ordered by hierarchical clustering based on the gene-level results. Associations that underwent validation by MCA are shown with a black edge, and successfully validated cases are represented with a black dot in the center. All results are derived by pooling data from at least two independent experiments (n=2-3).

Figure 3

a. Schematic view of the Multicolor Competition Assay (MCA). b. Validation of selected SLC/drug associations by MCA. Results are shown by gene tested, pooling data of 1-5 independent experiments (biological replicates) each performed in technical triplicates. Ratios of GFP+/mCherry+ populations normalized to day0 ratios are shown for the indicated SLC/drug combinations at the given timepoints for the two sgRNAs tested, with different point shapes corresponding to separate biological replicates. Bars correspond to mean of all measurements shown. Statistical significance was calculated by ANOVA using biological replicates followed by Dunnett’s test. Compounds tested: ART: Artesunate, DHA: Dihydroartemisinin, NIS: Nisoldipine, PEN: Pentamidine, MTX: Methotrexate, PDX: Pralatrexate, RTX: Raltitrexed, TOP: Topotecan, 5-AZA: 5-Azacytidine, DAC: Decitabine, ARA-C: Cytarabine, GEM: Gemcitabine, CDDP: Cisplatin. Controls: DMSO: Dimethyl sulfoxide, DMF: Dimethylformamide.

Figure 4

a. Cell viability assay comparing sensitivity to methotrexate of WT HAP1 cells and cells carrying frameshift mutations in the SLC19A1 gene (ΔSLC19A1_1, ΔSLC19A1_2). Average values of three measurements are shown for a representative experiment (n=1). b. LC-MS/MS-based assay to measure intracellular concentrations of methotrexate in WT HAP1 cells and cells carrying frameshift mutations in the SLC19A1 gene (ΔSLC19A1_1, ΔSLC19A1_2). Bars show average of three measurements for a representative experiment (n=1). c. Cell viability assay comparing sensitivity to artesunate of WT HAP1 cells and cells carrying frameshift mutations in the SLC16A1 gene (ΔSLC16A1_1, ΔSLC16A1_2). Average values of three measurements are shown for a representative experiment (n=1). d. Cell viability assay showing increased sensitivity of HAP1 SLC16A1 KO cells reconstituted with SLC16A1 cDNA compared to cells reconstituted with eGFP. Average values of three measurements are shown for a representative experiment (n=1). e. Confocal images of HAP1 cells lacking endogenous SLC16A1 and reconstituted with GFP or SLC16A1 cDNA. Green: eGFP or SLC16A1, Blue: DAPI. Scale bar: 20 μm. Representative images from one of two independent experiments performed. f. LC-MS/MS-based assay to measure intracellular concentrations of artesunate in WT HAP1 cells and cells with mutations in, or reconstituted with, the SLC16A1 cDNA. Bars show average of three measurements for a representative experiment (n=1). All experiments shown are representative of at least two independent measurements.

Figure 5

a. Venn diagram showing the compound subsets used for the chemoinformatic analysis after stripping of stereochemistry and removal of anorganic and duplicated compounds. b. Correlogram plot showing the 2D descriptors contribution to the PCA analysis (n=9597). c. Principal components analysis of compounds in the DrugBank set of reference as well as in the sets tested in this study based on 22 annotated 2D chemical descriptors. The adjunct density plots show the distribution of compounds for Dimension 1 and Dimension 2. Compounds with a molecular weight below 900 Da (defined as “small molecule” by DrugBank) are shown as circles, the remaining compounds as crosses. Sample size as in panel a. d. Principal components analysis of compounds in the DrugBank set of reference compared the SLC-associated (active, 47) and non-SLC-associated (inactive, 11) compounds based on 22 annotated 2D chemical descriptors. Zoomed-in version for clarity, the full plot is shown in Supplementary Fig. 5b.