A membrane transporter determines the spectrum of activity of a potent platinum–acridine hybrid anticancer agent

Cytotoxic drugs that are mechanistically distinct from current chemotherapies are attractive components of personalized combination regimens for combatting aggressive forms of cancer. To gain insight into the cellular mechanism of a potent platinum–acridine anticancer agent (compound 1), a correlation analysis of NCI-60 compound screening results and gene expression profiles was performed. A plasma membrane transporter, the solute carrier (SLC) human multidrug and toxin extrusion protein 1 (hMATE1, SLC47A1), emerged as the dominant predictor of cancer cell chemosensitivity to the hybrid agent (Pearson correlation analysis, p < 10–5) across a wide range of tissues of origin. The crucial role of hMATE1 was validated in lung adenocarcinoma cells (A549), which expresses high levels of the membrane transporter, using transporter inhibition assays and transient knockdown of the SLC47A1 gene, in conjunction with quantification of intracellular accumulation of compound 1 and cell viability screening. Preliminary data also show that HCT-116 colon cancer cells, in which hMATE1 is epigenetically repressed, can be sensitized to compound 1 by priming the cells with the drugs EPZ-6438 (tazemetostat) and EED226. Collectively, these results suggest that hMATE1 may have applications as a pan-cancer molecular marker to identify and target tumors that are likely to respond to platinum–acridines.

. Summary of RNA sequences used in RNAi experiments S3 Table S2. Summary of antibodies used in assays S4 Table S3. Ten NCI-60 cell lines most sensitive to compound 1/genetic backgrounds S5 Figure S1. Comparison of NCI-60 chemosensitivity profiles S6 Table S4. Results of NCI COMPARE analysis S7 Table S5. NCI-60/COMPARE analysis/summary of SLC genes S29 Figure S2. Expression of hMATE1 (SLC47A1) in normal human tissue and in cancer cells S30 Figure S3. SLC47A1 gene copy numbers and transcript levels in NCI-60 S31 Table S6. Summary of top 10 gene set overlaps in MSigDB for compound 1 S32 Figure S4. Western blot analysis of A549 cell lysate for hMATE1 expression Results for drug uptake experiments after hMATE1 knockdown S35 Table S7. Pattern comparisons for SLC47A1 expression in NCI CellMiner S36 Figure S6. Correlation between CPI methylation status and expression levels of SLC47A1 S37 Table S8. Summary of significant (p < 0.05) correlations identified between CPI methylation status and expression levels of the SLC47A1 gene S38 Table S9. Summary of correlations for chemosensitivity and omics data for compound 1 S39 Table S10. Summary of top 10 overlaps between all gene sets of the molecular signatures database (MSigDB) and the input gene set of hypermethylated genes negatively correlated with SLC47A1 expression. S40   Figure S1. Comparison of NCI-60 chemosensitivity profiles (averages of at least 2 assays) for cisplatin (CDDP, NSC 119875), doxorubicin (DOX, NSC 123127), topotecan (TOP, NSC 609699), and compound 1 (NSC # not disclosed).  -0.328 organic anion transporter * a N = 58. b * P < 0.05; ** P < 0.01; *** P < 0.001; ***** P < 0.00001.       Deposited as Excel spreadsheet Figure S6. A highly significant correlation (R = −0.32, p = 4.9  10 -25 ) between CPI methylation status and expression levels of the SLC47A1 gene is observed in 963 cancer cell lines of the Genomics of Drug Sensitivity in Cancer (GDSC) database (CellMinerCDB, version 1.1, discover.nci.nih.gov/cellminercdb). The data point for the colorectal cancer cell line HCT-116 used in this study is highlighted.  Table S9. Summary of correlations observed for chemosensitivity and omics data for compound 1.

Data in Comparison
Pearson's R P value NCI-60, logGI50 SLC47A1 transcript level 0.692 a < .00001 (*****) a NCI-60, logGI50 DNMT1 transcript level -0.378 a 0.0034 (**) a SLC47A1 transcript level .001 (***) b a NCI COMPARE analysis, n = 58. b NCI CellMiner analysis tool, database version 2.2, n = 60. Table S10. Summary of top 10 overlaps a between all gene sets of the molecular signatures database (MSigDB) b and the input gene set c of hypermethylated genes negatively correlated with SLC47A1 expression.  Assembly: Add 20 L Opti-Mem into wells B2-5, C2-6, and D2-5. Add 23.2 L of Opti-Mem into wells B6, C6, D6. Then add siRNA to B2-3, C2-3, and D2-3. Add scrambled RNA to B4-5, C4-5, and D4-5. Add diluted lipofectamine (siRNA:lipo 6:1) to B2-5, C2-5, and D2-5. Transfer cells to B2-5, C2-5, and D2-5 at a density of 5000 cells/well. Add same amount of medium into well B6-D6 as blank control. Add PBS into the outermost wells to prevent evaporation of transfection reactions. B2, C2, and D2 are siRNA For sensitization assays, HCT-116 cells were harvested from T-75 flasks, seeded on a 24-well plate with glass-like polymer bottom (P24-1.5P, Cellvis, Sunnyvale, CA) with 25,000 cells per well, and allowed to attached overnight. Single drugs or drug combinations were tested in this assay at final concentrations of 2.5 M EED226, 2.5 M EPZ-6438, 500 M valproic acid, and 10 M decitabine (see S49 assay layout 1, AL1, in the SI). Cells were incubated at 37 °C for 72 hours. Each well was replaced with fresh medium supplemented with 10 M compound 1, and incubation was continued for 4 additional hours. Each well was washed with 3 times with warm PBS, before cells were fixed with 0.5 mL of 4% formaldehyde at room temperature for 15 minutes. After 3 PBS washes, plates were immediately imaged or stored at 4 °C until analyzed. Subsequent incubations of HCT-116 cells at escalating doses of EED226 and EPZ-6438 were performed analogously (see AL2 in the SI). Representative conditions that were screened for expression levels of hMATE1 were determined by immunofluorescence with anti-hMATE1 antibody as described in RNAi knockdown experiments.

Assays and sample preparation for ICP-MS analysis
For transporter inhibition assays, 700,000 A549 cells in 2.5 mL of F12K media (ATCC 30-2004), supplemented with 10% FBS, and 10% penstrep, and 10% L-glutamine (Thermo Fisher, 25030-081), were seeded into T-25 flasks and allowed to attach overnight. Cells pre-treated with 100 nM pyrimethamine for 25 minutes and untreated cells were then dosed with 100 nM compound 1 for 3 hours.
After treatment, medium was aspirated, and cells were washed 3 times with fresh media. Trypsin was added to detach cells, and 3 mL of fresh media were added to each flask to collect the cell suspensions, which were pelleted by centrifugation at 250  g for 3 minutes. After the supernatant was aspirated, pellets were washed with 3 mL of PBS solution twice and centrifuged again at 250  g for 3 minutes.
Pellets were stored at -80 °C until analyzed by ICP-MS. The assay was performed in triplicate for each treatment group.
For uptake studies after hMATE1 (SLC47A1) knockdown, A549 cells were reverse-transfected with Silencer Select siRNA1 or Silencer Select Negative Control #1 scrambled RNA for 48 hours using the RNAiMAX system in Opti-Mem media. Media was replaced with fresh antibiotics-free DMEM/F12K medium and incubation was continued for an additional 24 hours. Cells were then incubated with 100 nM compound 1 at 37 °C for 4 hours, and cell pellets were prepared as described above. The assay was performed in triplicate for each treatment group. Microwave digestions and ICP-MS analysis for Pt were performed as described above.

Assays and sample preparation for cell viability screening
In pyrimethamine competition assays, A549 cells were seeded at a density of 5000 cells per well and allowed to attach for 24 hours. Cells were then pre-treated with 10 or 100 nM pyrimethamine for 20 minutes and subsequently incubated with 100 nM compound 1 or DMF-containing media (control) for 72 hours. No-treatment controls were also included. Assays were run in duplicate with 6 replicates per plate. Cell viability was assessed as described above.
Cell viability in RNAi knockdown assays was assessed by transfecting A549 cells on 96-well plates using a reverse transfection protocol. Briefly, Silencer siRNA, scrambled RNA (Silencer Negative Control #3 siRNA), and lipofectamine (RNAiMAX) were diluted with Opti-Mem prior to mixing in each well to generate a final siRNA concentration of 10 nM. Mixtures were incubated for 20 minutes at room temperature. Cells were then seeded into new wells in DMEM/F12 medium without antibiotics at a density of 5000 cells/well, incubated in the presence of transfection reagent for 24 hours at 37 °C in 5% CO2, and finally treated with compound 1 at fixed concentrations of 100 nM or 1 M (or DMF-containing media in control groups) for an additional 24 or 48 hours. Cell viability after 48 and 72 hours was assessed as described above.
In HCT-116 sensitization experiments, cells were seeded at a density of 1100 cells/well in 100 L of media and allowed to attach overnight. Medium in each well was replaced with fresh medium containing a combination of EED226 and EPZ-6438 to generate final concentrations of 2.5 and 5 M of each drug. Medium supplemented with epigenetic drugs was replaced every 24 hours and finally removed after 72 hours to begin treatment with compound 1 at concentrations of 1 M and 10 M for an additional 72 hours. Cell viability after 72 hours was assessed as described above.