Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1B(high) cells

Abstract

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

Figure 1. Cytotoxic treatment results in the relative enrichment of therapy resistant JARID1B^high melanoma cells

(A) WM3734 melanoma cells were persistently treated with cisplatin and analyzed for J/EGFP expression by flow cytometry at the indicated time points (left). Dead cells were detected by 7AAD staining. Control cells were analyzed for C/EGFP expression (right). Depicted is one representative of at least three independently performed experiments. (B) MTT cytotoxicity assay of WM3734 cells treated with increasing concentrations of cisplatin (left). Relative percentage of the J/EGFP^high subpopulation under escalating cisplatin treatment (right). Both panels show results from 72 hr of treatment. (C) Immunoblot of JARID1B protein expression in pre- and post-cisplatin treated WM3734 cells at the indicated time points. (D) Cytotoxicity assay and quantitation of the J/EGFP^high subpopulation after treatment of WM3734 cells for 72 hr with escalating vemurafenib concentrations (left and middle). Comparison of the J/EGFP expression levels of 7AAD⁺ and 7AAD cells (right). Error bars in the cytotoxicity assays represent SD. See also Figure S1.

Figure 2. The JARID1B^high subpopulation of melanoma cells is resistant to a broad panel of anti-cancer drugs

(A) Cytotoxicity assays of WM3734 cells after 72 hr of treatment with the indicated anti-cancer drugs (left). Flow cytometric determination of the relative percentage of the J/EGFP^high subpopulation under these treatments (middle). Typical examples for the corresponding drug-associated distribution of 7AAD and J/EGFP signals (right). (B) Frequency of J/EGFP^high WM3734 cells as measured by flow cytometry after incubation with the indicated compounds over 72 hr. Error bars represent SD. See also Figure S2.

Figure 3. The JARID1B^high subpopulation is enriched in melanomas resisting therapy and knockdown of JARID1B leads to increased drug sensitivity

(A) NSG mice xenografted with WM3734 melanoma cells were treated with vemurafenib or vehicle (n=7). Tumor weights were assessed after 11 days of treatment (left). Tumor residues were immunostained for JARID1B expression (middle) and nuclear staining signals of each tumor were scored at 40x magnification in 3 representative fields of vision (right, two-sided Wilcoxon two-sample test). (B) Immunstaining of JARID1B in matched pairs of melanoma samples before and relapse under vemurafenib treatment from three melanoma patients. (C) Two different JARID1B knockdown clones (sh_JARID1B_58 and 62) of WM35 melanoma cells and the sh_scrambled control were starved for 4 days at 0% FCS and subsequently stimulated by 10% FCS for 8 hr. The bar graph shows one example of five independent experiments of propidium iodide-based cell cycle analysis (left). The immunoblot shows pRB phosphorylation (right). (D) NSG mice were xenotransplanted with WM3734 melanoma cells stably knocked down for JARID1B (sh_JARID1B) or with control cells (sh_scrambled). Each mouse received 10⁴ cells (n=8). After tumors reached an average volume of ~200mm³, mice were treated with bortezomib (left, BOR), vemurafenib (right, VEM), or vehicle (PBS). Tumor volumes were measured at the indicated time points. Error bars represent SD. See also Figure S3.

Figure 4. Dynamics of the therapy resistant JARID1B^high phenotype

(A) WM3734^{JARID1Bprom-EGFP} cells were FACS-sorted according to their J/EGFP expression and the isolated subfractions were cultured as adherent monolayers over night. After subsequent treatment for 72 hr, the frequencies of J/EGFP^high and J/EGFP^low cells were again quantified by flow cytometry. Dead cells were detected by 7AAD staining. JARID1B mRNA expression was quantified by QPCR. (B) Flow cytometric detection of J/EGFP^high WM3734 cells after prolonged incubation with different concentrations of cisplatin and after drug recovery. (C) Colony forming assay of WM3734 cells that underwent cytotoxic pulse treatment prior to seeding. Cells were treated for 72 hr with cisplatin or vehicle. Surviving cells were subsequently embedded into soft agar (2000 cells/well). Colony sizes and numbers were digitally quantified and color-coded with dark blue colonies representing large colonies above 1000 pixels. Shown are results from two independent experiments, each performed in triplicate. Error bars represent SD.

Figure 5. Elevated mitochondrial bioenergy metabolism in the JARID1B^high subpopulation

(A) Immunoblotting of mitochondrial respiratory enzymes in FACS-sorted J/EGFP^high and J/EGFP^low WM3734 cells. Fold changes were normalized to GAPDH. (B) Relative mitochondrial ATP levels in control (CTRL) or JARID1B-transfected WM3734 cells were measured using the ATP sensor protein A_TEAM. Representative A_TEAM ratio images (top) and summarized A_TEAM ratio values in control (n=72) and JARID1B-transfected (n=47) cells (bottom). (C) Mean oxygen consumption of control- (black) or JARID1B-transfected (red) WM3734 cells presented as decline of trityl radical intensity per minute per 10⁶ cells (left). Mean oxygen concentration in presence of control or JARID1B-transfected WM3734 cells presented as trityl intensity per 10⁶ cells. Each data point is an average of 7 independent experiments. (D) Relative H₂O₂ levels were measured in JARID1B-transfected WM3734 cells using the H₂O₂ sensor protein Hyper. Columns represent averaged Hyper ratio values of control- (black, n=71) or JARID1B-transfected cells (red, n=48). To test the role of mitochondria as source of H₂O₂, cells were treated with 1 µM oligomycin (dark gray) or 1 µM rotenone (light gray). Numbers of cells averaged were: CTRL plus oligomycin (n=50), CTRL plus rotenone (n=42), JARID1B plus oligomycin (n=45) and JARID1B plus rotenone (n=52). In a confirmation experiment with WM3734 cells transfected with pCAGGS-JARID1B-IRES-RFP (right), positively transfected cells were selected for analysis based on the expression of RFP. Error bars represent SEM. See also Table S1.

Figure 6. Inhibition of the mitochondrial ATP synthase inhibits the JARID1B^high subpopulation and reveals long-term melanoma cell suppressive effects

(A) MTT reduction curve of regularly cultured WM3734 melanoma cells after oligomycin treatment. (B) Total cellular ATP levels were luminometrically determined in WM3734 cells after 24 hr of treatment with oligomycin at the indicated concentrations (left). Lactate production was measured in WM3734 cell culture supernatants after 24 hr of oligomycin. Ethanol (EtOH, diluted 1:1000) was used as vehicle control. Shown are means of three independently performed experiments. (C) Lactate measurement in a panel of genetically diverse melanoma cell lines under normoxic vs. hypoxic culture conditions after 24 hr of treatment. (D) Immunoblot of JARID1B protein expression in WM3734 cells treated with oligomycin for 72 hr at the indicated concentrations (top). Frequency of J/EGFP^high WM3734 cells under 72 hr of hypoxia vs. hypoxia plus oligomycin (oligo) treatment as determined by flow cytometry (middle). Immunoblots of endogenous JARID1B expression in MelJuSo and SKMel5 melanoma cells under hypoxia and hypoxia plus oligomycin treatment (bottom). (E) WM3734 cells were starved at 0% FCS and then released by 10% FCS for 8 and 16 hr. The bar graph shows one example of three independent experiments of propidium iodide-based cell cycle analysis. Dead cells (SubG1) were gated out. (F) Short-term effects of increasing concentrations of oligomycin on total WM3734 cell numbers as determined by crystal violet assays (left). Long-term growth reduction as measured by 3D soft agar colony formation assays (bi-daily treatment, right). Error bars represent SD. See also Figure S4.

Figure 7. The combination of anti-cancer agents plus inhibition of the mitochondrial ATP-synthase overcomes the enrichment of the JARID1B^high subpopulation in vitro

(A) Relative percentage of the J/EGFP^high subpopulation under combinatorial treatment of WM3734 cells for 72 hr with cisplatin (cis) and oligomycin as determined by flow cytometry (left). Ethanol (EtOH) was used as vehicle control. Immunoblots of the endogenous JARID1B protein expression of WM3734 cells under co-treatment with oligomycin at the indicated time points (right). (B) Cytotoxicity assays of melanoma cell lines after 72 hr of co-treatment with cisplatin (cis) and oligomycin (oligo). (C) Three-dimensional growth/invasion/survival assay of 1205Lu cells treated with vemurafenib (vem) and oligomycin (oligo) for 96 hr. Colony sizes were quantified digitally based on the relative number of pixels. Scale bar represents 500 µm. (D) Soft agar colony forming assay of WM3734 cells after 72-hr pulse treatment with oligomycin (left) and oligomycin plus cisplatin (right). Colonies were counted manually after 5 weeks of growth. Results shown are from one of two independent experiments. Error bars represent SD. See also Figure S5.

Figure 8. The effect of anti-cancer agents is increasedin vitroandin vivoby the combination with complex I inhibitors

(A) Flow cytometric pattern of 7AAD and J/EGFP signals in cultured WM3734 cells after 72 hr of treatment with cisplatin and rotenone. (B) SCID beige mice were xenotransplanted with 10⁵ SKMel28 human melanoma cells (left, n=7 per treatment group, n=6 in vehicle group), NSG mice with 5×10⁴ WM3734 cells (right, n=8 per group). SKMel28 tumors were treated when an average volume of ~120 mm³ was reached, WM3734 tumors at an average volume of 200 mm³. Tumor volumes were assessed every other day. Groups marked with # were terminated earlier due to excessive tumor growth. Error bars represent SEM. See also Figure S6.