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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Cancer Res. 2020 Jan 31;80(7):1387–1400. doi: 10.1158/0008-5472.CAN-19-1784

Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAFV600E-driven melanoma

Ye-Jin Kim 1, Tiffany Tsang 2, Grace R Anderson 3, Jessica M Posimo 1, Donita C Brady 1,4,*
PMCID: PMC7127963  NIHMSID: NIHMS1556301  PMID: 32005716

Abstract

The principal unmet need in BRAFV600E-positive melanoma is lack of an adequate therapeutic strategy capable of overcoming resistance to clinically approved targeted therapies against oncogenic BRAF and/or the downstream MEK1/2 kinases. We previously discovered that copper (Cu) is required for MEK1 and MEK2 activity through a direct Cu-MEK1/2 interaction. Repurposing the clinical Cu chelator tetrathiomolybdate (TTM) is supported by efficacy in BRAFV600E-driven melanoma models, due in part to inhibition of MEK1/2 kinase activity. However, the antineoplastic activity of Cu chelators is cytostatic. Here, we performed high-throughput small molecule screens to identify bioactive compounds that synergize with TTM in BRAFV600E-driven melanoma cells. Genetic perturbation or pharmacological inhibition of specific members of the BCL2 family of anti-apoptotic proteins (BCL-W, BCL-XL, and MCL-1) selectively reduced cell viability when combined with a Cu chelator and induced CASPASE-dependent cell death. Further, in BRAFV600E-positive melanoma cells evolved to be resistant to BRAF and/or MEK1/2 inhibitors, combined treatment with TTM and the clinically evaluated BCL2 inhibitor, ABT-263, restored tumor growth suppression and induced apoptosis. These findings further support Cu chelation as a therapeutic strategy to target oncogene-dependent tumor cell growth and survival by enhancing Cu chelator efficacy with chemical inducers of apoptosis, especially in the context of refractory or relapsed BRAFV600E-driven melanoma.

Introduction

Melanoma is driven in 40-50% of cases by activating mutations in the BRAF serine/threonine kinases(1,2). Over 90% of oncogenic BRAF mutations detected in melanoma are Val 600→Glu (V600E)(3,4). Activated BRAFV600E phosphorylates and activates MEK1/2, which subsequently phosphorylate and activate ERK1/2, resulting in hyperactivation of the evolutionarily conserved mitogen-activated protein kinase (MAPK) pathway to drive melanomagenesis(4,5). Thus, late-stage BRAFV600E-positive melanoma patients are typically treated with the FDA-approved combination of mutant-selective, ATP-competitive BRAF inhibitors (BRAFi, dabrafenib and vemurafenib) and allosteric MEK1/2 inhibitors (MEK1/2i, trametinib and cobimetinib)(6-9). Although this standard-of-care is initially effective, BRAFV600E-mutant melanoma patients have only modest improvements in median progression-free survival and eventually develop resistance(4,10,11). The limited clinical durability of the combination has bolstered research aimed at additional combination strategies to forestall resistance development, targeting multiple signaling pathways capable of driving resistance, or exploring alternative pharmacological accessible nodes within the MAPK pathway(4,10,11).

In search of identifying novel components of the canonical MAPK pathway, several groups have employed functional genomics approaches(12). Specifically, a whole genome RNAi screen revealed that the primary copper (Cu) transporter Ctr1 reduced ERK1/2 phosphorylation when knocked down in Drosophila S2 cells(13). We demonstrated that Cu directly binds to MEK1/2 and influences the strength of the RAF-MEK-ERK cascade (14). Leveraging the dependence of BRAF mutation-positive cancers on MEK1/2 for tumorigenesis(15), we found that decreasing the levels of CTR1 or introducing surface accessible mutations in MEK1 that disrupt Cu binding decreased BRAFV600E-driven signaling and tumor growth(16). Importantly, the Cu-selective chelator tetrathiomolybdate (TTM), used as an investigational treatment of Wilson disease(17), diminished tumorigenesis in models of BRAFV600E melanoma(18). Although TTM use has not been clinically explored BRAFV600E-driven melanoma, TTM has been assessed in breast cancers as an anti-angiogenic compound where patients have been treated safely for upwards of 65 months(19). Further, the combination of TTM, a well-tolerated and affordable drug, and vemurafenib led to a survival benefit in a murine model of metastatic melanoma, but failed to yield tumor regression (18).

In this study, we aimed to advance the therapeutic value of Cu chelation in BRAFV600E melanomas by identifying compounds that enhanced TTM efficacy. We performed high-throughput small molecule screens with a panel of bioactive compounds to explore collateral drug sensitives in combination with TTM. Here, we demonstrate that co-targeting select BCL2 proteins via BH3 mimetics synergizes with Cu chelators in both naïve and resistant forms of BRAFV600E melanoma cells. The findings presented here highlight the potential of inducing apoptosis and melanoma tumor suppression when Cu chelators are combined with BCL2is.

Materials and Methods

Reagents

A1210477 (ApexBio, B6011), ABT-199 (Selleck Chemicals, S8048), ABT-263 (M1637, AbMole), ABT-737 (Selleck Chemicals, S1002), Ammonium tetrathiomolybdate (TTM, Sigma-Aldrich, 323446), trametinib (Selleck Chemicals, S2673), WEHI-539 hydrochloride (ApexBio, A8634), vemurafenib (CT-P4032), and Z-DEVD-FMK (Selleck Chemicals, S7312) were purchased from indicated companies.

Cell lines

293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco). 451Lu parental cells and resistant derivatives, 451-Lu BRAFiR and 451-Lu MEKiR, were a kind gift from Jessie Villanueva (Wistar Institute) and maintained in DMEM supplemented with 5 % FBS with 1 µM vemurafenib or 1 µM trametinib(20,21). WM983B parental cells and a resistant derivative, WM983B BRAFiR, were a kind gift from Jessie Villanueva (Wistar Institute) and maintained in DMEM supplemented with 5 % FBS with 1 µM vemurafenib. Cell lines were not authenticated. Mycoalert testing was done to test for mycoplasma contamination of all cell lines. Derived cell lines were generated by stable infection with lentiviruses derived from the pSMARTvector inducible lentiviral shRNA plasmids (Dharmacon, see plasmids below) or retroviruses derived from the pBABE retroviral plasmid (see plasmids below) described below using established methods.

Plasmids

pSMARTvector inducible lentiviral shRNA plasmids were purchased from Dharmacon to express: non targeted control, human BCL2 target sequence #1 5’-TGACGCTCTCCACACACAT, human BCL2 target sequence #2 5’-AAGAAGGCCACAATCCTCC, human BCL-XL target sequence #1 5’-CAAACTGCTGCTGTGGCCA, human BCL-XL target sequence #2 5’-CTCCGATTCAGTCCCTTCT, human BCL-W target sequence #1 5’-AGCGGGTCTCGAACTCATC, human BCL-W target sequence #2 5’-CTGCTGTGGATCCAGTCAG, human MCL-1 target sequence #1 5’-CGAAGGAAGTATCGAATTT, human MCL-1 target sequence #2 5’-AGAGTGTATACAGAACGAA. GFP and shRNA expression was induced by adding 0.5 µg/ml (for A375) or 1 µg/ml (for WM88) of doxycycline hydrochloride (Dox, Fisher Scientific, AAJ67043AE) to DMEM supplemented with 10% FBS and 1% P/S, Gibco. pBABEpuro-HA-p61BRAFV600E, pBABEpuro-HA-MEK1C121S, and pBABEpuro-HA-PDGFRß were previously described(18).

Cell viability assay

5 x 103 of the indicated cells per well were plated in white 96-well plates (Fisher Scientific, 655098) and cultured for 12 hours prior to treatment with the indicated drugs for 72 hours or 96 hours. Cell viability was measured using CellTiter-Glo® cell viability assay (Promega, G7571). Normalized %ATP values were calculated by normalizing luminescence values for each drug treatment condition to vehicle (DMSO) treated wells. To generate IC50 curves, the nonlinear fit of Log(Drug) or Drug versus normalized response (%ATP Normalized to DMSO) with a variable slope was calculated in Prism8 (GraphPad). Each drug treatment condition was represented by at least three biological replicates plated in technical triplicate. The Bliss Indexes to test for synergy in drug combinations was performed as previously described(18,22).

Flow cytometry

2 x 105 of the indicated cells were seeded in 6-well plates (Genesee Scientific, 25-105) and treated as described, floating and attached cells were harvested, stained with Annexin V-FITC and Propidium Iodide using Annexin V-FITC apoptosis detection kit I (BD Biosciences, 556547) according to manufacturer’s protocols. Data were acquired using the Attune NxT flow cytometry (Thermo Scientific) and analyzed with FlowJo 8.7 (Tree Sar).

Immunoblot analysis

Indicated cells or xenograft derived tumor lysates were treated as indicated and then washed with cold 1X PBS (phosphate buffered saline) and lysed with cold RIPA buffer containing 1X EDTA-free Halt™ protease and phosphatase inhibitor cocktail (Thermo Scientific, 78441). The protein concentration was determined by BCA Protein Assay (Pierce) using BSA as a standard. Equal amount of lysates were resolved by SDS-PAGE using standard techniques, and protein was detected with the following primary antibodies: rabbit anti-phospho(Thr202/Tyr204)-ERK1/2 (1:1000; Cell Signaling Technology (CST), 9101), mouse anti-ERK1/2 (1:1000; CST, 9107), rabbit anti-phospho(Ser217/221)-MEK1/2 (1:1000; CST, 9154), mouse anti-MEK1/2 (1:1000; CST, 4694), rabbit anti-PARP (1:1000; CST, 9542), rabbit anti-cleaved PARP (1:1000; CST, 9541), rabbit anti-CASPASE-3 (1:1000; CST, 9662), mouse anti-β-actin (1:10000; CST, 3700), rabbit anti-BCL2 (1:1000; CST, 4223), rabbit anti-BCL-W (1:1000; CST, 2724), rabbit anti-BCL-XL (1:1000; CST, 2764), rabbit anti-MCL-1 (1:1000; CST, 94296), rabbit anti-CYTOCHROME C (1:1000; CST 11940), rabbit anti-HSP60 (1:1000; CST 4870), mouse anti-α-TUBULIN (1:1000; CST 3873), followed by detection with one of the horseradish peroxidase conjugated secondary antibodies: goat anti-rabbit IgG (1:5000; CST, 7074) or goat anti-mouse IgG (1:5000; CST, 7076), using SignalFire (CST) or SignalFire Elite ECL (CST) detection reagents. For detection of CYTOCHROME-C release, cells were treated as described, washed with cold 1X PBS, and mitochondrial and cytosolic fractions were separated using mitochondria/cytosol fractionation kit (BioVision K256) according to manufacturer’s protocols.

High throughput small molecule library screen

Drug screening was performed by the University of Pennsylvania High-Throughput Screening Core. To determine the IC20 for the indicated cell lines, 103 cells were plated per well in white 384-well plates, treated with eight concentrations of TTM (1:3 dilution, ranging from 0.0457-100μM), and cell viability was measured 96 hours later with the ATPlite™ cell viability assay (Perkin Elmer) on an EnVision Plate Reader (Perkin Elmer). To generate IC50 curves, the logistic regression curve of Log(Drug) versus normalized response (%ATP Normalized to DMSO) were fit in TIBCO Spotfire software. The IC20 was calculated using the following formula: IC_F=[(F/(100-F)^(1/H)]*IC50 where H= the Hill slope. For the subsequent drug screens, indicated cell lines were treated with 100nM of a compound from the SelleckChem Bioactive Compound Library with or without TTM at the IC20 for the respective cell line. DMSO and doxycycline were used as negative and positive controls respectively. Normalized percent inhibition (NPI) was calculated for all data points.

Mouse xenografts and drug treatments

All studies were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. 5x106 A375 ,451-Lu-BRAFiR, or 451-Lu-MEKiR or 8x106 451-Lu cells resuspended in 1X PBS were injected subcutaneously into flanks of SCID/beige mice (Charles River Laboratory) as previously described(23). When tumor volume reached ~100 mm3, mice were treated daily via oral gavage: vehicle [1% methylcullulose (Sigma-Aldrich, M0512), 1% dimethyl sulfoxide (DMSO, Sigma-Aldrich)]; 80 mg/kg TTM in vehicle; 25 mg/kg vemurafenib in 10% ethanol, 30% polyethylene glycol (PEG) 400 (EMD Millipore, 8.07485.1000) and 60% Phosal 50PG (Lipoid, 368315); 0.5 mg/kg trametinib in PEG; or 25 mg/kg ABT-263 in PEG.

Immunofluorescence

Tumor tissue sections were stained with either a rabbit anti-Ki67 antibody (1:100; Novus Biologicals, NB100-89717) followed by detection with a Alexa Fluor 647-conjugated anti-rabbit IgG (1:1000; Thermo Scientific, A27040) or DeadEnd™ Colorimetric TUNEL System (Promega, G3250), following the manufacturer’s protocol. Photographs were taken on a Leica DMI6000B inverted confocal microscope (40x) in a blinded fashion. Images were quantified using Image J software.

Results

TTM-mediated reductions in BRAFV600E-driven cell viability and MAPK signaling fail to induce apoptosis

To examine whether the Cu chelator efficacy could be improved in BRAFV600E-positive melanoma, we first treated BRAFV600E-mutant melanoma cells in vitro with increasing concentrations of TTM and measured cell viability. TTM reduced the number of viable 451-Lu, A375, WM88, and WM983B cells in a dose-dependent manner (Fig. 1A-E). However, TTM treatment failed to increase the percent of apoptotic cells (Fig. 1F-I), as measured by annexin-V and propidium iodide(PI)-positivity via flow cytometry(24), despite effectively decreasing ERK1/2 phosphorylation (Fig. 1J-M, Supplemental Fig. 1A-D). In agreement, cleavage of CASPASE-3 and PARP(25,26), well-known markers of cells undergoing apoptosis, were only observed at concentrations higher than the IC50 of TTM (Fig. 1J-M, Supplemental Fig. 1A-D). Similarly, treatment with either MAPKi (Supplemental Fig. 2A-J) was not sufficient to increase markers of apoptosis (Supplemental Fig. 2K-R). Thus, in vitro Cu chelators blunt MAPK pathway activation and inhibit the viability of BRAFV600E-driven melanoma cells but fail to induce apoptosis, akin to treatment with MAPKis, and support the rationale to identify other therapeutic combinations to improve Cu chelation therapy.

Figure 1. TTM reduces MAPK pathway activation and viability without inducing apoptosis in BRAFV600E-positive melanoma cells.

Figure 1.

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A,B,C,D, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells treated with indicated concentrations of tetrathiolmolybdate (TTM). n=3. E, Scatter dot plot of TTM IC50 in indicated cell lines ± s.e.m. F,G,H,I, Representative flow cytometry graphs and scatter dot plots of flow cytometry analysis of Annexin V-FITC and Propidium Iodide ± s.e.m. from indicated cell lines treated with vehicle (VEH) or TTM. n=3. J,K,L,M, Immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, Cleaved (Clv.) CASPASE-3, CASPASE-3, Clv. PARP, PARP, and β-actin from indicated cells treated with vehicle or TTM. n=3. Quantification: P-ERK1/2/T-ERK1/2 normalized to vehicle control and fold change Clv. CASPASE-3/CASPASE-3 and Clv. PARP/PARP.

BH3 mimetics synergize with TTM to reduce cell viability of BRAFV600E-driven melanoma cells

In order to identify collateral drug sensitivities with Cu chelators, we investigated combination of a minimally inhibitory dose of TTM with the Selleckchem bioactive compound library composed of a unique collection of 2123 bioactive chemical compounds, which are either FDA-approved or have known bioactivity and safety profiles (Fig. 2A). A375 and WM88 cells were treated with a low dose of each individual compound within the Selleckchem bioactive compound library and either vehicle or the respective IC20 of TTM (Fig. 2A). A 20 percent reduction in A375 and WM88 cell viability was achieved for the majority of the bioactive compounds, which reflected TTM efficacy alone, and indicated that most of the bioactive compounds displayed minimal efficacy alone at 100nM (Supplemental Fig. 3A, 3B). Therefore, initial hits were restricted to compounds that when combined with TTM yielded the intended 20 percent or greater inhibition of cell viability (Fig. 2B, 2C, Supplemental Fig. 3A, 3B). Further, synergistic combinations with TTM were identified by calculating the Bliss Index(22), which considers the expected additive effect of a drug combination compared to the observed effect, for each initial compound hit (NPI TTM + Compound ≥ 20) (Fig. 2D, 2E). Fifty and 233 bioactive compounds synergistically inhibited cell viability of A375 or WM88 cells, respectively, with a Bliss Index ≥ 1.5 (Fig. 2F, 2G). Only five bioactive compounds scored as hits (NPI TTM + Compound ≥ 20/ Bliss Index ≥ 1.5) in both BRAFV600E-positive melanoma cells (Fig. 2H, 2I). These findings establish the utility of employing high-throughput small molecule screens to identify synergistic drug combinations for potential cancer treatment and more importantly, suggest that several bioactive compounds may be useful to enhance Cu chelation therapy efficacy in BRAFV600E-mutant melanoma.

Figure 2. High throughput screen of bioactive compound library reveals BH3 mimetics as a synergistic combination with TTM in BRAFV600E-positive melanoma cells.

Figure 2.

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A, Schematic diagram of high throughput screen of 2123 Selleckchem bioactive compound library at 100nM alone or in combination with the IC20 of TTM (dashed red line) in A375 or WM88. B,C, Normalized Percent Inhibition (NPI) of TTM + Compound versus NPI Compound graph for indicated cells treated with Selleckchem bioactive compound library alone or in combination with IC20 of TTM. Hits (blue circles) are defined as NPI TTM + Compound ≥ 20 (dashed red line). D,E, Observed effect versus expected effect graph for indicated cells of Hits from B and C. Hits (blue circles) are defined as Bliss Index ≥ 1.5 (dashed red line). F,G, Graphical representation of Hits defined as NPI TTM + Compound ≥ 20 and Bliss Index ≥ 1.5 in indicated cells. H, Venn diagram relationship between Compound Hits. I, NPI of 5 overlapping Hit Compounds from H alone or in combination with IC20 of TTM (dashed red line). J,K, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with vehicle or indicated concentrations of drugs. L,M,N,O, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with VEH or indicated concentrations of drugs. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. One asterisk, P<0.05, Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3.

We were keenly interested in our finding that the pan BH3 mimetic ABT-737 synergistically inhibited the viability of both A375 and WM88 cells to the greatest extent when combined with TTM (Fig. 2I). The B-cell lymphoma-2 (BCL2) family consists of two types of proteins(27), anti-apoptotic (e.g., BCL2, BCL-XL, BCL-W, and MCL1)(28-30) and pro-apoptotic (e.g., BAK, BAX and BIM), which cooperate through homo- or heterodimer formation to retain the balance between cell survival and death(31-33). Drugs termed ‘BH3 mimetics’ that bind the surface groove of certain anti-apoptotic BCL2 proteins and thereby elicit apoptosis have been developed(34-40). Among them, successful clinical trials of venetoclax (ABT-199), a specific BCL2i, has led to its FDA-approval for chronic lymphocytic leukemia and clinical evaluation for treatment of other cancers(41-44). However, single agent inhibition of BCL2 is not sufficient to induce cell death of melanomas due in part to differential BCL2 protein expression and diversity(45-51). Similarly, ABT-199 failed to significantly reduce BRAF-mutant melanoma cell viability alone or in combination with TTM in the high throughput screen (Supplemental Fig. 3C). Inhibition of multiple anti-apoptotic BCL2 proteins is a major target for melanoma and other cancers(34,46).

Collateral inhibition of oncogenic MAPK signaling and BCL2 proteins with the pan BH3 mimetic, ABT-737, reduces targeted therapy resistance in BRAFV600E-driven melanoma(52-54). In support of these findings, combining MAPKis and navitoclax (ABT-263), an orally bioavailable analog of ABT-737 ( NCT01989585), is being tested clinically in BRAFV600E-positive melanoma. While not every BH3 mimetic in the high-throughput screen increased the efficacy of TTM, ABT-263 synergistically reduced A375 cell viability but was only additive in the WM88 cell line (Supplemental Fig. 3C). Combination of IC20 or IC50 doses of TTM and 100nM ABT-737 was sufficient to significantly reduce cell viability (Fig. 2J, 2K), but failed to increase the percent of annexin-V positive cells, cleaved-CASPASE-3, or cleaved-PARP (Supplemental Fig. 3D-G). Further, the addition of TTM to the combination of vemurafenib and ABT-737 or trametinib and ABT-737 significantly reduced A375 and WM88 cell viability (Fig. 2L-O). Treatment with TTM, ABT-737, and either MAPKi was either equivalent or superior to the combination of vemurafenib, trametinib, and ABT-737 (Fig. 2L-O). Finally, TTM significantly increased the capacity of the combination of the MAPKis and ABT-737 to reduce A375 cell viability (Fig. 2L). These findings indicate that Cu chelation therapy may be a viable alternative to either MAPKi when combined with a BH3 mimetic, and more importantly, could enhance the therapeutic benefit of the clinically investigated combination of the MAPKis and a BH3 mimetic.

Genetic loss of select anti-apoptotic BCL2 family proteins decreases TTM IC50 in BRAFV600E-driven melanoma cells

We next explored what molecular target(s) of ABT-737 impart(s) sensitivity to Cu chelators. ABT-737 selectively targets the anti-apoptotic proteins, BCL2, BCL-W, and BCL-XL but not, MCL1 or A1(37). Knockdown of BCL-XL, BCL-W, or MCL-1 with two independent doxycycline-inducible shRNAs increased TTM effectiveness in both A375 and WM88 cells (Supplemental Fig. 4A, 4B), as measured by a statistically significant shift in the IC50 of TTM in the presence of doxycycline (Fig. 3A-D). In contrast, depletion of BCL2 failed to decrease TTM IC50 (Fig. 3A-D). These results suggest that lowering the anti-apoptotic balance of BRAFV600E-mutant melanoma cells by targeting specific anti-apoptotic BCL2 proteins, BCL-W, BCL-XL, or MCL1, can enhance Cu chelator efficacy.

Figure 3. Targeting select BCL2 family proteins increases sensitivity to TTM in BRAFV600E-positive melanoma.

Figure 3.

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A,C, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells stably expressing two independent Dox-inducible shRNAs (#1 and #2) against indicated genes treated with indicated concentrations of TTM without or with Dox n=3. B,D, Scatter dot plot of TTM IC50 ± s.e.m in indicated cells cells stably expressing two independent Dox-inducible shRNAs against indicated genes treated without or with Dox. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3. E,G, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. F,H, Scatter dot plot of BH3 mimetic IC50 ± s.e.m in indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. n=3. I,J, Graphical representation of Bliss Index (observed effect versus expected effect) from A375 (E) and WM88 (G) at the indicated drug combinations. Synergistic combinations are indicated by Bliss Index values >1. Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001.

TTM enhances efficacy of select BH3 mimetics and induces apoptosis in BRAFV600E-driven melanoma cells

To further examine the generalizability of combining Cu chelators with BH3 mimetics in BRAFV600E mutation-positive melanoma, we tested whether treatment with TTM could lower the IC50 of a panel of four BH3 mimetics, ABT-737(37), ABT-199 (BCL2i)(38), WEHI-539 (BCL-XLi)(39), and A1210477 (MCL1i)(40). ABT-737 IC50 was significantly decreased by ~3-fold in A375 cells and ~10-fold in WM88 cells when co-treated with the highest concentration of TTM (Fig. 3E-H). However, the BRAF mutation-negative melanoma cell lines, WM3311 and WM3743, did not show a similar dose-dependence when TTM and ABT-737 were co-administered (Supplemental Fig. 4C, 4D), suggesting that BRAFV600E-mutant melanomas are selectively sensitive to the combination. The efficacy of WEHI-539 and A1210477 was also substantially enhanced with TTM (Fig. 3E-H). TTM only slightly decreased the IC50 of ABT-199 in either A375 or WM88 cells (Fig. 3E-H). Further, combination of TTM with ABT-737, WEHI-539, or A1210477 synergistically inhibited A375 and WM88 cell viability, while combining TTM with ABT-199 was additive by Bliss Indexes (Fig. 3I, 3J). Collectively, the above results suggest that the combined effects of TTM and BH3 mimetics on BRAF-mutant melanoma cell viability are driven by targeting BCL-W, BCL-XL, and/or MCL1, but not BCL2.

Melanomas are known to harbor elevated levels of the anti-apoptotic BCL2 proteins(55,56), which underlies intrinsic resistance to BH3 mimetics. Mechanistically, treatment of BRAF-mutant melanoma cells with TTM was sufficient to selectively reduce ERK1/2 phosphorylation and the expression of at least two BCL2 proteins (Fig. 4A, 4B, Supplemental Fig. 5A, 5B). The differential expression of BCL-W, BCL-XL, and/or MCL1 correlated with significantly increased cytosolic CYTOCHROME-C in A375 or WM88 cells treated with both TTM and ABT-737 (Fig. 4C, 4D, Supplemental 5C, 5D), indicating that mitochondrial outer membrane permeabilization (MOMP) was triggered. In turn, co-treatment with TTM and ABT-737 increased the population of cells undergoing apoptotic cell death by 2- or 3-fold (Fig. 4E, 4F). Finally, cleaved CASPASE-3 and PARP levels, which are initiated by MOMP-mediated CYTOCHROME-C release, were elevated following co-treatment with TTM and any of the three BH3 mimetics, ABT-737, WEHI-539, and A1210477 (Fig. 4G-J, Supplemental Fig. 5E-G). As expected, preincubation of A375 or WM88 cells with the irreversible CASPASE-3i Z-DEVD-FMK blunted the induction of markers of apoptosis in response to the combination of TTM and ABT-737 (Fig. 4E-H, Supplemental Fig. 5E, 5F). Taken together, these results revealed that Cu chelation can enhance the apoptotic activity of BH3 mimetics in a CASPASE-dependent manner in BRAFV600E-driven melanoma cells.

Figure 4. TTM reduces BCL2 protein expression and lowers the apoptotic threshold when combined with BH3 mimetics in BRAFV600E-positive melanoma.

Figure 4.

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A,B, Immunoblot detection of phosphorylated (P)-MEK1/2, total (T)-MEK1/2, P-ERK1/2, T-ERK1/2, BCL2, BCL-W, BCL-XL, MCL1, and β-actin from indicated cells treated with vehicle (VEH) or the indicated concentrations of TTM. n=3. C,D, Immunoblot detection of CYTOCHROME-C, HSP60, and α-TUBULIN of mitochondrial or cytosolic fractions from indicated cells treated with vehicle or the indicated concentrations of drugs. E,F, Scatter dot plot of flow cytometry analysis of Annexin V-FITC and Propidium Iodide ± s.e.m. stained cells pretreated without or with Z-DEVD-FMK followed by treatment with vehicle or indicated drugs. n=3. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. Two asterisks, P<0.01, Four asterisks, P<0.0001. n=3. G,H, Immunoblot detection of Cleaved (Clv.) CASPASE-3, CASPASE-3, Clv. PARP, PARP, and β-actin from indicated cells pretreated without or with Z-DEVD-FMK followed by treatment with vehicle or indicated drugs. I,J, Immunoblot detection of Clv.CASPASE-3, CASPASE-3, Clv. PARP, PARP, and β-actin from indicated cells treated with vehicle or indicated drugs. Quantification: P-MEK1/2/T-ERK1/2, P-ERK1/2/T-ERK1/2, BCL2 family protein/β-actin, CYTOCHROME-C/HSP60, and CYTOCHROME-C/α-TUBULIN normalized to vehicle control. Fold change Clv. CASPASE-3/CASPASE-3 and Clv. PARP/PARP.

TTM treatment with ABT-737 sensitizes trametinib or vemurafenib-resistant BRAFV600E-positive melanoma cells

To interrogate the utility of combining Cu chelation with BH3 mimetics to restore apoptotic cell death in the setting of MAPKi acquired resistance, we employed BRAFi or MEK1/2i resistant variants of the BRAFV600E-mutant cell lines 451-Lu and WM983B (20,21). Treatment with TTM, trametinib, or vemurafenib reduced the IC50 of ABT-737 in parental 451-Lu and WM983B cells (Fig. 5A, 5B, Supplemental Fig. 6A, 6B). Whereas in 451-Lu-BRAFiR and WM983B-BRAFiR cells, both TTM and trametinib markedly decreased ABT-737 IC50 (Fig. 5C, 5D, Supplemental Fig. 6C, 6D), while only TTM was sufficient to sensitize the MEK1/2i resistant 451-Lu cells to the BH3 mimetic (Fig. 5E, 5F). Further, co-treatment with TTM and ABT-737 was the only synergistic combination in both the parental and resistant cell lines based on Bliss Indexes (Fig. 5G-I, Supplemental Fig. 6E, 6F). As such, TTM treatment enhanced the efficacy of a BH3 mimetic in the setting of MAPKi resistance.

Figure 5. TTM sensitizes vemurafenib or trametinib-resistant BRAFV600E-positive melanoma cells to a BCL2 protein inhibitor.

Figure 5.

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A,C,E, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells treated without or with indicated concentrations of TTM, vemurafenib (VEM), or trametinib (TRAM) and increasing concentrations of ABT-737. n=3. B,D,F Scatter dot plot of ABT-737 IC50 ± s.e.m in indicated cells treated without or with indicated concentrations of TTM, VEM, or TRAM and increasing concentrations of ABT-737. n=3. Results were compared using a one-way ANOVA followed by a Dunnett’s multi-comparisons test. n=3. G,H,I, Graphical representation of Bliss Index (observed effect versus expected effect) from indicated cells at the indicated drug combinations. Synergistic combinations are indicated by Bliss Index values >1. J,K,L,M,N,O, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with VEH or indicated concentrations of TTM, VEM, TRAM, ABT-737, or the indicated combinations for 72 hours. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. n=3. One asterisk, P<0.05, Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001.

Combining the Cu chelator and BH3 mimetic in the clinically relevant setting of MAPKi resistance revealed that the combination was more efficacious than the FDA-approved combination of vemurafenib and trametinib in both the naïve and MAPKi resistant 451-Lu and WM983B cells (Fig. 5L-O, Supplemental Fig. 6G-J). In addition, TTM significantly increased the efficacy of the MAPKis and BH3 mimetic in WM983B cells (Supplemental Fig. 6G, 6H). Most importantly, TTM treatment significantly improved the efficacy of ABT-737 combined with the MAPKis in each of the MAPKi resistant BRAF mutation-positive melanoma cells (Fig. 5L-O, Supplemental Fig. 6I, 6J). Thus, Cu chelators may be advantageous in improving the response to the combination of a BRAFi, MEK1/2i, and BH3 mimetic being tested clinically and more importantly be an additional treatment option in the context of MAPKi resistance.

TTM dampens the apoptotic threshold in response to BH3 mimetics by limiting anti-apoptotic protein expression in MAPKi resistant BRAFV600E-positive melanoma cells

Next, we tested and found in parental cells, MAPK signaling was abrogated by either TTM or MAPKi treatment, but unlike TTM that significantly decreased the expression of BCL-W, BCL-XL, and/or MCL1, trametinib failed to impact the levels of BCL2 proteins (Fig. 6A, Supplemental Fig. 7A). Interestingly, vemurafenib significantly decreased the expression of BCL2 and BCL-W in 451-Lu cells only (Fig. 6A, Supplemental Fig. 7A). Thus, surprisingly the pattern of BCL2 protein expression was divergent amongst the 451-Lu or WM983B cells treated with TTM or the MAPKis (Fig. 6A, Supplemental Fig. 7A-C). The BRAFi resistant 451-Lu and WM983B cells(20,21), which harbor upregulation of receptor tyrosine kinases as a resistance mechanism, and MEK1/2i resistant 451Lu cells(20,21), which harbor a Q60P activating mutation in MEK2 and BRAF amplification as a resistance mechanism, displayed elevated MEK1/2 and ERK1/2 phosphorylation (Fig. 6A-C, Supplemental Fig. 7A-D). Increased MAPK signaling in the BRAFi resistant variants was dampened by TTM and trametinib treatment but not the mutant selective BRAFi vemurafenib (Fig. 6B, Supplemental Fig. 7B-D). Of clinical relevance, TTM treatment of the dual MAPKi resistant 451-Lu cells was sufficient to reduce ERK1/2 phosphorylation, along with BCL-W, BCL-XL, and MCL1 expression, which may explain their maintained sensitivity to the combination of TTM and ABT-737 in these cells (Fig. 5E, 5F, 6C, Supplemental Fig. 7D). These findings indicate that the cooperativity between the Cu chelator and BH3 mimetic in MAPKi resistant BRAF mutation-positive melanoma cells may be driven in part by diminished anti-apoptotic protein expression.

Figure 6. TTM reduces BCL2 protein expression and lowers the apoptotic threshold when combined with BH3 mimetics in MAPK inhibitor resistant BRAFV600E-positive melanoma.

Figure 6.

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A,B,C, Immunoblot detection of phosphorylated (P)-MEK1/2, total (T)-MEK1/2, P-ERK1/2, T-ERK1/2, BCL2, BCL-W, BCL-XL, MCL1, and β-actin from indicated cells treated with vehicle (VEH) or the indicated concentrations of TTM, trametinib (TRAM), vemurafenib (VEM), or ABT-737. n=3. D,E,F, Scatter dot plot of flow cytometry analysis of Annexin V-FITC and Propidium Iodide stained ± s.e.m. from indicated cells treated with indicated drugs. n=3. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. Two asterisks, P<0.01, Four asterisks, P<0.0001. n=3. G,H,I, Immunoblot detection of Cleaved (Clv.) CASPASE-3, CASPASE-3, Clv. PARP, PARP, and β-actin from indicated cells treated with vehicle, TTM, TRAM, VEM, and/or ABT-737. Quantification: Quantification: P-MEK1/2/T-ERK1/2, P-ERK1/2/T-ERK1/2, or BCL2 family protein/β-actin, normalized to vehicle control. Fold change Clv. CASPASE-3/CASPASE-3 and Clv. PARP/PARP.

To address whether the threshold for apoptosis induction is tipped when MAPKi resistant BRAFV600E-mutant melanoma cells are treated with the combination of ABT-737 and TTM or a MAPKi, we measured the percent of apoptotic cells and the levels of markers of apoptosis. Co-treatment with TTM and ABT-737 significantly elevated the number of annexin-V positive parental and BRAFi resistant cells, while vemurafenib failed to increase the apoptotic response of ABT-737 in the 451-Lu BRAFiR and WM983B BRAFiR cells (Fig. 6D, 6E, Supplemental Fig. 7E, 7F). While the dual combination of trametinib and ABT-737 was sufficient to increase the percent of apoptotic parental and 451-Lu BRAFiR cells to a similar degree to co-treatment with TTM and ABT-737, apoptotic cell death was only observed in MEK1/2i resistant 451-Lu cells when TTM and ABT-737 were co-administered (Fig. 6F). Further, co-treatment of TTM with ABT-737 was the only combination to result in elevated cleavage of CASPASE-3 and PARP in both the parental and MAPKi resistant 451-Lu cells (Fig. 6G-I, Supplemental Fig. 7G-I). These results provide a rationale for a co-treatment strategy with a Cu chelator and BH3 mimetics in the context of MAPKi-resistant forms of BRAFV600E melanoma.

Finally, to extend our findings to in vitro models with well-characterized mechanisms of resistance to MAPKi, A375 cells were engineered to stably expressing the p61BRAFV600E splice variant, MEK1C121S, or PDGFRβ(18). As expected, each protein conveyed resistance to vemurafenib, while only p61BRAFV600E and MEK1C121S imparted resistance to trametinib(18), as measured by an increase in the IC50 of the inhibitors (Supplemental Fig. 8A, 8B). In contrast, TTM reduced the number of viable A375 cells expressing these MAPK-dependent drivers of MAPKi resistance (Supplemental Fig. 8A, 8B). Mechanistically, we previously showed that TTM treatment elicits a reduction in ERK1/2 phosphorylation in A375 cells stably expressing either the p61BRAFV600E splice variant, MEK1C121S, or PDGFRβ (18). TTM was the only compound capable of significantly increasing the cleavage of CASPASE-3 and PARP when combined with ABT-737 in each cell line (Supplemental Fig. 8C, 8D). In summary, these data indicate that MAPK-dependent forms of resistance can be countered by a Cu chelator by mechanistically inhibiting MEK1/2 kinase activity and apoptosis induction can be achieved when the Cu chelator is combined with BH3 mimetics.

TTM in combination with ABT-263 suppresses growth of naïve and MEK1/2i-resistant BRAFV600E-positive melanoma xenografts

To evaluate the therapeutic potential of combining TTM with BH3 mimetics in vivo, we utilized mouse xenograft tumor models of parental and MAPKi-resistant BRAF mutation-positive melanoma. Instead of using ABT-737 for in vivo experiments, we evaluated its clinically relevant, orally available analog navitoclax, ABT-263(36). Mice bearing ~100mm3 A375 or 451-Lu parental tumors were randomly assigned to one of five treatment arms: i) vehicle, ii) TTM, iii) TTM and ABT-263, iv) BRAFi, MEK1/2i, and ABT-263, and v) BRAFi, MEK1/2i, ABT-263, and TTM. These treatments arms were prioritized based on their ability to exam the benefit of combining a Cu chelator and a BH3 mimetic, along with a Cu chelator and the clinically investigated combination of MAPKis and a BH3 mimetic. The growth of established xenograft tumors derived from 451-Lu and A375 cells was reduced when mice were treated with TTM alone and was enhanced when TTM was combined with ABT-263 (Fig. 7A-F). Further, growth of the parental tumors was severely reduced when mice were treated for 30 days with the combination of MAPKis and ABT-263 and this reduction in A375 tumor growth was significantly accentuated when TTM was also administered (Fig. 7A-F). While the in vivo tumor growth response to the combination of MAPKis and ABT-263 was attenuated in BRAFi resistant 451-Lu cells, the inclusion of TTM delayed tumor growth and significantly improved the time to endpoint tumor volume (Fig. 7G-I). Finally, we demonstrate that xenograft tumors derived from MEK1/2i resistant 451-Lu cells failed to respond to the combination of MAPKis and ABT-263, but remained sensitive to TTM and ABT-263 dual treatment, which also improved the anti-tumorigenic properties of the MAPKis (Fig. 7J-L). The body weights of mice, monitored as an indicator of drugs toxicity, were similar compared to vehicle group (Supplemental Fig. 9A-D). In concordance with reduced tumor growth kinetics, co-treatment with either TTM and ABT-263 or MAPKis and ABT-263 significantly decreased Ki67 positive staining and increased TUNEL positive staining of parental BRAF-mutant melanoma tumors (Fig. 7M-T, Supplemental Fig. 9E-L). While the addition of TTM to the combination of the MAPKis and ABT-263 did not increase tumor cell apoptosis (Fig. 7Q-T, Supplemental Fig. 9I-L), cell proliferation was dampened in the parental and MAPKi resistant tumors (Fig. 7M-P, Supplemental Fig. 9E-H). As further evidence of apoptosis, cleaved CASPASE-3 and PARP were elevated in parental tumor tissues isolated from mice treated with the dual combination of TTM and ABT-263, the triple combination of MAPKis and ABT-263, and quadruple combination of TTM, MAPKis, and ABT-263 (Supplemental Fig. 9M, 9N). This observation was restricted to the MAPKi resistant tumors tissue isolated from mice treated with the aforementioned dual and quadruple combinations of TTM and ABT-263 (Supplemental Fig. 9O, 9P). Taken together these findings support our conclusion that the efficacy of Cu chelators can be enhanced with chemical inducers of apoptosis and this combination can cooperate with MAPKis to reduce tumor cell growth and survival, especially in the context of refractory or relapsed BRAFV600E-driven melanoma.

Figure 7. Cotreatment with TTM and ABT-263 suppresses in vivo growth of parental and MAPK inhibitor resistant BRAFV600E-positive melanoma.

Figure 7.

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A,D,G,J, Mean tumor volume (mm3) ± s.e.m. versus time (days) in mice (n=5 or 6) injected with indicated cells and treated with indicated drugs until tumor volume 1.0cm3 or for 30 days. B,E,H,K, Kaplan-Meier analysis of percentage of mice (n=5 or 6) with tumor volume ≥ 1.0cm3 versus time injected with indicated cells treated with indicated drugs. C,F, Scatter dot plot of tumor volume (mm3) ± s.e.m. at endpoint of 1.0cm3 or at 30 days in mice (n=5 or 6) injected with indicated cells and treated with indicated drugs. Results were compared using unpaired student’s t-test. n=5 or 6. I,L, Scatter dot plot of days to endpoint tumor volume ± s.e.m. of 1.0cm3 or 30 days of mice (n=5 or 6) injected with indicated cells treated with indicated drugs. Results were compared using unpaired student’s t-test. n=5 or 6. M,N,O,P,Q,R,S,T Scatter dot plot of percent (%) Ki67 or TUNEL positive cells per tumor ± s.e.m. from mice injected with indicated cells treated with indicated drugs. Results were compared using unpaired student’s t-test. n=5 or 6. One asterisk, P<0.05, Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001. n=5 or 6.

Discussion

Here, we identified several compounds with the capacity to enhance TTM efficacy in BRAFV600E-driven melanoma, but focused our efforts on an inhibitor of BCL2 anti-apoptotic proteins. While the combination of ABT-737 and a BRAFi induces apoptosis in BRAFV600E-mutant melanoma cells(52-54), it is not effective in BRAFi resistant cells and required additional targeted or conventional chemotherapeutics to tip the apoptotic threshold towards cell death(54). The absence of efficacy achieved with ABT-737 and BRAFi in the context of resistance may be driven by a unique repertoire of BCL2 proteins in melanocytes(45,46,49-51,54,56). Additionally, pro-apoptotic BH3-only proteins expression patterns or their different binding affinity to the anti-apoptotic proteins can induce different dependencies on BCL2 proteins(34,45,57). Recently Lee et al. defined that BCL-XL and MCL-1 dual targeting is critical to blunt melanoma cell survival, but it remains to be determined whether their combination targeting would reduce MAPKi-resistant melanoma cell viability(58).

Mechanistically, genetic knockdown of BCL-XL, BCL-W, or MCL1, but not BCL2, revealed that several BCL2 proteins were involved in the synergistic reduction in cell viability and induction of apoptotic cell death elicited by co-targeting the MAPK pathway with TTM and anti-apoptotic machinery with BH3 mimetics (Fig. 3, 4, Supplemental Fig. 4, 6). Mechanistically, the Cu chelator lowered the threshold for apoptotic cell death by limiting the expression of several BCL2 proteins (Fig. 4, Supplemental Fig. 7). Encouragingly, BRAFV600E-mutant melanoma tumor growth was effectively suppressed and controlled when TTM was combined with ABT-263 without negatively impacting animal welfare, suggesting that Cu chelation could be added to current efforts to evaluate MAPKi with navitoclax in BRAF mutation-positive melanoma ( NCT01989585) (Fig. 7, Supplemental Fig 9). Our results provide the possibility that BH3 mimetics can be an effective therapeutic with less adverse effects when dosed with a Cu chelator based on the synergistic combination, which is not achieved with vemurafenib or trametinib (Fig. 3, 5, Supplemental Fig. 5). Finally, in the setting of MAPKi-resistant BRAFV600E-driven melanoma, TTM in combination with ABT-737 in vitro or ABT-263 in vivo reduced cell viability, decreased tumor growth kinetics, reduced tumor proliferation, and triggered apoptotic cell death (Fig. 5, 6, 7, Supplemental Fig. 6-9). These findings highlight a need for additional exploration of the interplay between Cu-dependent MAPK signaling and cellular intrinsic apoptotic pathways. Thus, we provide a proof-of-concept that Cu chelation therapy synergistically improves BH3 mimetic efficacy by inducing apoptosis and supports the utility of this combination in BRAF mutation-positive melanoma patients that do not respond to targeted therapy or immunotherapy, have severe side effects, or acquire resistance.

Supplementary Material

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Acknowledgements

We thank J. Villanueva (The Wistar Institute) for cell lines, S. Cherry and D.C. Schultz of the University of Pennsylvania High-Throughput Screening Core for technical support, I.A. Asangani, L. Busino, T.P. Gade, B.J. Kim, S.W. Ryeom, and E.S. Witze (University of Pennsylvania) for technical support, discussions, and/or review of the manuscript, and D. Sneddon for administrative support. This work was supported by the Pew Charitable Trust Pew Scholars Program in Biomedical Science Award #50359 (D.C. Brady) and by a pilot project from Tara Miller Foundation at University of Pennsylvania/Wistar Institute supported by Skin Cancer SPORE Career Enhancement Program Award to D.C. Brady (P50CA16452). T. Tsang is supported by NCI NRSA Fellowship (F31) F31CA243294. G.R. Anderson is supported by NCI Predoctoral to Postdoctoral Transition Fellowship (F99/K00) K00CA222728. J.M. Posimo is supported by American Cancer Society Postdoctoral Fellowship 131203PF1714701CCG. This work is dedicated in the memory of Maria Whitehead.

Footnotes

Disclosure of Potential Conflicts of Interest: D.C. Brady holds ownership in Merlon Inc. D.C. Brady is an inventor on the patent application 20150017261 entitled “Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway”. No potential conflicts of interest were disclosed by the other authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1556301-supplement-1.pdf (1.8MB, pdf)
2
NIHMS1556301-supplement-2.pdf (1.6MB, pdf)
3
NIHMS1556301-supplement-3.pdf (1.1MB, pdf)
4
NIHMS1556301-supplement-4.pdf (612.4KB, pdf)
5
NIHMS1556301-supplement-5.pdf (956.9KB, pdf)
6
NIHMS1556301-supplement-6.pdf (1MB, pdf)
7
NIHMS1556301-supplement-7.pdf (1.1MB, pdf)
8
NIHMS1556301-supplement-8.pdf (32MB, pdf)

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