Simultaneously Inhibiting BCL2 and MCL1 Is a Therapeutic Option for Patients with Advanced Melanoma
"> Figure 1
<p>Differential expression of apoptotic-related mRNA and proteins in The Cancer Genome Atlas Network (TCGA) cutaneous melanoma dataset and the effects of knocking down B-cell CLL/lymphoma 2 (BCL2) or myeloid cell leukemia sequence 1 (MCL1). (<b>a</b> and <b>b</b>) Comparison between two genotypic groups: BRAF-hotspot mutated (MUT) (<span class="html-italic">n</span> = 110) and BRAF-wild-type (WT) (harboring RAS hotspot mutated, any NF1 mutated, and triple wild type <span class="html-italic">n</span> = 122). (<b>a</b>) mRNA expression values for BCL2, CASP8, PDCD4, and MCL1. (<b>b</b>) Relative reverse phase protein array (RPPA) protein expression values for PDCD4, CASP8, and BCL2. MCL1 was not included on the RPPA panel. Each dot represents an individual sample, and the horizontal line represents the mean. (<b>c</b>) and (<b>d</b>) show the effects of BCL2 or MCL1 knockdown in A375 cells. Cells were treated with the indicated drugs for 48 h. Knocking down BCL2 (shBCL2) sensitized cells to MCL1 inhibitor S63845 and knocking down MCL1 (shMCL1) sensitized cells to BCL2 inhibitor ABT-199. Y-axis shows percentage of relative viability and X-axis indicates the BH3 mimetics used. ** indicates <span class="html-italic">p</span> < 0.01; *** indicates <span class="html-italic">p</span> < 0.001. Error bars represent +/− SEM. (<b>e</b>) Immunoblots to confirm the knockdown.</p> "> Figure 2
<p>The combination of the BCL2 inhibitor ABT-199 with the MCL1 inhibitor S63845 had high efficacy in advanced melanomas in vitro. (<b>a</b>) ATP assays for various subtypes of melanoma patient samples and normal human melanocytes upon indicated treatments for 48 h. The viability of the DMSO control for each cell line was set to 100%. The viability of almost all combinational treatments was statistically significantly different from those of control or single drugs. For clarity and readability, we listed <span class="html-italic">p</span> values of all comparisons in <a href="#app1-cancers-12-02182" class="html-app">Table S4</a> instead of showing an asterisk on figure. (<b>b</b>) Summary of ATP assay data of fifteen melanoma cell lines and patient samples and one primary melanocyte line treated with vehicle, single drug or combination of S63845 + ABT-199 at a dose of 2.5 μM. Black dotted line indicates 50% relative viability. (<b>c</b>) Dot plot of IC50 values for combination treatment in BRAF-V600E-MUT and BRAF-WT lines. Each dot represents one cell line. (<b>d</b>) Dot plot with the Combination-Index (CI) of the drug combination at 2.5 μM dose calculated using Compusyn software (version 1) (<a href="http://www.combosyn.com/" target="_blank">http://www.combosyn.com/</a>) CI values < 0.5 (red line) indicate very strong synergism. Smaller CI values indicate stronger synergy. For visual clarity, the * is not shown in panels a and b. (<b>e</b>) Immunoblot with lysates collected after 48 h treatment with DMSO, single drugs, or combination at 0.625 μM for each drug, and probed for poly ADP-ribose polymerase (PARP). The ratio of cleaved/full-length PARP in DMSO control was set as 1 within each cell line, and the relative ratios in all conditions were normalized to DMSO controls and listed at the bottom. The combination increased the ratio of cleaved/full-length PARP in both cell lines, indicating that apoptosis was activated. Molecular weight markers are in kDa. *** indicates <span class="html-italic">p</span> < 0.01.</p> "> Figure 3
<p>The treatment of ABT-199 plus S63845 significantly inhibited tumor growth without affecting mouse weight. A BRAF-WT line, MB3616, was used in the xenograft study, and tumor volume (<b>a</b>) and weight of the mice (<b>b</b>) were measured. The combination’s inhibiting effects on tumor volume was statistically significant, compared to vehicle or the single drugs (<span class="html-italic">p</span> < 0.001) (<b>a</b>). (<b>c</b>) Shows representative bright-field images of Ki67 and Cleaved Caspase 3 staining from tumor sections derived from mouse xenografts experiments. Scale bar, 50 μm. The summary quantifications are in (<b>d</b>) for Ki67 and (<b>e</b>) for cleaved Caspase 3 positive area. The effects of the combination were statically significant, compared to vehicle or individual treatments, and we only show the least significant p value of the comparisons. * indicates <span class="html-italic">p</span> < 0.05; *** indicates <span class="html-italic">p</span> < 0.001. Error bars represent +/− SEM.</p> "> Figure 4
<p>The combination of ABT-199 with S63845 significantly inhibited sphere-forming capacity of the Melanoma Initiating Cells. Melanoma cells were subjected to the primary sphere assay (<b>a</b>–<b>c</b>). Spheres were treated with the indicated compounds either alone, or in combination, for 48 h, and the number of surviving spheres were counted and quantified (<b>a</b>). (<b>b</b>) Shows example images by phase contrast microscopy. (<b>c</b>) Dot plot of normalized primary sphere (expressed as percentage) for combination treatment in BRAF-V600E MUT and WT lines. Secondary sphere assay was conducted with surviving cells from each treatment conditions from the primary sphere assay (<b>d</b>–<b>f</b>). (<b>d</b>) Quantification of the number of secondary spheres; (<b>e</b>) the images of a representative secondary sphere, and (<b>f</b>) dot plot of the relative number of secondary spheres in the combination wells for BRAF-V600E MUT and WT lines. In all melanoma lines, the combination treatment significantly reduced the primary and secondary sphere formation compared with all other treatments (DMSO or single drug). For visual clarity, we have not marked the significance in panel (<b>a</b>) and (<b>d</b>). * indicates <span class="html-italic">p</span> < 0.05. Error bars represent +/− SEM. For clarity and readability of panels a and b, we listed <span class="html-italic">p</span> values in <a href="#app1-cancers-12-02182" class="html-app">Table S5</a>. Scale bar = 100 μm.</p> "> Figure 5
<p>The combination-induced cell death was partially dependent on NOXA, BCL2-like 11 (apoptosis facilitator) (BIM) or BID. ATP assays with knockdown (KD) or knock out (KO) of indicated cells to test if the KD/KO protects against combination-induced cell death. Immunoblot to show the knockdown or knockout of NOXA, BIM or BID. (<b>a</b>) KD lines for NOXA, BIM, or BID in A375. (<b>b</b>) KD lines for NOXA in SKMEL-28. (<b>c</b>) BIM KO lines in WM852c. * indicates <span class="html-italic">p</span> < 0.05; ** indicates <span class="html-italic">p</span> < 0.01. Error bars represent +/− SEM.</p> "> Figure 6
<p>Combination therapy of S64315/MIK665 (the clinic-ready version of S63845) with ABT-199 has a synergistic effect in treating melanoma samples of diverse genetic backgrounds and is comparable to the effects of S63845 with ABT-199. (<b>a</b>) ATP assays of melanoma cell lines upon indicated treatments for 48 h. The viability of the DMSO control for each cell line was set to 100%. Both the combinations (S63845 + ABT-199; upper panel and S64315 + ABT-199, lower panel) had similar efficacy in reducing the cell viability of representative melanoma lines. (<b>b</b>) Summary of ATP assay data of six melanoma cell lines, including patient derived cell lines, treated with single drug or a combination of S63845 + ABT-199 or S64315 + ABT-199. All drugs were used at a dose of 625 nM. For visual clarity, the * is not shown in the figures. Both combinations were highly synergistic at sub-micromolar doses. Error bars represent +/− SEM. For clarity and readability, we listed <span class="html-italic">p</span> values in <a href="#app1-cancers-12-02182" class="html-app">Table S6</a>.</p> ">
Abstract
:1. Introduction
2. Results
2.1. TCGA mRNA and Protein Expression Analyses Suggest BCL2 as a Potential Therapeutic Target for BRAF-WT Melanomas
2.2. The Combination of the BCL2 Inhibitor ABT-199 with the MCL1 Inhibitors S63845 Has High Efficacy in BRAF-WT Melanomas In Vitro
2.3. The Combination of ABT-199 with S63845 Effectively Slowed Tumor Growth In Vivo
2.4. The Combination of ABT-199 with S63845 Significantly Inhibited Sphere-Forming Capacity of the Melanoma Initiating Cells
2.5. The Effects of ABT-199 + S63845 Is Partially Dependent on Pro-Apoptotic BCL2 Family Members NOXA, BIM, and BID
2.6. S64315 Has Similar Synergistic Effects as S63845, When Combined with ABT-199
3. Discussion
4. Materials and Methods
4.1. Analysis of the TCGA Cutaneous Melanoma Dataset
4.2. Reagents and Drug Treatments
4.3. Melanoma Cell Lines, Either Long-Established Conventional Lines or Newly Established Patient Lines
4.4. ATP Viability Assay, Primary and Secondary Sphere Assays
4.5. Immunoblot
4.6. Creation of Short Hairpin RNA Transduced Cell Lines and CRISPR/Cas9-Mediated BIM Knockout Cell Lines
4.7. Mouse Xenograft Studies
4.8. Immunohistochemistry (IHC)
4.9. Calculation of IC50 and Combination Index (CI) Values
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
MCL1 | Myeloid cell leukemia sequence 1 |
BCL2 | B-cell CLL/lymphoma 2 |
BIM | BCL2-like 11 (apoptosis facilitator) |
PARP | Poly ADP-ribose polymerase 1 |
NOXA | Phorbol-12-myristate-13-acetate-induced protein 1 |
BID | BH3 Interacting Domain Death Agonist |
CASP8 | Caspase 8 |
PDCD4 | Programmed cell death protein 4 |
CRISPR | Clustered regularly interspaced short palindromic repeats |
BRAF | B-Raf proto-oncogene |
BH3 | Bcl-2 Homology 3 |
ABT-199 | Abbott Laboratories, a Bcl-2 selective BH3 mimetic |
FDA | U.S. Food and Drug Administration |
RAS | Rat sarcoma family of oncogenes |
NF1 | Neurofibromin 1 |
NRAS | Neuroblastoma RAS Proto Oncogene |
SEM | Standard error of the mean |
MEK | Mitogen-activated protein kinase family |
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Mukherjee, N.; Amato, C.M.; Skees, J.; Todd, K.J.; Lambert, K.A.; Robinson, W.A.; Van Gulick, R.; Weight, R.M.; Dart, C.R.; Tobin, R.P.; et al. Simultaneously Inhibiting BCL2 and MCL1 Is a Therapeutic Option for Patients with Advanced Melanoma. Cancers 2020, 12, 2182. https://doi.org/10.3390/cancers12082182
Mukherjee N, Amato CM, Skees J, Todd KJ, Lambert KA, Robinson WA, Van Gulick R, Weight RM, Dart CR, Tobin RP, et al. Simultaneously Inhibiting BCL2 and MCL1 Is a Therapeutic Option for Patients with Advanced Melanoma. Cancers. 2020; 12(8):2182. https://doi.org/10.3390/cancers12082182
Chicago/Turabian StyleMukherjee, Nabanita, Carol M. Amato, Jenette Skees, Kaleb J. Todd, Karoline A. Lambert, William A. Robinson, Robert Van Gulick, Ryan M. Weight, Chiara R. Dart, Richard P. Tobin, and et al. 2020. "Simultaneously Inhibiting BCL2 and MCL1 Is a Therapeutic Option for Patients with Advanced Melanoma" Cancers 12, no. 8: 2182. https://doi.org/10.3390/cancers12082182