Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Apr 24;10(5):342.
doi: 10.1038/s41419-019-1568-3.

BCL-XL and MCL-1 are the key BCL-2 family proteins in melanoma cell survival

Erinna F Lee  1   2   3 , Tiffany J Harris  4 , Sharon Tran  4   5 , Marco Evangelista  4 , Surein Arulananda  4   5 , Thomas John  4   5 , Celeste Ramnac  4   5 , Chloe Hobbs  4   5 , Haoran Zhu  6   7 , Gency Gunasingh  8 , David Segal  6 , Andreas Behren  4   5 , Jonathan Cebon  4   5 , Alexander Dobrovic  4   5 , John M Mariadason  4   5 , Andreas Strasser  6   9 , Leona Rohrbeck  6   10 , Nikolas K Haass  8 , Marco J Herold  6   9 , W Douglas Fairlie  11   12   13
Affiliations

BCL-XL and MCL-1 are the key BCL-2 family proteins in melanoma cell survival

Erinna F Lee et al. Cell Death Dis. .

Abstract

Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy. Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs. Hence, alternative treatments are still required. Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting. To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures. None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK. Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma. Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines. Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma.

PubMed Disclaimer

Conflict of interest statement

The Walter and Eliza Hall Institute has previously been involved in collaborations with Abbvie and Genentech to develop and characterize BH3-mimetic drugs. The Institute and some authors have or may receive payments in respect of Venetoclax. The Walter and Eliza Hall Institute also has an on-going collaboration with Servier to investigate the use of MCL-1 inhibitors for cancer therapy.

Figures

Fig. 1
Fig. 1. Expression levels of BCL-2 protein family members in melanoma cell lines and patient tissue.
a Cell lysates from established, and patient-derived melanoma lines were analyzed by Western blot and probed for both pro-survival and pro-apoptotic BCL-2 family proteins. Blots were re-probed for β-actin as a loading control. b Representative samples from IHC analysis of melanoma tissue microarray showing examples of high and low expression of MCL-1, BCl-XL and BCL-2
Fig. 2
Fig. 2. Melanoma cells are insensitive to BH3-mimetic drugs as single agents.
Selective antagonism of BCL-XL (A1331852), BCL-2 (ABT-199) or MCL-1 (S63845), as well as co-targeting of BCL-XL, BCL-2 and BCL-W (ABT-263) fails to kill the majority of melanoma cells unless treated with high concentrations. Cell viability was determined after 24 h treatment by CellTiter-Glo luminescent assay. Data represent mean ± standard deviation from N = 3 separate assays. See Supplementary Figure 1 for corresponding data with other cell lines
Fig. 3
Fig. 3. Co-antagonism of several BCL-2 pro-survival proteins has greater effect on cell viability than targeting each protein alone.
a Antagonism of MCL-1 plus BCL-XL (S63845 + either A1331852 or ABT-263) is more effective than the combination targeting MCL-1 plus BCL-2 (S63845 + ABT-199) in all melanoma cell lines tested. Cell viability was determined after 24 h treatment by CellTiter-Glo luminescent assay. See Supplementary Figure 2 for data with other cell lines. Data represent mean ± standard deviation from N = 3–4 separate assays. b Heatmap of EC50 values for each cell line and drug combination. The S63845 plus ABT-199 combination was less effective than the other two combinations. See Supplementary Table 2 for actual EC50 values for all treatments
Fig. 4
Fig. 4. Combinations of BH3-mimetics induce apoptosis in melanoma cells as determined by Annexin V / propidium iodide staining.
Co-targeting of MCL-1 plus BCL-XL is more effective than the co-targeting of MCL-1 plus BCL-2, consistent with the CellTiter-Glo assay data. For most cell lines, potent melanoma cell killing was observed after 24 h treatment when S63845 was combined with either ABT-263 or A1331852 at a concentration where both drugs had no major effect on their own (i.e., 1 μM). Melanoma cell killing was inhibited by the addition of the pan-caspase inhibitor Q-VD-OPh, consistent with induction of apoptosis. Data represent mean ± standard deviation from N = 3 separate assays
Fig. 5
Fig. 5. BH3-mimetics combinations are synergistic in 3D cultures.
a A02, b A375, c LM-MEL-28 cells were allowed to form spheroids over 72 h and then treated for 24 h with drugs or combinations as indicated. Bright field images (left panel) and fluorescence images for DRAQ7 staining (right panel) are shown for each combination. The panel in the top right (red box) represents the vehicle only control. Spheroids were imaged with an IN Cell Analyzer 2200/Nikon ×4/0.20, Plan Apo, CFI/60. Note differences in DRAQ7 intensity and distribution. Data is representative from N = 3 independent experiments
Fig. 6
Fig. 6. Deletion of BFL-1 together with co-antagonism of other pro-survival BCL-2 proteins has minor impact on melanoma cell killing.
a Inhibition of both MCL-1 and BFL-1 in combination with either BCL-XL and BCL-2 antagonism increases melanoma cell killing in M14 cells, though not dramatically. b EC50 values for BH3-mimetic drug combinations in each BFL-1-deleted (BCL2A1 sgRNA) and control (BCL2A1 wt) cell line. The effect of BFL-1 deletion was slightly more significant in M14 cells compared to the LM-MEL-28 cells. Data represent mean ± standard deviation from N = 3 separate assays
Fig. 7
Fig. 7. Co-treatment of melanoma cell lines with S63845 and either a BRAF inhibitor or a proteasome inhibitor has enhanced effect over treatment with each drug alone in certain melanoma cell lines.
Melanoma cell lines were treated with (a), PLX4032 or (b), bortezomib, alone or in combination with S63845 for 72 h before analysis by CellTiter-Glo viability assay. Data represent mean ± standard deviation from N = 3 separate assays
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Soengas MS, Lowe SW. Apoptosis and melanoma chemoresistance. Oncogene. 2003;22:3138–3151. - PubMed
    1. Chapman PB, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N. Engl. J. Med. 2011;364:2507–2516. - PMC - PubMed
    1. Colombino M, et al. BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma. J. Clin. Oncol. 2012;30:2522–2529. - PubMed
    1. Wagle N, et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol. 2011;29:3085–3096. - PMC - PubMed
    1. Luke JJ, Flaherty KT, Ribas A, Long GV. Targeted agents and immunotherapies: optimizing outcomes in melanoma. Nat. Rev. Clin. Oncol. 2017;14:463–482. - PubMed

Publication types

MeSH terms

-