Abstract
B-RAF is mutated to a constitutively active form in 8% of human cancers including 50% of melanomas. In clinical trials, the RAF inhibitor, PLX4032 (vemurafenib), caused partial or complete responses in 48–81% of mutant B-RAF harboring melanoma patients. However, the average duration of response was 6–7 months before tumor regrowth, indicating the acquisition of resistance to PLX4032. To understand the mechanisms of resistance, we developed mutant B-RAF melanoma cells that displayed resistance to RAF inhibition through continuous culture with PLX4720 (the tool compound for PLX4032). Resistance was associated with a partial reactivation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, recovery of G1/S cell-cycle events, and suppression of the pro-apoptotic B-cell leukemia/lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins, Bcl-2-interacting mediator of cell death-extra large (Bim-EL) and Bcl-2 modifying factor (Bmf). Preventing ERK1/2 reactivation with MEK (mitogen-activated protein/extracellular signal-regulated kinase kinase) inhibitors blocked G1-S cell-cycle progression but failed to induce apoptosis or upregulate Bim-EL and Bmf. Treatment with the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid, led to de-repression of Bim-EL and enhanced cell death in the presence of PLX4720 or AZD6244 in resistant cells. These data indicate that acquired resistance to PLX4032/4720 likely involves ERK1/2 pathway reactivation as well as ERK1/2-independent silencing of BH3-only proteins. Furthermore, combined treatment of HDAC inhibitors and MEK inhibitors may contribute to overcoming PLX4032 resistance.
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Abbreviations
- ERK1/2:
-
extracellular signal-regulated kinase 1/2
- BH3:
-
Bcl-2 homology domain 3
- Bim-EL:
-
Bcl-2-interacting mediator of cell death-extra large
- Bmf:
-
Bcl-2 modifying factor
- HDAC:
-
Histone deacetylase
- SAHA:
-
suberoylanilide hydroxamic acid
- MEK:
-
mitogen-activated protein/extracellular signal-regulated kinase kinase
- HER2:
-
human epidermal receptor 2
- BCR-ABL:
-
Breakpoint cluster region-v-abl abelson murine leukemia viral oncogene homolog 1
- EGFR:
-
epidermal growth factor receptor
- PTEN:
-
phosphatase and tensin homolog
- PDGFRβ:
-
platelet-derived growth factor receptor beta
- IGF1R:
-
insulin-like growth factor 1 receptor
- MAP3K:
-
mitogen-activated protein kinase kinase kinase
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- siRNA:
-
small interfering ribonucleic acid
- FAK:
-
focal adhesion kinase
- Rb:
-
retinoblastoma 1
- EdU:
-
5-ethynyl-2′-deoxyuridine
- DMSO:
-
dimethyl sulfoxide
- Bcl-2:
-
B-cell leukemia/lymphoma 2
- Bcl-xl:
-
B-cell lymphoma-extra large
- Mcl-1:
-
myeloid cell leukemia sequence 1
- Bad:
-
Bcl-xL/Bcl-2-associated death promoter
- qRT-PCR:
-
quantitative real-time polymerase chain reaction
- eGFP:
-
enhanced green fluorescence protein
- RTK:
-
receptor tyrosine kinase
- FoxO1:
-
forkhead box O1
- FoxO3a:
-
forkhead box O3a
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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Acknowledgements
We thank Melanie Mayberry for the sequencing of WM793-Res cells, Dr Gideon Bollag (Plexxikon, Berkeley, CA) for providing PLX4720, Dr Meenhard Herlyn (Wistar Institute, Philadelphia, PA) for WM793 cell line and Dr Antoni Ribas (UCLA, CA) for M238 cell line. This work was supported by NIH grants R01-GM067893 and R01-CA125103 (AE Aplin) and an Outrun the Sun fellowship (Y Shao). The Kimmel Cancer Center is supported by National Cancer Institute Support Grant 1P30CA56036.
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Shao, Y., Aplin, A. BH3-only protein silencing contributes to acquired resistance to PLX4720 in human melanoma. Cell Death Differ 19, 2029–2039 (2012). https://doi.org/10.1038/cdd.2012.94
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DOI: https://doi.org/10.1038/cdd.2012.94
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