Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma
Xiao-Hong Ma, … , Constantinos Koumenis, Ravi K. Amaravadi
Xiao-Hong Ma, … , Constantinos Koumenis, Ravi K. Amaravadi
Published February 24, 2014
Citation Information: J Clin Invest. 2014;124(3):1406-1417. https://doi.org/10.1172/JCI70454.
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Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma

Abstract

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAFV600E melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAFV600E melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

Authors

Xiao-Hong Ma, Sheng-Fu Piao, Souvik Dey, Quentin Mcafee, Giorgos Karakousis, Jessie Villanueva, Lori S. Hart, Samuel Levi, Janice Hu, Gao Zhang, Rossitza Lazova, Vincent Klump, John M. Pawelek, Xiaowei Xu, Wei Xu, Lynn M. Schuchter, Michael A. Davies, Meenhard Herlyn, Jeffrey Winkler, Constantinos Koumenis, Ravi K. Amaravadi

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Figure 2

BRAFi induces cytoprotective autophagy.

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BRAFi induces cytoprotective autophagy. (A) Dose-dependent growth impair...
(A) Dose-dependent growth impairment of 6 BRAF mutant cell lines treated in 6 replicates with PLX4720 (72-hour MTT assay). Results are mean ± SEM. (B) Immunoblots and gel density quantifications (mean ± SEM for 3 separate experiments) directed against autophagy markers in 48-hour cell lysates from the indicated cell lines treated with vehicle or PLX4720. (C) A375P mCherry-GFP-LC3 cells were treated with DMSO, 1 μM PLX4720, 500 nM rapamycin, or 10 μM HCQ. Representative merged images of red and green channels after 8 hours of treatment are shown. Original magnification, ×40. Autophagic flux (measured by red puncta) and distal blockade of autophagy (measured by yellow puncta) was quantified (mean ± SD of triplicate experiments). (D) Immunoblotting against autophagy markers (mean ± SEM) in untreated A375PshNT and 3 distinct A375PshBRAF clones. (E) Percent growth inhibition after 48 hours of 10 μM HCQ treatment compared with control in A375PshNT and A375PshBRAF cells (MTT assay). Results are mean ± SEM. (F) A375P cells were treated with vehicle, the BRAF inhibitor GSK2118436, the MEK inhibitor GSK1120212, 10 μM HCQ, or the indicated combinations. Shown are 48-hour immunoblots directed against the indicated proteins. *P < 0.05.