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. 2014 Jul;4(7):816-27.
doi: 10.1158/2159-8290.CD-13-0424. Epub 2014 Apr 25.

A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors

David J Konieczkowski  1 , Cory M Johannessen  1 , Omar Abudayyeh  2 , Jong Wook Kim  1 , Zachary A Cooper  3 , Adriano Piris  4 , Dennie T Frederick  5 , Michal Barzily-Rokni  2 , Ravid Straussman  2 , Rizwan Haq  6 , David E Fisher  6 , Jill P Mesirov  2 , William C Hahn  1 , Keith T Flaherty  7 , Jennifer A Wargo  3 , Pablo Tamayo  2 , Levi A Garraway  8
Affiliations

A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors

David J Konieczkowski et al. Cancer Discov. 2014 Jul.

Abstract

Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma.

Significance: Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-κB that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.

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Conflict of interest statement

Conflicts of interest: L.A.G. is a consultant for Foundation Medicine, Novartis, Boehringer Ingelheim, Millennium/Takeda, and Onyx Pharmaceuticals; L.A.G. is an equity holder in Foundation Medicine. L.A.G. receives research support from Novartis. W.C.H. is a consultant for Novartis and Blueprint Medicines.

Figures

Figure 1
Figure 1. Association of expression classes with differential MAPK pathway inhibitor sensitivity in BRAFV600-mutant melanoma in vitro
(a) Sensitivity to PLX4720 (RAFi) across a collection of BRAFV600-mutant melanoma cell lines. (b) Relationship between PLX4720 sensitivity and MITF-high versus NF-κB-high classes. (c) Transcriptional class distinction in BRAFV600-mutant melanoma short-term cultures. (d) Relationship in short-term cultures between expression class and MAPK pathway inhibitor sensitivity. Graph shows GI50 values relative to median GI50 of the sensitive short-term cultures. PLX4720, RAFi; AZD6244, MEKi; VTX11E, ERKi.
Figure 2
Figure 2. MITF-low/NF-κB-high transcriptional classis present and associated with resistance to MAPK pathway inhibition in human tumors
(a) Transcriptional class distinction in BRAFV600-mutant melanoma tumor samples. (b) Examples of AXL and MITF staining in pre-treatment melanoma biopsies (40× magnification). (c) Comparison of progression-free survival between MITF-positive/AXL-negative and MITF-negative/AXL-positive classes. asterisk, p=0.0313, two-tailed t test.
Figure 3
Figure 3. AXL is not necessary for maintenance of intrinsic resistance
(a) Effects of AXL overexpression on survival of drug-sensitive BRAFV600-mutant melanoma cell lines following 4d treatment with PLX4720 (RAFi, 2 μM), AZD6244 (MEKi, 200 nM), PLX4720 + AZD6244, or VTX11E (ERKi, 2 μM). MEK1 is a negative control; RAF1 is a positive control for RAFi resistance. Data are mean ± standard deviation. Asterisks beneath graph indicate p<0.01 (two-tailed t test) relative to the same cell line, expressing MEK1, and treated with the same drug. (b) Effects of AXL overexpression on phosphorylation of Akt and maintenance of ERK phosphorylation following overnight treatment with MAPK pathway inhibitors. D, DMSO; P, 2 μM PLX4720; A, 200 nM AZD6244; P+A, PLX4720 + AZD6244; E, VTX11, 2 μM. MEK1 is a negative control; RAF1 is a positive control for pERK reactivation following RAFi treatment. (c) Effects of AXL inhibitors on induction of pAkt and rescue of pERK following AXL overexpression. R428, 500 nM; XL184, 3 μM; XL880, 100 nM; in the presence or absence of 2 μM PLX4720. shAXL is a positive control. (d) Effects of AXL inhibitors on pAkt and pERK levels in intrinsically resistant cell lines in the presence or absence of PLX4720. (e) Effects of AXL inhibitors on intrinsic resistance to PLX4720.
Figure 4
Figure 4. Establishment of transcriptional class distinction in melanocytes
(a) Effects of aberrant MAPK pathway activation on melanocyte whole-genome expression profiles. (b) Effects of aberrant MAPK pathway activation on markers of the MITF-high and NF-κB-high classes; E, VTX11E (ERKi); P, PLX4720 (RAFi); A, AZD6244 (MEKi), all overnight at 2 μM. (c) Effects of chronic BRAFV600E expression of markers of the MITF-high and NF-κB-high classes. Experiments were performed in TICVA medium (+) or Ham's F10 (-) as indicated. (d) Effect of MITF overexpression on MAPK pathway-induced expression changes. n.s.: non-specific band (e) Relationship between MITF expression levels and MITF amplification in melanoma cell lines.
Figure 5
Figure 5. Plasticity of transcriptional class distinction in melanoma cell lines
(a) Effect of TNFα (25 ng/mL), with or without concomitant IκBα super-repressor expression, on RelA phosphorylation and expression of resistance markers, MITF, and MITF target genes. n.s.: non-specific band. (b) Effect of TNFα (25 ng/mL) on MITF transcriptional activity, as measured in an SK-MEL-5 melanoma cell line stably expressing a TRPM1-luciferase MITF reporter construct. Asterisks beneath graph indicate p<0.001 (two-tailed t test) relative to no TNFα treatment. (c) Effect of TNFα (30 ng/mL) on sensitivity to the indicated MAPK pathway inhibitors. PLX4720, 2 μM; AZD6244, 200 nM; VTX11E, 2 μM. Asterisks beneath graph indicate p<0.001 (two-tailed t test) relative to the same cell line and same MAPK inhibitor without TNFα. (d) Comparison of expression of MITF, AXL, and associated marker genes in parental (sensitive) and cultured-to-resistant melanoma cell lines, with or without 24 hr treatment with PLX4720 (2 μM). PR1 and PR100 denote independent derivations of a resistant subclone. (e) Model of transcriptional state distinction in melanoma.
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