COP1 Deficiency in BRAFV600E Melanomas Confers Resistance to Inhibitors of the MAPK Pathway

Abstract

Aberrant activation of the mitogen-activated protein kinase (MAPK) cascade promotes oncogenic transcriptomes. Despite efforts to inhibit oncogenic kinases, such as BRAFV600E, tumor responses in patients can be heterogeneous and limited by drug resistance mechanisms. Here, we describe patient tumors that acquired COP1 or DET1 mutations after treatment with the BRAF^V600E inhibitor vemurafenib. COP1 and DET1 constitute the substrate adaptor of the E3 ubiquitin ligase CRL4^COP1/DET1, which targets transcription factors, including ETV1, ETV4, and ETV5, for proteasomal degradation. MAPK-MEK-ERK signaling prevents CRL4^COP1/DET1 from ubiquitinating ETV1, ETV4, and ETV5, but the mechanistic details are still being elucidated. We found that patient mutations in COP1 or DET1 inactivated CRL4^COP1/DET1 in melanoma cells, stabilized ETV1, ETV4, and ETV5, and conferred resistance to inhibitors of the MAPK pathway. ETV5, in particular, enhanced cell survival and was found to promote the expression of the pro-survival gene BCL2A1. Indeed, the deletion of pro-survival BCL2A1 re-sensitized COP1 mutant cells to vemurafenib treatment. These observations indicate that the post-translational regulation of ETV5 by CRL4^COP1/DET1 modulates transcriptional outputs in ERK-dependent cancers, and its inactivation contributes to therapeutic resistance.

Keywords: BRAF; COP1; DET1; melanoma; vemurafenib.

Figure 1

Deletion or mutational inactivation of COP1 mediates resistance to MAPK pathway inhibitors. (A) DNA sequence of the COP1^K539E missense mutation. (B) Domain organization of the COP1 protein indicating the mutations found in melanoma patients treated with Vemurafenib. (C) Western blots of A375 cells (COP1^WT) and two COP1-deficient clones generated using CRISPR/Cas9 technology (COP1^KO.1 and COP1^KO.2). (D) Chemical genomic screen strategy using COP1^WT and COP1^KO A375 cells. (E) The graph indicates the mean viability (MV) differential (y axis) between COP1^WT and COP1^KO A375 cells treated with the compounds in (d; x axis). (F) The graph shows the qIC50 for different MAPK pathway inhibitors in A375 cells. (G) Western blots of A375 cells. (H) The graphs indicate A375 cell proliferation. The results are representative of 3 independent experiments.

Figure 2

COP1^K539E fails to promote ETV5 degradation upon ERK inhibition. (A) Global proteomic analysis of A375 cells before and after a 1 h treatment with 1 µM ERK inhibitor Vertex11e. The graph indicates the change in abundance (y axis) for detected peptides (x axis). The circles indicating peptides unique to ETV5 are colored blue. (B,C) The graphs show the change in ETV5 protein abundance in A375 cells after treatment with 1 µM Vertex11e. The circles in (B) indicate the results from individual biological replicates (n = 2). The bars in (C) represent the mean of two biological replicates. (D) Western blots of A375 cells treated with 1 µM Vertex11e for 1 h. (E) COP1 is immunoprecipitated (IP) from lysates of WT or COP1^K539E A375 cells using an anti-COP1 antibody (28A4, Genentech) and magnetic protein A/G beads. The input lysates and immunoprecipitates were subsequently blotted against ETV5. COP1^KO A375 cells were used. (F–H) Western blots of transfected 293T cells. WT and mutant genes are overexpressed using a PRK5-based mammalian expression, introduced into 293T cells via Lipofectamine transfection reagent. (I) Western blots of COP1^K539E A375 cells expressing doxycycline-inducible ^FlagCOP1. Cells were treated with doxycycline for 24 h. (J) The graphs indicate A375 cell proliferation. The results are representative of 2 independent experiments. * Non-specific band.

Figure 3

COP1^K539E A375 melanoma cells exhibit altered expression of genes regulating cell survival and differentiation. (A,B) Heatmaps show gene expression for the top 50 differentially expressed genes between COP1^WT and COP1^K539E A375 cells (A) and differentially expressed genes involved in melanocyte differentiation (B). (C) Boxplots of the gene signature score using the melanoma subtypes defined in Tsoi et al. [36] across WT and COP1 SNP genotypes. (D) Box and whisker plots of gene expression in A375 cells. Black circles indicate 3 biological replicates. (E) Western blots of A375 and Cloudman S91 cells.

Figure 4

Analysis of the chromatin in COP1^K539E A375 cells. (A) Scheme for ChIP-seq analysis of COP1^WT and COP1^K539E A375 cells. Cells were treated with 1 µM Vertex11e. (B) The bars indicate the number of ETV5 binding sites showing differential occupancy in the A375 cells indicated. (C) The histogram shows the distribution of ETV5 peaks relative to transcription start sites (TSS) in COP1^K539E A375 cells. (D) The graph indicates transcription factor motifs associated with ETV5 distal binding sites in COP1^K539E A375 cells. The p-values are determined by the hypergeometric test. (E) Gene ontology (GO) terms associated with ETV5 distal binding sites in COP1^K539E A375 cells. The p-values are determined by the hypergeometric test. (F) Traces indicate normalized H3K4me3 ChIP-seq reads at the BCL2A1 promoter in A375 cells. The location of the BCL2A1 coding region is shown in purple at the top. (G) Traces show normalized ETV5 (blue), H3K4me3 (red), and H3K27ac (orange) ChIP-seq reads around the BCL2A1 gene. Hi-ChIP arcs (magenta).

Figure 5

Elevated BCL2A1 expression in COP1 mutant cells drives resistance to MAPK pathway inhibitors. (A) Western blots of A375 cells. (B) Graphs indicate A375 cell proliferation. Results are representative of 2 independent experiments. (C) Relative viability of A375 cells upon treatment with different inhibitors of the BCL2 pro-survival proteins, GDC-0199 targeting BCL2, A-1331852 targeting BCL-XL, and AMG-176 and S63845 targeting MCL1, alone or in combination with the MEK inhibitors cobimetinib (GDC-0973). The black line shows the response to cobimetinib at 0.18 μM, yellow-red shades show the response to the BCL2-family inhibitor at respective concentrations (see x-axis), and purple-blue shades show the response to the corresponding combination of drugs.