BRAF^V600E-PROTAC versus inhibitors in melanoma cells: Deep transcriptomic characterisation

Abstract

Aims: This study compares the suppression of Mitogen-activated protein kinase (MAPK) signalling and early resistance potential between a proteolysis-targeting chimera (PROTAC) and inhibitors targeting BRAF^V600E.

Methods: We performed a detailed in silico analysis of the transcriptomic landscape of the A375 melanoma cell line treated with a PROTAC and BRAF^V600E inhibitors from RNA sequencing data. The study assessed gene dysregulation, MAPK and Phosphoinositide-3-kinase (PI3K/AKT) pathway inhibition, and cell survival. Key genes uniquely dysregulated by PROTAC treatment were validated by qPCR. Furthermore, analysis was performed to evaluate dedifferentiation and early resistance signatures to understand melanoma drug-induced plasticity.

Results: PROTAC-treated cells showed significantly lower MAPK pathway activity, strong cell cycle arrest and elevated apoptotic gene expression compared to inhibitor-treated cells, with no effect on the PI3K/AKT pathway. A high microphtalmia-associated transcription factor (MITF)/Tyrosine-Protein Kinase Receptor (AXL) ratio in PROTAC-treated cells indicated reduced early drug resistance. BRAF degradation induced a melanocytic-transitory phenotype. Although PROTAC and inhibitor treatments caused overlapping transcriptomic changes, key differences were observed. PROTAC treatment enriched processes such as epithelial‒mesenchymal transition, inflammatory responses, and Tumor necrosis factor-Alpha (TNF-α) and IL2/STAT5 signalling.

Conclusion: PROTAC-targeting BRAF^V600E demonstrates enhanced MAPK suppression, reduced early resistance and distinct transcriptional effects compared to traditional inhibitors. It represents a promising strategy for overcoming resistance in melanoma treatment.

Keywords

BRAF ^V600E / differentiation state / MAPK pathway / melanoma / MITF / PROTAC

References

[1] McArthur G, Ribas A. Targeting oncogenic drivers and the immune system in melanoma. J Clin Oncol. 2013;31(4):499-506.

[2] Wellbrock C, Arozarena I. The complexity of the ERK/MAP-kinase pathway and the treatment of melanoma skin cancer. Front Cell Dev Biol. 2016;4:33.

[3] Czarnecka AM, Bartnik E, Fiedorowicz M, Rutkowski P. Targeted therapy in melanoma and mechanisms of resistance. Int J Mol Sci. 2020;21(13):4576.

[4] Corrales E, Levit-Zerdoun E, Metzger P, et al. PI3K/AKT signaling allows for MAPK/ERK pathway independency mediating dedifferentiation-driven treatment resistance in melanoma. Cell Commun Signal. 2022;20(1):187.

[5] Proietti I, Skroza N, Bernardini N, et al. Mechanisms of acquired BRAF inhibitor resistance in melanoma: a systematic review. Cancers. 2020;12(10):2801.

[6] Welsh SJ, Rizos H, Scolyer RA, Long GV. Resistance to combination BRAF and MEK inhibition in metastatic melanoma: where to next? Eur J Cancer. 2016;62:76-85.

[7] Posternak G, Tang X, Maisonneuve P, et al. Functional characterization of a PROTAC directed against BRAF mutant V600E. Nat Chem Biol. 2020;16(11):1170-1178.

[8] McKay M, Ritt D, Morrison D. RAF inhibitor-induced KSR1/B-RAF binding and its effects on ERK cascade signaling. Curr Biol. 2011;21(7):563-568.

[9] Maik-Rachline G, Seger R. The ERK cascade inhibitors: towards overcoming resistance. Drug Resist Updat. 2016;25:1-12.

[10] He M, Cao C, Ni Z, et al. PROTACs: great opportunities for academia and industry (an update from 2020 to 2021). Signal Transduct Target Ther. 2022;7(1):181.

[11] Paiva S, Crews C. Targeted protein degradation: elements of PROTAC design. Curr Opin Chem Biol. 2019;50:111-119.

[12] Li K, Crews C. PROTACs: past, present and future. Chem Soc Rev. 2022;51(12):5214-5236.

[13] Chirnomas D, Hornberger K, Crews C. Protein degraders enter the clinic—a new approach to cancer therapy. Nat Rev Clin Oncol. 2023;20(4):265-278.

[14] Ohoka N, Suzuki M, Uchida T, et al. Development of a potent small-molecule degrader against oncogenic BRAF(V600E) protein that evades paradoxical MAPK activation. Cancer Sci. 2022;113(8):2828-2838.

[15] Alabi S, Jaime-Figueroa S, Yao Z, et al. Mutant-selective degradation by BRAF-targeting PROTACs. Nat Commun. 2021;12(1):920.

[16] Marini E, Marino M, Gionfriddo G, et al. Investigation into the use of encorafenib to develop potential PROTACs directed against BRAF(V600E) protein. Molecules. 2022;27(23):8513.

[17] Love M, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.

[18] Kuleshov MV, Jones MR, Rouillard AD, et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016;44(W1):W90-W97.

[19] Keenan AB, Torre D, Lachmann A, et al. ChEA3:transcription factor enrichment analysis by orthogonal omics integration. Nucleic Acids Res. 2019;47(W1):W212-W224.

[20] Abu-Jamous B, Kelly S. Clust: automatic extraction of optimal co-expressed gene clusters from gene expression data. Genome Biol. 2018;19(1):172.

[21] Lê S, Josse J, Husson F. FactoMineR: an R package for multivariate analysis. J Stat Software. 2008;25(1):1-18.

[22] Lee E, Chuang H, Kim J, Ideker T, Lee D. Inferring pathway activity toward precise disease classification. PLoS Comput Biol. 2008;4(11):e1000217.

[23] Kolde R. Pheatmap: Pretty Heatmaps. R Package Version 1.0.12. 2019.

[24] Santos A, Wernersson R, Jensen L. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes. Nucleic Acids Res. 2015;43(Database issue):D1140-D1144.

[25] Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402-408.

[26] van Dam S, Võsa U, van der Graaf A, Franke L, de Magalhães JP. Gene co-expression analysis for functional classification and gene-disease predictions. Brief Bioinform. 2018;19(4):575-592.

[27] Wagle M, Kirouac D, Klijn C, et al. A transcriptional MAPK pathway activity score (MPAS) is a clinically relevant biomarker in multiple cancer types. NPJ Precis Oncol. 2018;2(1):7.

[28] Caunt C, Keyse S. Dual-specificity MAP kinase phosphatases (MKPs):shaping the outcome of MAP kinase signalling. FEBS J. 2013;280(2):489-504.

[29] Locard-Paulet M, Palasca O, Jensen L. Identifying the genes impacted by cell proliferation in proteomics and transcriptomics studies. PLoS Comput Biol. 2022;18(10):e1010604.

[30] Najem A, Soumoy L, Sabbah M, et al. Understanding molecular mechanisms of phenotype switching and crosstalk with TME to reveal new vulnerabilities of melanoma. Cells. 2022;11(7):1157.

[31] Tsoi J, Robert L, Paraiso K, et al. Multi-stage differentiation defines melanoma subtypes with differential vulnerability to drug-induced iron-dependent oxidative stress. Cancer Cell. 2018;33(5):890-904.e5.

[32] Fallahi-Sichani M, Moerke NJ, Niepel M, Zhang T, Gray NS, Sorger PK. Systematic analysis of BRAF(V600E) melanomas reveals a role for JNK/c-Jun pathway in adaptive resistance to drug-induced apoptosis. Mol Syst Biol. 2015;11(3):797.

[33] Müller J, Krijgsman O, Tsoi J, et al. Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nat Commun. 2014;5:5712.

[34] Ruiz EM, Alhassan SA, Errami Y, et al. A predictive model of adaptive resistance to BRAF/MEK inhibitors in melanoma. Int J Mol Sci. 2023;24(9):8407.

[35] Kurimchak AM, Herrera-Montávez C, Montserrat-Sangrà S, et al. The drug efflux pump MDR1 promotes intrinsic and acquired resistance to PROTACs in cancer cells. Sci Signal. 2022;15(749):eabn2707.

[36] Alabi S, Crews C. Major advances in targeted protein degradation: PROTACs, LYTACs, and MADTACs. J Biol Chem. 2021;296:100647.

[37] Lu J, Qian Y, Altieri M, et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem Biol. 2015;22(6):755-763.

[38] Subbiah V, Baik C, Kirkwood J. Clinical development of BRAF plus MEK inhibitor combinations. Trends Cancer. 2020;6(9):797-810.

[39] Lin A, Sahun M, Biscop E, et al. Acquired non-thermal plasma resistance mediates a shift towards aerobic glycolysis and ferroptotic cell death in melanoma. Drug Resist Updat. 2023;67:100914.

[40] Simmons J, Neuendorf H, Boyle G. BRN2 and MITF together impact AXL expression in melanoma. Exp Dermatol. 2022;31(1):89-93.

[41] Yang C, Tian C, Hoffman TE, Jacobsen NK, Spencer SL. Melanoma subpopulations that rapidly escape MAPK pathway inhibition incur DNA damage and rely on stress signalling. Nat Commun. 2021;12(1):1747.

[42] Eccles M, He S, Ahn A, et al. MITF and PAX3 play distinct roles in melanoma cell migration; outline of a “genetic switch” theory involving MITF and PAX3 in proliferative and invasive phenotypes of melanoma. Front Oncol. 2013;3:229.

[43] Verastegui C, Bille K, Ortonne J, Ballotti R. Regulation of the microphthalmia-associated transcription factor gene by the Waardenburg syndrome type 4 gene, SOX10. J Biol Chem. 2000;275(40):30757-30760.

[44] Pastwińska J, Karaś K, Karwaciak I, Ratajewski M. Targeting EGFR in melanoma—the sea of possibilities to overcome drug resistance. Biochim Biophys Acta. 2022;1877(4):188754.

[45] Bai X, Fisher D, Flaherty K. Cell-state dynamics and therapeutic resistance in melanoma from the perspective of MITF and IFNgamma pathways. Nat Rev Clin Oncol. 2019;16(9):549-562.

RIGHTS & PERMISSIONS

2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.