BRAF Inhibitor Resistance Mediated by the AKT Pathway in an Oncogenic BRAF Mouse Melanoma Model
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BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model Daniele Pernaa,b,1,2, Florian A. Karrethb,1,3, Alistair G. Rusta, Pedro A. Perez-Mancerab, Mamunur Rashida, Francesco Iorioc,d, Constantine Alifrangisc, Mark J. Arendse, Marcus W. Bosenbergf, Gideon Bollagg, David A. Tuvesonb,h,4, and David J. Adamsa,4 aExperimental Cancer Genetics and cCancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom; bCancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, United Kingdom; dEuropean Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, Cambridge CB10 1SA, United Kingdom; eUniversity of Edinburgh Division of Pathology, Edinburgh Cancer Research Centre, Institute of Genetics & Molecular Medicine, Western General Hospital, Edinburgh, EH4 2XR, United Kingdom; fDepartment of Dermatology, Yale University, New Haven, CT 06520; gPlexxikon Inc., Berkeley, CA 94710; and hCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 Edited by Neal G. Copeland, Houston Methodist Research Institute, Houston, TX, and approved December 30, 2014 (received for review September 20, 2014) BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors netic lesions including increased expression of the insulin-like growth elicit a transient anti-tumor response in ∼80% of BRAFV600-mutant factor 1 receptor (IGF1R) and expression of hepatocyte growth melanoma patients that almost uniformly precedes the emergence factor (HGF) by stromal cells has been linked to BRAF inhibitor of resistance. Here we used a mouse model of melanoma in which resistance (12–14). Similarly, activation of the PI3K pathway V618E melanocyte-specific expression of Braf (analogous to the hu- through loss of PTEN, or deregulation of other genes (e.g., AKT3, V600E man BRAF mutation) led to the development of skin hyper- BCL2A1, IGF1,andPIB5PA), has been shown to result in re- pigmentation and nevi, as well as melanoma formation with sistance to BRAF inhibitor-induced apoptosis (15–19). Additional incomplete penetrance. Sleeping Beauty insertional mutagenesis mediators of resistance have also been proposed, including up- in this model led to accelerated and fully penetrant melanomagenesis V618E regulation of the tyrosine kinase platelet-derived growth factor re- and synchronous tumor formation. Treatment of Braf trans- ceptor beta (PDGFRB), possibly through MAPK- or PI3K-related poson mice with the BRAF inhibitor PLX4720 resulted in tumor mechanisms (8). regression followed by relapse. Analysis of transposon insertions We generated a mouse model of BrafV618E-induced mela- identified eight genes including Braf, Mitf,andERas (ES-cell noma (analogous to BRAFV600E in humans; transcript expressed Ras) as candidate resistance genes. Expression of ERAS ENSMUSE00000277767 in mouse) and used it in combination in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thy- moma viral oncogene homolog 1), a phenotype reverted by com- Significance binatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associ- Using Sleeping Beauty transposon mutagenesis in a melanoma ated with phosphorylation/inactivation of BAD, and that the re- model driven by oncogenic BRAF (B-Raf proto-oncogene, serine/ sistance of hepatocyte growth factor-treated human melanoma threonine kinase), we identified both known and novel candi- cells to PLX4720 can be reverted by treatment with the BAD-like date genes that mediate resistance to the BRAF inhibitor BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD PLX4720. We validate ES-cell expressed Ras as a novel pro- pathway in resistance to BRAF inhibition and illustrate an in vivo moter of BRAF inhibitor resistance and propose that AKT (v-akt approach for finding drug resistance genes. murine thymoma viral oncogene homolog 1)-mediated in- activation of BAD (BCL2-associated agonist of cell death) con- melanoma | drug resistance | BRAF inhibitors | mouse models stitutes a pathway that may contribute to hepatocyte growth factor-mediated therapy resistance. Our work establishes he discovery that ∼50–60% of melanomas carry BRAFV600E Sleeping Beauty mutagenesis as a powerful tool for the iden- Tpoint mutations (1) prompted the generation of compounds tification of novel resistance genes and mechanisms in genet- specifically targeting this hyperactive mutated kinase. One such ically modified mouse models. compound, PLX4032, has shown unprecedented therapeutic effi- Author contributions: D.P., F.A.K., D.A.T., and D.J.A. designed research; D.P., F.A.K., F.I., cacy in clinical trials and was therefore FDA-approved for clinical and C.A. performed research; P.A.P.-M., M.W.B., and G.B. contributed new reagents/analytic therapy under the name vemurafenib. Despite its remarkable ef- tools; D.P., F.A.K., A.G.R., M.R., F.I., and M.J.A. analyzed data; and D.P., F.A.K., D.A.T., and ficacy, almost all patients receiving BRAF inhibitor treatment re- D.J.A. wrote the paper. lapsed after weeks to months of therapy (2–5). Acquired resistance Conflict of interest statement: G.B. is an employee of Plexxikon Inc., which owns and to BRAF inhibitors has since been a major focus of research markets vemurafenib. and two major paths to resistance have emerged: MAPK-dependent This article is a PNAS Direct Submission. and MAPK-independent mechanisms. MAPK-dependent mecha- Freely available online through the PNAS open access option. nisms primarily involve reactivation of the MAPK pathway to Data deposition: The sequence reported in this paper has been deposited in the European substitute for the inhibition of BRAFV600E. This may be achieved Nucleotide Archive (accession no. ERP002600). 1 through mechanisms including expression of alternative splicing D.P. and F.A.K. contributed equally to this work. forms of BRAFV600E, amplification of BRAFV600E, the acquisition of 2Present address: Biopharm Discovery Process, GlaxoSmithKline, Stevenage SG1 2NY, United Kingdom. activating mutations in NRAS or MEK (MAP2K1), or loss of NF1. 3Present address: Meyer Cancer Center, Weill Cornell Medical College, New York, Similarly, COT (MAP3K8) overexpression has been shown to drive NY 10021. resistance through ERK activation independent of RAF signaling 4To whom correspondence may be addressed. Email: [email protected] or da1@sanger. (6–11). Elevated signaling through the PI3K/AKT pathway, with or ac.uk. without concomitant MAPK reactivation, represents an alternative This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. path to resistance. Indeed, AKT signaling mediated by several ge- 1073/pnas.1418163112/-/DCSupplemental. E536–E545 | PNAS | Published online January 26, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1418163112 Downloaded by guest on September 25, 2021 with Sleeping Beauty insertional mutagenesis to identify mecha- rare and suggests a requirement for additional genetic alter- PNAS PLUS nisms of resistance to BRAF inhibition using PLX4720, a vemur- ations for melanomagenesis. Those melanomas that did form afenib analog. As in patients with tumors carrying BRAFV600 were generally hypopigmented and primarily dermal (Fig. S2G). mutations, melanomas in our mouse model showed an initial re- There was little or no epidermal involvement, and most tumors sponse to BRAF inhibitor treatment, characterized by rapid tumor had a spindle-cell morphology. Tumors with predominant epi- regression, followed by tumor recurrence after weeks to months thelioid cell features were rarely observed. Melanoma cells dis- of therapy. Analysis of transposon insertion sites in naïve and played characteristic enlarged and pleomorphic nuclei, sometimes PLX4720-resistant melanomas identified the proto-oncogene with prominent eosinophilic atypical nucleoli and numerous mi- ERas, and seven other genes, as putative mediators of resistance. totic figures (Fig. S2H). Larger lesions showed superficial ulcer- We show that ERAS expression confers resistance associated with ation, but with no or very little evidence of an intraepidermal inactivation of the proapoptotic protein BAD in an AKT/PI3K- invasive component (radial/horizontal growth phase) (Fig. S2I). dependent manner, and that BAD also contributes to BRAF Infiltration of melanoma cells through the underlying skeletal inhibitor resistance in the context of activated HGF signaling. muscle and into s.c. adipose tissue was commonly observed (Fig. These data illustrate the human relevance of genes/pathways S2J). Occasionally melanomas showed foci of lymphocyte aggre- found through insertional mutagenesis screens for drug re- gates and scattered tumor-infiltrating lymphocytes (Fig. S2K). sistance mediators. Residual pigmented melanocytes were observed in a subset of melanomas (Fig. S2L). Despite their predominantly amelanotic Results nature, tumors expressed the melanocyte lineage markers S100A Targeted Expression of Oncogenic BrafV618E Induces Skin Hyper- and MITF (Fig. S2 M and N). At necropsy draining lymph pigmentation, Nevi, and Melanoma. We targeted the endogenous nodes appeared brown in color and histological analysis murine Braf locus by introducing a stop element (LoxP-Stop- revealed the presence of lightly pigmented as well as unpig- LoxP or LSL cassette) into intron 2 and a V618E mutation into mented melanoma cells with pleomorphic nuclei and also exon 15 (Fig. 1A). Our model is similar to other