The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF.

Following mutations in BRAF and NRAS, the RAC1 c.85C>T single-nucleotide variant (SNV) encoding P29S amino acid change represents the next most frequently observed protein-coding hotspot mutation in melanoma. However, the biologic and clinical significance of the RAC1 P29S somatic mutation in approximately 4% to 9% of patients remains unclear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (vemurafenib and dabrafenib). Enforced expression of RAC1 P29S in sensitive BRAF-mutant melanoma cell lines confers resistance manifested by increased viability, decreased apoptosis, and enhanced tumor growth in vivo upon treatment with RAF inhibitors. Conversely, RNAi-mediated silencing of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to vemurafenib and dabrafenib. Our results suggest RAC1 P29S status may offer a predictive biomarker for RAF inhibitor resistance in melanoma patients, where it should be evaluated clinically.

[1]  G. Bokoch,et al.  Characterization of Rac and Cdc42 Activation in Chemoattractant-stimulated Human Neutrophils Using a Novel Assay for Active GTPases* , 1999, The Journal of Biological Chemistry.

[2]  Yu Shyr,et al.  Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. , 2012, The New England journal of medicine.

[3]  Kam Y. J. Zhang,et al.  Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity , 2008, Proceedings of the National Academy of Sciences.

[4]  J. Sosman,et al.  Preexisting MEK1 exon 3 mutations in V600E/KBRAF melanomas do not confer resistance to BRAF inhibitors. , 2012, Cancer discovery.

[5]  P. Tamayo,et al.  Integrative functional genomics identifies RINT1 as a novel GBM oncogene , 2012, Neuro-oncology.

[6]  K. Flaherty,et al.  Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. , 2012, The New England journal of medicine.

[7]  D. Schadendorf,et al.  A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition. , 2013, Cancer discovery.

[8]  Dirk Schadendorf,et al.  Improved survival with MEK Inhibition in BRAF-mutated melanoma for the METRIC Study Group , 2012 .

[9]  A. Nicholson,et al.  Mutations of the BRAF gene in human cancer , 2002, Nature.

[10]  Young Lim Choi,et al.  Transforming mutations of RAC guanosine triphosphatases in human cancers , 2013, Proceedings of the National Academy of Sciences.

[11]  R. Sullivan,et al.  Resistance to BRAF-targeted therapy in melanoma. , 2013, European journal of cancer.

[12]  A. Hauschild,et al.  Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial , 2012, The Lancet.

[13]  Kate Owen,et al.  Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation , 2016, eLife.

[14]  Y. Matsuo,et al.  False leukemia–lymphoma cell lines: an update on over 500 cell lines , 2003, Leukemia.

[15]  Dennis C. Friedrich,et al.  MAP kinase pathway alterations in BRAF-mutant melanoma patients with acquired resistance to combined RAF/MEK inhibition. , 2014, Cancer discovery.

[16]  Antoni Ribas,et al.  A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition. , 2014, Cancer discovery.

[17]  Alan Hall,et al.  Rho GTPases: biochemistry and biology. , 2005, Annual review of cell and developmental biology.

[18]  L. Garraway,et al.  C-RAF mutations confer resistance to RAF inhibitors. , 2013, Cancer research.

[19]  A. Hauschild,et al.  Improved survival with vemurafenib in melanoma with BRAF V600E mutation. , 2011, The New England journal of medicine.

[20]  S. Nelson,et al.  Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation , 2010, Nature.

[21]  A. McKenna,et al.  The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma. , 2014, Cancer discovery.

[22]  W. Sellers,et al.  MEK1 mutations confer resistance to MEK and B-RAF inhibition , 2009, Proceedings of the National Academy of Sciences.

[23]  L. Chin,et al.  Melanoma: from mutations to medicine. , 2012, Genes & development.

[24]  Rajiv Narayan,et al.  A melanocyte lineage program confers resistance to MAP kinase pathway inhibition , 2013, Nature.

[25]  J. Utikal,et al.  Improved survival with MEK inhibition in BRAF-mutated melanoma. , 2012, The New England journal of medicine.

[26]  A. McKenna,et al.  The Mutational Landscape of Head and Neck Squamous Cell Carcinoma , 2011, Science.

[27]  Ultan McDermott,et al.  Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. , 2008, Cancer research.

[28]  A. Sivachenko,et al.  A Landscape of Driver Mutations in Melanoma , 2012, Cell.

[29]  M. Kazanietz,et al.  Rac signaling in breast cancer: a tale of GEFs and GAPs. , 2012, Cellular signalling.

[30]  Antoni Ribas,et al.  Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. , 2014, Cancer discovery.

[31]  Matthew J. Davis,et al.  RAC1P29S is a spontaneously activating cancer-associated GTPase , 2013, Proceedings of the National Academy of Sciences.

[32]  S. Larson,et al.  Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation. , 2011, The Journal of clinical investigation.

[33]  V. Sondak,et al.  PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression. , 2011, Cancer research.

[34]  Matthew J. Davis,et al.  Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma , 2012, Nature Genetics.

[35]  Tom Misteli,et al.  RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E) , 2011, Nature.

[36]  A. Bucheit,et al.  Emerging insights into resistance to BRAF inhibitors in melanoma. , 2014, Biochemical pharmacology.

[37]  T. Golub,et al.  Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion , 2012, Nature.