P58: Pigment epithelia-derived factor drives invasion in NF1-mutant malignant melanoma

Charlotte Lovatt,¹ Megan Williams,¹ Alex Gibbs,¹ Mukhtar Abdullahi,¹ Huw Morgan,¹ Simone Lanfredini,¹ Carlotta Olivero,¹ G. Spurlock,² Sally Davies,² Charlotte Philpott,² Hannah Tovell,² Peter Turnpenny,³ Dilair Baban,⁴ Sam Knight,⁴ Julian Sampson,² Hilda Berm,⁵ Eric Legius,⁵ Meena Upadhyaya² and Girish Patel¹

¹European Cancer Stem Cell Research Institute Cardiff University, Cardiff, UK; ²Division of Cancer and Genetics Cardiff University, Cardiff, UK; ³Royal Devon and Exeter NHS Foundation Trust, Exeter, UK; ⁴The Wellcome Trust Centre for Human Genetics, Oxford, UK; and ⁵Department of Genetics University of Leuven, Leuven, Belgium

Inherited loss of NF1 in neurofibromatosis type 1 is associated with melanocyte proliferation and migration to form café au lait macules (CALMS). More recently, NF1 mutations have been identified as the third most common driver mutation in malignant melanoma (MM). Even alongside the most common MM BRAF mutation, loss-of-function NF1 mutations portend a worse prognosis due to more invasive disease. To understand the molecular basis for CALMS and invasive malignancy a comparative transcriptomic analysis was undertaken of normal vs. CALMS melanocytes from three distinct genodermatoses: classic NF1 loss (CALMS and internal malignancy, n = 3), three base-pair loss (CALMS but no internal malignancy, n = 3) and Legius syndrome (CALMS but no internal malignancy, n = 1). Based on our findings, we examined the protective effect of pigment epithelia-derived factor (PEDF) on MM cellular models as part of an international consortia, melanocyte culture, microarray, bioinformatic analysis and loss of heterozygosity single-nucleotide polymorphism analysis. Validation of findings by reverse-transcriptase polymerase chain reaction, NF1 knockdown with small interfering RNA and PEDF overexpression in normal melanocytes and characterized MM cell lines was carried out. Functional studies included cell proliferation, migrations and invasion. CALMS melanocytes uniformly demonstrated loss of heterozygosity. Bioinformatic analysis of CALM vs. within-patient normal melanocytes confirmed not only activation of the RAS–mitogen-activated protein kinase (MAPK) pathway in keeping with these RASopathies, but also identified PEDF as uniformly discordantly downregulated. NF1 knockdown in normal melanocytes and MM (including NF1 driver mutant and BRAF co-mutant) cells, led to increased proliferation, migration and invasion, which was reversed in PEDF co-transduced cells. The phosphoinositide 3 kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) pathway was uniquely activated in classic neurofibromatosis CALMS only, suggesting that this oncogenic pathway is associated with the tumour-generating phenotype. Melanocytes in CALMS arise from loss of heterozygosity associated with activation of the RAS–MAPK and PI3K–AKT–mTOR pathway in the case of classic neurofibromatosis. Somatic mutations in MM similarly drive these pathways leading to discordant downregulation of PEDF that in turn leads MM invasiveness. Thus, PEDF represents a novel therapy for MM with NF1 mutation.