The NF1 somatic mutational landscape in sporadic human cancers

Abstract Background Neurofibromatosis type 1 (NF1: Online Mendelian Inheritance in Man (OMIM) #162200) is an autosomal dominantly inherited tumour predisposition syndrome. Heritable constitutional mutations in the NF1 gene result in dysregulation of the RAS/MAPK pathway and are causative of NF1. The major known function of the NF1 gene product neurofibromin is to downregulate RAS. NF1 exhibits variable clinical expression and is characterized by benign cutaneous lesions including neurofibromas and café-au-lait macules, as well as a predisposition to various types of malignancy, such as breast cancer and leukaemia. However, acquired somatic mutations in NF1 are also found in a wide variety of malignant neoplasms that are not associated with NF1. Main body Capitalizing upon the availability of next-generation sequencing data from cancer genomes and exomes, we review current knowledge of somatic NF1 mutations in a wide variety of tumours occurring at a number of different sites: breast, colorectum, urothelium, lung, ovary, skin, brain and neuroendocrine tissues, as well as leukaemias, in an attempt to understand their broader role and significance, and with a view ultimately to exploiting this in a diagnostic and therapeutic context. Conclusion As neurofibromin activity is a key to regulating the RAS/MAPK pathway, NF1 mutations are important in the acquisition of drug resistance, to BRAF, EGFR inhibitors, tamoxifen and retinoic acid in melanoma, lung and breast cancers and neuroblastoma. Other curiosities are observed, such as a high rate of somatic NF1 mutation in cutaneous melanoma, lung cancer, ovarian carcinoma and glioblastoma which are not usually associated with neurofibromatosis type 1. Somatic NF1 mutations may be critical drivers in multiple cancers. The mutational landscape of somatic NF1 mutations should provide novel insights into our understanding of the pathophysiology of cancer. The identification of high frequency of somatic NF1 mutations in sporadic tumours indicates that neurofibromin is likely to play a critical role in development, far beyond that evident in the tumour predisposition syndrome NF1

Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader

Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC₅₀ of 3–10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (<i>D</i>_max of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function

Pigment epithelium derived factor drives melanocyte proliferation and migration in neurofibromatosis café au lait macules

Background: RASopathies, which include neurofibromatosis type 1 (NF1), are defined by Ras/mitogen‐activated protein kinase (Ras/MAPK) pathway activation. They represent a group of clinically related disorders often characterised by multiple Café au Lait Macules (CALMs). Objectives: To determine, using in depth transcriptomic analysis of NF1 melanocytes from CALM and unaffected skin, (1) the gene(s) responsible for melanocyte proliferation and migration, and (2) the activated signalling pathway(s) in NF1 melanoma. Methods: Classical NF1 (n = 2, who develop tumours) and 3bp deletion NF1 (p. Met992del, who do not develop tumours) (n = 3) patients underwent skin biopsies from CALM and unaffected skin. Melanocytes were isolated and propagated, with five replicates from each tissue sample. DNA and RNA were extracted for mutational analysis and transcriptomic profiling with six replicates per sample. Mechanistic determination was undertaken using melanocyte and melanoma cell lines. Results: All CALMs in NF1 were associated with biallelic NF1 loss, resulting in amplification of Ras/MAPK and Wnt pathway signalling. CALMs were also associated with reduced SERPINF1 gene expression (and pigment epithelium‐derived factor (PEDF) levels, the reciprocal protein), a known downstream target of the master regulator of melanocyte differentiation microphthalmia‐associated transcription factor (MITF), leading to increased melanocyte proliferation, migration and invasion. In classical NF1 and melanoma, but not 3bp deletion NF1, there was also activation of the PI3K/AKT pathway. Pigment epithelium‐derived factor was found to reduce cell proliferation and invasion of NF1 melanoma. Conclusions: Melanocyte proliferation and migration leading to CALMs in NF1 arises from biallelic NF1 loss, resulting in RAS/MAPK pathway activation, and reduced expression of the tumour suppressor PEDF. Activation of the PI3K/AKT pathway in classical NF1 and NF1 melanoma may facilitate tumour growth