Nevospheres from neurocutaneous melanocytosis cells show reduced viability when treated with specific inhibitors of NRAS signaling pathway.
BACKGROUND
Neurocutaneous melanocytosis (NCM) is characterized by clonal nevomelanocytic proliferations in the CNS and skin. Given the scarcity of effective therapeutic targets, testing new drugs requires a reliable and reproducible in vitro cellular model of the disease.


METHODS
We generated nevomelanocytic spheroids in vitro from lesions of the spinal cord, brain, and skin from 4 NCM patients. Nevomelanocytic cells were grown as monolayers or spheroids and their growth characteristics were evaluated. Cultured cell identity was confirmed by demonstration of the same NRAS mutation found in the original lesions and by immunophenotyping. Nevomelanocytic spheroids were treated with inhibitors of specific mediators of the NRAS signaling pathway (vemurafenib, MEK162, GDC0941, and GSK2126458). Drug sensitivity and cell viability were assessed.


RESULTS
Cultured cells were growth-factor dependent, grew as spheroids on Geltrex matrix, and maintained their clonogenicity in vitro over passages. Skin-derived cells formed more colonies than CNS-derived cells. Inhibitors of specific mediators of the NRAS signaling pathway reduced viability of NRAS mutated cells. The highest effect was obtained with GSK2126458, showing a viability reduction below 50%.


CONCLUSIONS
NRAS mutated cells derived from clinical NCM samples are capable of continuous growth as spheroid colonies in vitro and retain their genetic identity. Drugs targeting the NRAS signaling pathway reduce in vitro viability of NCM cells. NCM lesional spheroids represent a new and reliable experimental model of NCM for use in drug testing and mechanistic studies.
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Dipanjan
D
Basu
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Cláudia M
CM
Salgado
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Bruce S
BS
Bauer
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Donald
D
Johnson
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Veronica
V
Rundell
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Marina
M
Nikiforova
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Yasmin
Y
Khakoo
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Lorelei J
LJ
Gunwaldt
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Ashok
A
Panigrahy
Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (D.B., C.M.S., M.R.M.); Department of Plastic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (L.J.G.); Department of Radiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania (A.P.); Division of Plastic and Reconstructive Surgery, NorthShore University HealthSystem, Northbrook, Illinois (B.S.B., D.J., V.R.); Division of Molecular Genomic Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (M.N.); Department of Pediatrics and Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (Y.K.); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Y.K.).
Miguel
M
Reyes-Múgica
D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles / Q000494:pharmacology*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D049109:Cell Proliferation / Q000187:drug effects; D002648:Child; D002675:Child, Preschool; D005455:Fluorescent Antibody Technique; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007223:Infant; D008297:Male; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011446:Prospective Studies; D015398:Signal Transduction / Q000187:drug effects; D012878:Skin Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D018874:Spheroids, Cellular / Q000187:drug effects / Q000473:pathology*; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D008565:Membrane Proteins; C581313:binimetinib; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human
NRAS; giant congenital melanocytic nevi; neurocutaneous melanocytosis; targeted NRAS therapy; tumor spheroids