Journal: Cancer Research

Article Title: Blocking Nitrosylation Induces Immunogenic Cell Death by Sensitizing NRAS -Mutant Melanoma to MEK Inhibitors

doi: 10.1158/0008-5472.CAN-24-0693

Figure Lengend Snippet: NOS inhibition enhances MEKi-mediated ERK dephosphorylation. A, Phosphorylation status of 43 kinases in YUGASP cells treated with NOSi, 2 nmol/L Tr, and NOSi + Tr sequential combination. Selected kinases implicated in the MEK-ERK signaling pathway in melanoma are shown. B, Representative Western blots showing ERK dephosphorylation upon NOS and MEK inhibition. YUDOSO and YUGASP cells were treated with NOSi for 48 hours, followed by Tr for various time points (1, 6, and 24 hours). Although Tr alone dephosphorylated ERK, NOSi + Tr enhances ERK dephosphorylation for up to 24 hours. WM1366 cells demonstrated similar results for up to 72 hours (Supplementary Fig. S2A–S2C). C, pERK signal quantification of B , normalized to vinculin and then to untreated control. D and E, Expression ( D ) and relative quantification ( E ) of pERK and downstream oncogenic targets in WM1366 cells treated with NOSi, Tr, or both. F, IHC staining of pERK and 14-3-3 proteins in NRAS -mutant melanoma tumors treated with NOSi, Tr, or both (from ). Scale bar, 200 µm. G, Relative quantification of pERK and 14-3-3 in >10 images from n > 3 mice/group from F . Each dot represents one image. H–J, Orthogonal validation of NOS modulation in MEKi sensitization. H, Stable NOS2 (iNOS) overexpression using a tet-inducible lentiviral pLIX403-hNOS2 vector enhanced the survival of WM1366 NRAS -mutant melanoma cells in a doxycycline dose–dependent manner. Tr IC 50 values are indicated in parentheses. I and J, CRISPR-mediated NOS1 ( nNOS) and NOS2 ( iNOS ) knockdown, using two individual gRNAs (gRNA1 and gRNA2) and their combination (gRNA1 + gRNA2). Nontargeting scramble control (NTC) gRNA was used as a negative control. NOS1 and NOS2 knockdown reduced the survival of WM1366 cells and sensitized them to MEKi (Tr; IC 50 in parentheses). K, Enhanced pERK levels and the expression of several ERK targets in response to NOS2 overexpression induced at 24 hours after doxycycline treatment. L, Relative quantification of K normalized to GAPDH. M and N, Immunoblots showing the ERK phosphorylation and the expression status of representative ERK targets upon nNOS ( M ) and iNOS ( N ) knockdown. O, Signal intensities relative to GAPDH of M and N . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant; two-way ANOVA. AU, arbitrary units.

Article Snippet: For orthogonal assessments, NOS2 ( iNOS ) was exogenously overexpressed using the pLIX403-hNOS2 lentiviral vector (110800, Addgene), which employs a tetracycline-inducible system.

Techniques: Inhibition, De-Phosphorylation Assay, Phospho-proteomics, Western Blot, Control, Expressing, Quantitative Proteomics, Immunohistochemistry, Mutagenesis, Biomarker Discovery, Over Expression, Plasmid Preparation, CRISPR, Knockdown, Negative Control