Feed-forward Cysteine Regulation Maintains Melanoma Differentiation State and Limits Metastatic Spread

The inherent ability of melanoma cells to alter the differentiation-associated transcriptional repertoire to evade treatment and facilitate metastatic spread is well accepted and has been termed phenotypic switching. However, how these facets of cellular behavior are controlled remains largely elusive. Here we show that cysteine availability, whether from lysosomes (CTNS-dependent) or exogenously derived (SLC7A11-dependent or as N-acetylcysteine), controls melanoma differentiation-associated pathways by acting on the melanocyte master regulator MITF. Functional data indicate that low cysteine availability reduces MITF levels and impairs lysosome functions, which affects tumor ferroptosis sensitivity but improves metastatic spread in vivo. Mechanistically, cysteine-restrictive conditions reduce acetyl-CoA levels to decrease p300-mediated H3K27 acetylation at the melanocyte-restricted MITF promoter, thus forming a cysteine feed-forward regulation that controls MITF levels and downstream lysosome functions. These findings collectively suggest that cysteine homeostasis governs melanoma differentiation by maintaining MITF levels and lysosome functions, which protect against ferroptosis and limit metastatic spread. To compare the gene expression profiling between lysohigh and lysolow cells, G361 lysohigh and lysolow cells grown in DMEM growth media reaching around 80 % confluence were used for RNA extraction and RNA was isolated using TRIzol reagent. Following RNA quality assessment, library preparation,RNA sequencing, raw sequencing reads were mapped to GRCh38.90 using HiSat2.168 and quantitated based on genes as counts per million reads (CPM) using SeqMonk v48.2. Differential gene expression across each of the two states Lysohigh and Lysolow were calculated as average Log2fold (L2F) changes with significance (p) based on unpaired two-sided students’ t-test