Production of melanin pigment is the archetypal readout of differentiation in normal melanocytes. In histological assessments of melanomas, which arise from melanocytes, we found a high degree of intratumoral heterogeneity (ITH) for pigment production, even in overtly pigmented tumors. Using a novel flow cytometry-based approach to prospectively separate cells according to pigment content from pigmented patient melanomas and cell lines, we found that low-pigment cells are abundant and have enhanced clonogenic potential in vitroand tumorigenic potential in vivo, compared to high-pigment cells. Moreover, low- but not high-pigment cells generated progeny that recapitulated the pigment heterogeneity of the original tumor or cell line.
Transcriptome profiling of low- and high-pigment cell subsets suggested drivers of the heritable functional distinctions between them. By gene ontology and Gene Set Enrichment Analysis, high-pigment cells showed increased expression of markers of melanocytic differentiation and increased oxidative phosphorylation. In contrast, low-pigment cells displayed gene expression patterns associated with ribosome biogenesis and upregulation of Myc-driven transcription. Treatment of heterogeneously pigmented cell lines with the RNA Polymerase Iinhibitor CX5461, which inhibits ribosome biogenesis, increased pigmentation and senescence markers while markedly decreasing clonogenicity.
Our data are inconsistent with proposed plastic relationships in melanoma between high- and low-pigment melanoma cells. Rather, we find evidence in pigmented melanomas of relatively flat cellular hierarchies, with low-pigment cells at a typically broad apex of the hierarchy that both self-renew as well as generate high-pigment cells that are irreversibly non-clonogenic. We also find that this may be exploited therapeutically by inhibiting mechanisms such as ribosome biogenesis that apparently sustain the highly clonogenic, low-pigment state.