Targeting CD20-expressing malignant melanoma cells augments BRAF inhibitor killing

Melanoma is a common skin cancer of pigment cells that can be fatal. We developed a method for growing melanoma cancer cells in three-dimensional culture to recreate the melanoma cancer cell-cell interactions, leading to the identification of a CD20+ melanoma cancer stem cell population, as previously postulated. This population persists after conventional treatment targeting the commonest mutation in melanoma, which could be responsible for the inevitable relapse seen in patients. Herein, we show that combining BRAF-targeting therapy with currently available anti-CD20 antibody treatment greatly increased melanoma cell killing, eliminating the CD20+ melanoma cancer stem cell population. This study provides novel insights into melanoma biology that could significantly improve clinical outcomes for patients with melanoma. Background Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however, high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. As MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesized the presence of an intrinsically resistant MM cell subpopulation.Objectives To identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and possibly responsible for MM recurrence.Methods Using melanoma cell lines, we defined culture conditions for the reproducible three-dimensional growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterize cell populations between adherent and nonadherent culture, and cells expressing or not expressing CD20. Furthermore, we defined an in vitro assay to evaluate the killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20.Results We described the culture conditions that promote MM cells to form melanospheres with a reproducible colony-forming efficiency rate of 0.3-1.3%. RNA sequencing of melanosphere vs. conventional MM cell cultures (n = 6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy and, consistent with this finding, demonstrated a Forkhead box protein M1 transcriptomic profile (n = 6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+ BRAF mutant MM cells.Conclusions In patients with MM that harbour a CD20+ subpopulation, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and prevent disease recurrence.