BACKGROUND: Targeted therapies have improved outcomes in oncogene-driven melanoma, but resistance invariably occurs. Drug resistance can be preceded by drug-tolerant persisters cells (DTPs) which share characteristics with cancer stem cells (CSC) that allow them to enter a dormant state under drug treatment, eventually resulting in stable genetic resistance. As the anti-parasitic ivermectin has been shown to target CSCs, we explored whether it also targets DTPs.
RESULTS: To generate DTPs, BRAF-mutant A375 cells were treated with 10 µM vemurafenib. To investigate the ability of ivermectin to inhibit DTPs, cells were treated with a combination of ivermectin and vemurafenib for 3- and 7-days. The combination led to a greater inhibition of cell proliferation compared to vemurafenib alone, and fewer surviving DTPs. Adding ivermectin after DTP induction also reduced cell survival , suggesting an ability to target already tolerant cells. In melanoma, DTPs consist of subpopulations with distinct transcriptional profiles, including starved melanoma-like cell (SMC) and neural crest stem cell-like (NCSC) states. Consistent with previous reports, NCSC markers NGFR, GFRA2 and SLITRK6 and the SMC marker CD36 were significantly upregulated in DTPs surviving vemurafenib treatment. NGFR, GFRA2 and SLITRK6 were downregulated (82-fold, 327-fold and 14-fold) with combination treatment at Day 3, and further downregulated at Day 7 compared to vemurafenib alone. CD36 was also downregulated with combination treatment (2-fold) at Day 7. The stem cell marker SOX-2 showed downregulation similar to NCSC markers. Expression of NGFR and CD36 proteins at the cell surface was also upregulated in DTPs following vemurafenib treatment, but downregulated after treatment with the combination. To investigate whether ivermectin can inhibit DTP emergence or evolution during long-term drug treatment, cells were treated with either vemurafenib alone or the combination for 12 weeks and assessed weekly using the Phenix High Content Imaging System. Over the 12-week period, vemurafenib-treated cells resumed growth under treatment whereas cells treated with the combination failed to proliferate.
CONCLUSION: These results suggest that the combination of ivermectin and vemurafenib could be an effective strategy to eliminate DTPs. By inhibiting DTPs, combination treatment may help prevent progression to stable resistance, ultimately enhancing oncogene-driven melanoma outcomes.