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Purpose To investigate the roles of melanoma-associated macrophages in melanoma resistance to BRAF inhibitors (BRAFi). Experimental Design An in vitro macrophage and melanoma cell co-culture system was used to investigate whether macrophages play a role in melanoma resistance to BRAFi. The effects of macrophages in tumor resistance were examined by proliferation assay, cell death assay and western blot analyses. Furthermore, two mouse preclinical models were used to validate whether targeting macrophages can increase the anti-tumor activity of BRAFi. Finally, the number of macrophages in melanoma tissues was examined by immunohistochemistry. Results We demonstrate that in BRAF mutant melanomas, BRAFi paradoxically activate the MAPK pathway in macrophages to produce VEGF, which reactivates the MAPK pathway and stimulates cell growth in melanoma cells. Blocking the MAPK pathway or VEGF signaling, then reverses macrophage-mediated resistance. Targeting macrophages increases the anti-tumor activity of BRAFi in mouse and human tumor models. The presence of macrophages in melanomas predicts early relapse after therapy. Conclusions Our findings demonstrate that macrophages play a critical role in melanoma resistance to BRAFi, suggesting that targeting macrophages will benefit patients with BRAF mutant melanoma.

An unusually long repeated DNA sequence was identified in cloned DNA, three kb 3' to the human beta-globin gene. Other members of this repeated sequence family were isolated from a human genomic DNA library and characterized by Southern blotting techniques, electron microscopy, and solution hybridization. The copy located next to the beta-globin gene was found to be 6.4 +/- 0.2 kb long and continuous over that length. This repeated sequence family comprises about 1% of the human genome and contains 3000-4800 copies of moderate sequence divergence which are interspersed with other less-highly repeated DNA. The 6.4 kb repeated unit does not appear to be composed of any smaller tandemly repeated subunits, nor is it expressed at a high level in bone marrow cell RNA.