All mice were maintained in a specific pathogen-free barrier facility at The University of Texas MD Anderson Cancer Center. B6 nude mice were purchased from the Taconic Farms. Furthermore, a subset of responding melanoma patients showed similar changes in the tumor microenvironment following BRAF inhibitor treatment, providing a strong rationale to explore the use of combination treatments involving MAPK pathway inhibition and T cell-based immunotherapy.Ĭ57BL/6, C57BL/6J-Tyr-2J/J albino and pmel-1 TCR transgenic mice on a C57BL/6 background were purchased from the Jackson Laboratory. We found that ACT with melanoma-specific T cells was much more effective in the context of concurrent BRAF inhibition, which led to increased T-cell infiltration of tumors that could be attributed largely to decreased VEGF production by the tumor cells. In the current pre-clinical study, we assessed whether the addition of a selective BRAF(V600E) inhibitor could improve the efficacy of T-cell based immunotherapy in vivo. Perhaps most importantly, the exquisite specificity of recently developed small molecule inhibitors that target mutated oncogenes have shown little or no detrimental effects on immune cells that also utilize the MAPK pathway ( 23, 25). In addition, a study by Jiang et al showed that the paradoxical activation of MAPK promoted programmed death ligand 1 (PD-L1) expression in melanoma cells resistant to BRAF inhibition ( 24). Recent in vitro experiments showed that blocking of MAPK signaling in melanoma cells could increase the expression of melanocyte differentiation antigens (MDA), leading to improved recognition by MDA-specific T cells ( 22, 23). For example, knockdown of BRAF(V600E) in melanoma cell lines has been shown to decrease the production of immunosuppressive soluble factors such as IL-10, vascular endothelial growth factor (VEGF) and IL-6 ( 21). This approach appears particularly promising due to the emerging link between MAPK pathway activation in cancer and the suppression of anti-tumor immunity. Hence, to improve long term clinical responses and avoid selection of drug-resistant tumors, combinational therapies that target multiple pathways have been proposed ( 3, 4, 20).Īlthough therapeutic approaches that combine small molecule-based inhibition of multiple signal transduction pathways has been an area of ongoing investigation, one alternative involves the combination of BRAF inhibitors with immune-based therapies. The mechanisms that cause resistance are diverse, and include MAPK pathway re-activation by alternate means ( 14– 19). However, complete and durable remissions were rarely observed in these patients, and disease relapses accompanied by BRAF inhibitor resistance typically occurred within a year ( 12, 13). Recent clinical trials have demonstrated that over half of melanoma patients with BRAF(V600E)-expressing tumors experience objective clinical responses to selective inhibitors of BRAF. Thus, BRAF(V600E) being so widely expressed, has provided a strong rationale for the development and clinical application of small molecule-based pharmaceutical inhibitors that selectively target BRAF(V600E) to treat patients with metastatic melanoma, whose treatment options are limited ( 8– 11). Although over 50 distinct mutations in BRAF have been described to date, a valine to glutamic acid substitution at amino acid position 600(V600E), is by far the most frequent, comprising more than 70% of BRAF mutations in melanoma ( 1, 7). As a component of the RAS-RAF-MEK-MAPK signal transduction pathway, BRAF is also mutated to a constitutively activated form in many other cancers, including thyroid, colorectal, and hairy cell leukemia ( 1– 6). The identification of activating point mutations of the BRAF gene, present in approximately half of all human cutaneous melanomas, has proven to be a milestone for contributing not only to our understanding of melanoma biology but for changing the treatment and clinical outcomes of the disease ( 1).
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