DX400 murine PD-1 antibody was obtained through an MTA with Merck. dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and MHC expression, and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested combination of dabrafenib, trametinib with anti-PD1 therapy in SM1 tumors, Framycetin and observed superior anti-tumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAFmutant metastatic melanoma. Introduction The recent breakthroughs brought by the clinical use of immune checkpoint inhibition in cancer provide an exciting promise of long-term responses in clinically significant numbers of patients (1-5). Strategies to extend this low frequency event to the majority of patients have become the focus of cancer immunotherapy research. In BRAF mutant melanoma, the combination of Framycetin BRAF inhibitors and immunotherapy has been tested in both preclinical models and clinical trials (6-9). This is based on the targeting of the BRAFV600E driver mutation, present in approximately 50% of metastatic melanomas, and the immunosensitization effects of BRAF inhibitors through increased antigen presentation (10-12), antigen-specific T cell recognition(10, 13), homing of immune effector cell to the tumors (12, 14, 15) and improved T cell effector functions(6, 16). However, the benefit of this combination in preclinical models has been modest (6-9), while substantial liver toxicity was observed in the first clinical trial combining the BRAF inhibitor vemurafenib and the CTLA4 blocking antibody ipilimumab (17). Both the improved effector function and the toxicities were attributed to the paradoxical activation of the MAPK pathway by vemurafenib in BRAF wild type cells (18). MEK inhibitors, on the other hand, can potentiate the antitumor effects in the melanoma cells (19) and reduce toxicity associated with BRAF inhibitors (18), given their ability to inhibit MAPK signaling in cells with and without a BRAF mutation (20). In addition, MEK inhibitors have exhibited potential of immunosensitization by up-regulation of tumor antigen expression and presentation (10, 21), serving as a rational addition to the BRAF inhibitor and immunotherapy combination. However, there is theoretical concern that a MEK inhibitor could dampen immune effector functions, given that studies have shown impaired T cell proliferation and functions with MEK inhibition (10, 22). Alternatively, when combining with BRAF inhibitors, MEK inhibitors might balance the potential overreacting effector cells to avoid exhaustion, and improve the tumor Framycetin microenvironment by influencing the cytokine production and immune suppressive cell populations in the tumor microenvironment (20). Using a syngeneic BRAFV600E mutant melanoma mouse model (6), we tested the hypothesis that this addition of Rabbit polyclonal to AnnexinA10 a MEK inhibitor would enhance the immunosensitization effects of BRAF inhibition, with increased antitumor activity and decreased toxicity. Results Enhanced antitumor activity with pmel-1 adoptive cell transfer (ACT), dabrafenib and/or trametinib We derived a BRAFV600E mutant murine melanoma Framycetin SM1, syngeneic to fully immune-competent C57BL/6 mice, from a spontaneously arising melanoma in BRAFV600E transgenic mice (6). Besides the presence of the BRAFV600E transversion, SM1 also has CDKN2A gene deletion and BRAF and MITF gene amplification, and is only moderately sensitive to vemurafenib (6). In this study, we first confirmed the downstream MAPK pathway inhibition of SM1 after treatment with dabrafenib, trametinib, or the combination by down-regulated phosphorylated ERK (Fig. 1A). To further explore the drug effects on effector T cells, we treated gp10025-33-activated pmel-1 mouse splenocytes with serial dilutions of dabrafenib, trametinib, or dabrafenib plus trametinib. Western blot analysis at 24 hours of treatment showed paradoxical activation of the MAPK pathway with dabrafenib alone at medium and high concentrations, evidenced by increased phosphor-ERK (Fig S1A). Trametinib alone or with dabrafenib blocked the MAPK pathway even at low doses. However, cell viability (MTS) assay with concentration up to dabrafenib 40M, and trametinib 2M did not show any decreased cell viability at 72 hours (Fig S1B). Open in a separate windows Fig. 1 Enhanced antitumor activity with pmel-1 adoptive cell transfer (ACT) plus dabrafenib (D) and/or trametinib (T)(A) Western blot analysis of MAPK pathway. SM1 cells were treated with serial dilutions of D, T, or D+T for 1 and 24 hours..
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- The authors wish to acknowledge The Childrens Memorial Health Institute, Warsaw, Poland for the access to Q-TOF LC/MS; mass spectrometer purchase was supported by European POIG
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