Abstract 3510: Paradoxical MAPK activation and beneficial effects of vemurafenib on T-cell phenotype resulting in improved functionality in vivo
Authors: Koya Richard C. , Mok Stephen , Otte Nicholas , Chodon Thinle , Comin-Anduix Begonya , Blacketor Kevin , Tumeh Paul C. , Ribas Antoni 
Published: August 2012

Abstract
Adoptive T cell transfer (ACT) based immunotherapy for melanoma can induce remarkable and highly durable tumor responses, which may last many years. Vemurafenib (Vmf) is a potent inhibitor of BRAF mutated at V600E with response rates of up to 80% in patients with metastatic melanoma. However, drug resistance develops in most of patients leading to response durations of only several months. We previously showed that the combined approach of BRAF inhibition with adoptive cell immunotherapy leads to an improved outcome in vivo. Here we show that Vmf also directly and independently affects T lymphocytes, resulting in favorable anti-tumoral phenotypic changes. We had created a transplantable murine melanoma cell-line driven by V600E BRAF oncogene (SM1) derived from a spontaneously arising melanoma in transgenic mice harboring the V600E BRAF mutation under the control of tyrosinase promoter. SM1 cells stably expressing the ovalbumin (OVA) model antigen were implanted in C57BL6 mice. Daily i.p. Vmf combined with ACT of OVA-specific TCR transgenic cells generated by retroviral transduction demonstrated superior tumor control of the combined treatment in comparison to each treatment alone. We also confirmed better outcomes with this combination in the pmel-1 model, which is based on the ACT of TCR transgenic cells against the endogenously expressed and relevant melanoma antigen, gp100. We then cultured primary T cells in the presence of Vmf with a broad range of concentration (0.1 to up to 100 uM). There was no evidence of cytotoxicity, but interestingly, T cells differentiated into a phenotype resembling T central memory (CM) cells (CD44+, CD62L+) in a dose dependent manner as assessed by flow-cytometry. CM T cells were shown to induce superior anti-tumoral responses in comparison to ACT of Effector T cells in murine models. As expected for CM T cells, further analysis of 24 h collection supernatants from in vitro cognate peptide stimulated T cells showed dose-dependent lower interferon-gamma secretion with Vmf as analyzed by ELISA. Pmel-1 T cells were then analyzed by Immunoblotting for phosphorylation status (activation) of protein kinases at 1, 5, 15, 30 min and at 24h after Vmf treatment (concentrations from 1 to 15 uM). Vmf induced increased levels of pERK and pMEK. Co-immunoprecipitation studies and kinase assays further demonstrated a role of C-Raf in this paradoxical activation of the MAPK pathway in T cells induced by Vmf. Furthermore, in vivo studies in C57BL6 mice treated with Vmf daily or vehicle control for 3 weeks demonstrated skewing of CD3+ cells towards a phenotype resembling central memory T cells (CD44+, CD62L+, LY-6C+). Taken all our data together, we provide further support for the rationale of combining BRAF targeted therapy and adoptive T cell immunotherapy and the testing of such combinations in patients with V600E BRAF mutant metastatic melanoma.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3510. doi:1538-7445.AM2012-3510