Abstract PL03-03: The rational combination of BRAF inhibition with immunotherapy for the treatment of metastatic melanoma
Authors: Lizée Gregory , Liu Chengwen , Hwu Patrick 
Published: August 2012

Abstract
Patients with metastatic melanoma have a median survival of less than one year and new treatments are clearly needed. Targeted therapy with BRAF inhibitors (BRAFi) in patients whose tumors contain mutated BRAF has been promising with response rates of over 50%. However, the responses are transient with the median duration of response under 7 months. In contrast, immunotherapies such as interleukin-2 or anti-CTLA-4 can induce long term survival in some patients, but the overall response rates are low. The combination of targeted therapies with immunotherapies has the potential to result in higher, more durable clinical responses and improved survival in patients with metastatic cancer. Besides inducing the release of antigens following tumor destruction, targeted therapies may also reverse the immunosuppressive microenvironment found at the tumor site.
Rationally combining immunotherapy with targeted therapy requires in-depth knowledge of the effects of the targeted therapies on immune effector cells and the immune tumor microenvironment. We have taken 3 appoaches in defining the interaction of mutated BRAF and BRAF inhibitors with the immune system by studying: 1) the direct effects of mutated BRAF on the tumor immune microenvironment 2) the in vivo consequences of BRAF inhibition on anti-tumor T-cell responses in murine models and 3) T-cell function in patients receiving BRAF inhibitors.
To determine the role of mutated BRAF on the tumor microenvironment, we performed RNA analysis of melanocytes expressing control, wild type BRAF or mutated BRAF genes. This revealed the preferential upregulation of several immune molecules in cells expressing mutated BRAF, including IL-1. In turn, we found that IL-1 could induce the expression of immune inhibitory molecules on tumor-associated fibroblasts and myeloid cells such as PDL1 (program death ligand-1). Furthermore, fibroblasts exposed to IL-1 were capable of inhibiting T-cell function in vitro. Inhibition of the MAPK pathway using a BRAF-specific inhibitor resulted in the reversal of these immune inhibitory effects.
We next evaluated the effects of BRAF inhibition on T-cell migration to the tumor site. Mice bearing a BRAF mutated tumor were treated with antigen-specific T-cells, a BRAF inhibitor or a combination of the two agents. Tumor regression was highest in mice receiving both T-cells and the BRAF inhibitor. T-cells were monitored in vivo using a sensitive modified luciferase gene, and were found to migrate more efficiently to the tumor following addition of the BRAF inhibitor. This was partially due to the downregulation of VEGF expression by BRAF inhibition, since tumors which constitutively expressed VEGF driven by a viral promoter failed to demonstrate enhanced T-cell migration following addition of the BRAF inhibitor in vivo. In addition, anti-VEGF therapy also resulted in enhanced migration of T-cells to the tumor site. We are currently investigating the mechanisms by which VEGF inhibits T-cell migration to tumor.
Finally, we have investigated the effects of BRAF inhibitors on T-cell function in patients. Peripheral blood mononuclear cells were isolated from patients before and after initiation of therapy with a BRAF inhibitor. Functional T-cell assays were performed to assess the amount of interferon gamma produced following stimulation with class I and class II restricted pools of peptides from recall antigens, in order to assess the function of CD8+ and CD4+ T-cells, respectively. We found that therapy with the BRAF inhibitor did not adversely affect T-cell function in these patients with metastatic melanoma. Luminex assays were performed from sera following therapy with the BRAF inhibitor to evaluate the expression of multiple cytokines. No changes in circulating cytokines were observed following therapy with BRAFi, except for a slight elevation in circulating TNF-alpha levels.
In summary, we have found that: 1) mutated BRAF induces an immunosuppressive tumor microenvironment which can be reversed by BRAFi; 2) BRAFi enhances the migration of T-cells to tumor; and 3) therapy with BRAFi does not inhibit immune function in patients with metastatic melanoma. This work further underscores the potential synergy in combining BRAFi with immunotherapies.
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 PL03-03. doi:1538-7445.AM2012-PL03-03