Abstract 1015: Dual BRAF and VEGFA targeting in melanoma elicits antitumor immune response that is enhanced by PD-1 blockade
Authors: Comunanza Valentina , Martin Valentina , Doronzo Gabriella , Nicolantonio Federica Di , Sangiolo Dario , Bussolino Federico 
Published: August 2018

Abstract
The introduction of BRAF inhibitors (BRAFi) has improved response rate and overall survival of metastatic melanoma patients compared to standard chemotherapy. However, acquired drug resistance occurs in nearly all patients. The comprehension of cellular and molecular mechanisms underlying BRAF inhibitor resistance could help to identify novel actionable pathways in the treatment of BRAF dependent tumors.VEGFA is an attractive target for combinatorial cancer therapy and we have recently demonstrated that targeting VEGFA in melanoma and CRC xenografts could enhance the antitumor effect of BRAFi by normalizing the tumor vasculature, recruiting M1 macrophages and inducing a remodeling of the extracellular matrix characterized by a reduction in collagen I and in cancer-associated fibroblasts. While the previous proof of concept was obtained within an immunodeficient model, here we investigated the therapeutic effect of VEGFA targeting in association with PLX4720 (BRAFi) in a dedicated immunocompetent model. D4M cells, a BRAFV600E-mutant melanoma murine cell line, were subcutaneously injected in syngeneic C57BL/6J mice. We demonstrated that the association of BRAFi with DC101 (antibody anti VEGFR2) had a weak activity while we observed a synergistic antitumor effect when combined with B20 (murine anti-VEGFA neutralizing antibody). Although targeted inhibition of either BRAF or VEGFA delayed tumor growth, only combined inhibition of both pathways could induce regression of initial tumor size, with an evident apoptotic effect, and delayed the onset of acquired resistance to BRAF target therapy. Since VEGFA has a well characterized tumor immune-suppressive role, we further investigated whether contrasting VEGFA along with simultaneous BRAF inhibition could modulate both innate and adaptive immunity. Both flow cytometry and immunofluorescence analysis of tumors demonstrated that the combinatorial regimen activates the host immune system, inducing the tumor infiltration by cytotoxic CD8+ lymphocytes, macrophages with tumor suppressive features and NK cells. Moreover, the association between BRAF targeting and VEGFA removal reduced the number of circulating CD11b+Ly6ClowLy6G+ polymorphonuclear MDSCs (PMN-MDSCs). Based on this observation, we hypothesized that the therapeutic effect obtained by the simultaneous VEGFA blockade and BRAFi could be further exploited as a favorable platform for the association with immune-checkpoint inhibitor targeting PD-1. Although the addition of anti-PD-1 antibody improved the antitumor activity of either PLX4720 or B20, we observed striking tumor volume regression only when combining PD-1 blockade with dual BRAF and VEGFA inhibition. Our results offer the rationale for novel combinatorial approaches including co-targeting of signaling molecules, tumor angiogenesis and immune system.
Citation Format: Valentina Comunanza, Valentina Martin, Gabriella Doronzo, Federica Di Nicolantonio, Dario Sangiolo, Federico Bussolino. Dual BRAF and VEGFA targeting in melanoma elicits antitumor immune response that is enhanced by PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1015.