Impact of IFN-γ resistance & MAPK inhibition on the immune surveillance of malignant melanoma - relevance for immune-based therapies

Abstract

The BRAFV600E mutation, expressed in approximately 50% of melanomas, mediates constitutive activation of the BRAF-MEK-ERK (MAPK) signaling pathway and therefore tumor proliferation. Rapid and high rate of clinical responses can be achieved by applying MAPK inhibitors (MAPKi), such as BRAFi and MEKi as single agents or in combination. Nevertheless, disease progresses in the majority of treated patients due to resistance. Combining targeted therapy with immune checkpoint blockade is proposed to improve the long-term survival of patients. However, to which extent BRAFi may affect melanoma immunogenicity over time remains largely unknown. Moreover, a considerable number of patients does not respond to immune checkpoint blockade pointing to immune escape mechanisms. Since IFN-γ is known to play an essential role in tumor control mediated by CD8+ T cells, which are the anti-tumor effectors in immunomodulating antibody therapy, the first aim of the thesis was to explore mechanisms contributing to insensitivity of melanoma cells towards IFN-γ. The second objective was to investigate the impact of MAPK inhibition on the recognition of human melanoma cells by autologous CD8+ T cells and NK cells, in order to provide mechanistic insights and valuable suggestions concerning treatment strategies for malignant melanoma. To accomplish this, several melanoma patient models consisting of tumor tissue, corresponding cell lines, autologous CD8+ T cells and NK cells were used in the studies. The first article included in this work demonstrates that IFN-γ-resistant melanoma clones can evolve in the course of disease under the selective pressure of an effective anti-tumor immune response in patients receiving different types of immunotherapy. Here, loss of chromosomal material and subsequent inactivating mutations in genes of the type II IFN signaling pathway (e.g. JAK1, JAK2) were found to be causative for the insensitivity of melanoma cells towards IFN-γ-mediated antitumorigenic effects. Moreover, it was demonstrated that IFN-γ resistant cells can further evolve into HLA class I negative populations due to silencing of genes involved in antigen presentation, the re-expression of which could no longer be induced by IFN-γ. This mechanism can lead to melanoma cell escape from CD8+ T cell recognition rendering immunotherapeutic approaches ineffective. These results indicate that screening of tumor lesions for genetic defects in the IFN-γ signaling pathway should be considered in selecting patients for immunotherapy. The two following publications deal with the effects of MAPKi on the recognition of different patient-derived melanoma cell lines by NK cells and T cells. Article II presented in this thesis demonstrates that already after 2-3 days of BRAFi treatment melanoma cell variants evolve, which escape NK cell-mediated recognition by strongly decreasing the surface expression of ligands for NK cell activating receptors. However, ligand downregulation and thus impaired NK cell recognition of BRAFi- treated melanoma cells could be counteracted by simultaneous application of the HDAC inhibitor, sodium butyrate, favoring combination therapy that in addition might combine the direct pro-apoptotic effects of both drugs towards melanoma cells. The third article demonstrates the evolution of melanoma cross-resistance to the pre- existing tumor-specific T cell repertoire during prolonged BRAFi treatment. While efficiently recognizing short-term (3, 7 days) BRAFi-treated melanoma cells, antigen- specific CD8+ T cells were less responsive towards long-term (14, 21 days) exposed tumor cells due to a time-dependent downregulation of target antigens. Thus, MAPKi strongly alters the tumor antigen expression profile over time favoring outgrowth of melanoma variants cross-resistant to both T cells and targeted therapy, suggesting that MAPKi treatment preceding T cell-based immunotherapy might be disadvantageous for melanoma treatment. Therefore, to induce long-lasting benefit and to avoid cross-resistance it seems reasonable to complement targeted therapy by an early sequential or simultaneous immunotherapy, but only if no genetic defects in IFN-γ signaling pathway and/or antigen presentation are present within the tumor.