The profound but frequently transient clinical responses to BRAF inhibitor (BRAFi) treatment in melanoma emphasize the need for combinatorial therapies. Multiple clinical trials combining BRAFi and immunotherapy are under way to further enhance therapeutic responses. However, to which extent BRAF inhibition may affect melanoma immunogenicity over time remains largely unknown. To support the development of an optimal treatment protocol, we studied the impact of prolonged BRAFi exposure on the recognition of melanoma cells by T cells in different patient models. We demonstrate that autologous CD8 tumor-infiltrating lymphocytes (TILs) efficiently recognized short-term (3, 7 days) BRAFi-treated melanoma cells but were less responsive towards long-term (14, 21 days) exposed tumor cells. Those long-term BRAFi-treated melanoma cells showed a non-proliferative dedifferentiated phenotype and were less sensitive to four out of five CD8 T cell clones, present in the preexisting TIL repertoire, of which three recognized shared antigens (Tyrosinase, Melan-A and CSPG4) and one being neoantigen-specific. Only a second neoantigen was steadily recognized independent of treatment duration. Notably, in all cases the impaired T cell activation was due to a time-dependent downregulation of their respective target antigens. Moreover, combinatorial treatment of melanoma cells with BRAFi and an inhibitor of its downstream kinase MEK had similar effects on T cell recognition. In summary, MAP kinase inhibitors (MAPKi) strongly alter the tumor antigen expression profile over time, favoring evolution of melanoma variants cross-resistant to both T cells and MAPKi. Our data suggest that simultaneous treatment with MAPKi and immunotherapy could be most effective for tumor elimination.
IntroductionThe BRAFV600 mutation, expressed in approximately 50% of melanomas, mediates constitutive activation of the BRAF-MEK-ERK in the MAPK signalling pathway and therefore tumour proliferation. Rapid and high rate of clinical responses can be achieved by targeting this axis using BRAF V600 inhibitor (BRAFi) as single therapy or the combination of BRAFi and MEKi (BRAFi/MEKi). But still disease progresses in the majority of treated patients due to acquired resistance in tumour cells. Combining targeted therapy with immunotherapy is proposed to improve the long-term outcomes of patients. However, to which extent BRAFi may affect melanoma immunogenicity over time remains largely unknown. To support the development of an optimal treatment protocol, we studied the impact of prolonged BRAFi or BRAFi/MEKi exposure on the recognition of melanoma cells by T cells in different patient models in vitro.Material and methodsT-cell activation by inhibitor-treated autologous tumour cells was determined by IFNγ-ELISPOT/ELISA or intracellular cytokine staining. If available, tumor-infiltrating lymphocytes (TILs) representing the T cell repertoire of the tumor-microenvironment were used in functional assays. Otherwise, tumor-reactive T cells were obtained from the peripheral blood. BRAFi- or BRAFi/MEKi-treated melanoma cells were analysed for the expression of T cell targeted tumour antigens and components in the antigen processing and presentation machinery via qRT-PCR, Western blot, or flow cytometry.Results and discussionsRecurrently, we observed evolution of melanoma cross-resistance to the pre-existing tumor-specific T cell repertoire during prolonged BRAFi treatment in different patient models. While efficiently recognising short-term BRAFi-treated melanoma cells, TILs were less responsive towards long-term exposed tumour cells. Similar results were obtained with four out of five CD8+ T-cell clones due to a time-dependent downregulation of their respective target antigens (three shared antigens, one neoantigen). Only a second neoantigen was steadily recognised independent of treatment duration. Moreover, BRAFi/MEKi treatment recapitulated the evolution of T cell resistance detected under single BRAFi treatment.ConclusionInhibiting the BRAF-MEK-ERK axis in melanoma cells strongly alters the tumour antigen expression profile over time, favouring evolution of melanoma variants cross-resistant to both T cells and MAPKi. Our data suggest that simultaneous treatment with MAPKi and immunotherapy could be most effective for tumour elimination.
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