Melanoma Differentiation Trajectories Determine Sensitivity toward Pre-Existing CD8+ Tumor-Infiltrating Lymphocytes.

The highly plastic nature of melanoma enables its transition among diverse cell states to survive hostile conditions. However, the interplay between specific tumor cell states and intratumoral T cells remains poorly defined. With MAPK inhibitor‒treated BRAFV600-mutant tumors as models, we linked human melanoma state transition to CD8+ T cell responses. Repeatedly, we observed that isogenic melanoma cells could evolve along distinct differentiation trajectories on single BRAF inhibitor (BRAFi) treatment or dual BRAFi/MEKi treatment, resulting in BRAFi‒induced hyperdifferentiated and BRAFi/MEKi‒induced dedifferentiated resistant subtypes. Taking advantage of patient-derived autologous CD8+ tumor-infiltrating lymphocytes (TILs), we demonstrate that progressive melanoma cell state transition profoundly affects TIL function. Tumor cells along the hyperdifferentiation trajectory continuously gained sensitivity toward tumor-reactive CD8+ TILs, whereas those in the dedifferentiation trajectory acquired T cell resistance in part owing to the loss of differentiation antigens. Overall, our data reveal the tight connection of MAPKi‒induced temporary (drug-tolerant transition state) and stable (resistant state) phenotype alterations with T cell function and further broaden the current knowledge on melanoma plasticity in terms of sculpting local antitumor immune responses, with implications for guiding the optimal combination of targeted therapy and immunotherapy.

Evolution of melanoma cross-resistance to CD8+ T cells and MAPK inhibition in the course of BRAFi treatment.

The profound but frequently transient clinical responses to BRAFV600 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 BRAFV600 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.