Differential and Inducible Expression of Immune-Regulatory Molecules in Melanoma Cells: A Case for Combination Therapy

Introduction

Metastatic melanoma is responsible for the highest number of skin cancer-related deaths in the USA. Checkpoint inhibitor therapy using anti-CTLA-4 and anti-PD-1, individually and in combination, has resulted in a significant partial and complete remission rate in melanoma patients. Various checkpoint molecules, including include CTLA-4, PD-1, HVEM, VISTA, 41-BB, OX-40 and CD226 are expressed not only on T cells, antigen presenting cells, and tumor cells, but also in other body tissues, indicating potential and actual cross reactivity of these molecules when used as clinical targets. The role and expression of these immunomodulating targets is critical for proper immunotherapeutic application and remains to be elucidated.

Materials and methods

We isolated and characterized five primary patient derived melanoma cell lines: MEL-2, MEL-V, 3MM, KFM and GLM2 and screened these cells for the expression of a comprehensive panel of twenty-five co-stimulatory and co-inhibitory molecules by RT-PCR. Cells were analyzed under control conditions, as well as in the presence of the BRAF^V600E inhibitor, vemurafenib (PLX4032), and specific cytokines.

Results and discussion

Under standard conditions, we identified significant heterogeneity in the expression of immunomodulatory molecules by the various melanoma cell lines compared to normal adult melanocytes. Significantly, inhibitory molecules including PD-1, VISTA and LAIR1 and stimulatory molecules including 4-1BB, HVEM and ICOS were differentially regulated in these cell lines by metabolic stress brought on by starvation conditions. Some of these molecules were restored to basal levels of expression following 24 hour treatment with 10μM vemurafenib. However, the treatment led to concurrent upregulation of molecules such as LAG3, BTLA, CD226 and TIM1, suggesting a compensatory mechanism that could aid melanoma cell adaptation and escape from immune recognition/destruction. Exposing melanoma cells to classical, activated-dendritic cell cytokines, IL-6 and IL-12, also led to a differential expression of these molecules correlating with unique stimulatory and inhibitory molecule expression profile of each primary cell line.

Conclusion

Our results underscore the importance of understanding the profile of co-stimulatory and co-inhibitory molecules expressed on tumor cells. With eighty percent of melanoma patients exhibiting the BRAF^V600E mutation, we make a case for designing a combinatorial therapeutic regimen, with targeted immunotherapies as well as targeting specific genetic lesions with small molecule inhibitors.

Acknowledgement - This work was funded by a grant from the Empire Clinical Research Investigator Program to Metropolitan Hospital - Dr. Raj Tiwari, PI