Novel Immune Targets in Melanoma by Profiling of Co-Activators and Co-Receptors

Author Type(s)

Faculty

Document Type

Abstract

Publication Date

7-1-2021

Journal Title

Cancer Research

Department

Surgery

Second Department

Pathology, Microbiology and Immunology

Abstract

Melanoma, the most aggressive form of skin cancer, is still a challenge despite the advent of recent immunotherapies using checkpoint inhibitors. The presence of checkpoint molecules on immune cells serve to regulate optimal immune responses, however, when present on tumor cells, they serve as immune evasion molecules. As such, this presents challenges to the specific inhibitors being used in relation to dose, side effects, and specificity. We hypothesize that tumor cell profiling of co-activators and co-inhibitors will serve as an important step in the identification and use of specific checkpoint inhibitors in personalized medicine. To this end, we used five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 as a model and screened them for the expression of a comprehensive list of twenty-nine co-stimulatory and co-inhibitory molecules. We compared the differentially expressed molecules on tumor cells with the expression of the co-stimulatory and co-inhibitors on normal non-transformed adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. The expression of these molecules by immunohistochemistry in patient melanoma issues provided additional validation. Analysis of protein expression in various cancers from the Human protein atlas was conducted so as to evaluate the expression of these immune regulators across cancers. HVEM expression in melanoma was high. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient-derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. Since BRAFV600E is the most common mutation, and some response to small molecule inhibitors has been clinically verified, we undertook an analysis of the expression of immune regulators with overall survival using the cancer genome atlas. We also evaluated the interferon regulatory factor binding site so as to molecularly validate that expression of these molecules is dependent on secreted cytokine in the tumor microenvironment. We conclude that the BTLA-HVEM axis is a significant novel target in melanoma that can be used in combinatorial therapy with small molecule inhibitors of cell survival. Our results advocate the need for profiling of immune modulators on tumor cells prior to immunotherapy.

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