Date of Award

4-11-2019

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Basic Medical Sciences

First Advisor

Dr. Raj K. Tiwari

Abstract

Melanoma is the most aggressive form of skin cancer with an estimated 96,480 newly diagnosed cases in the United States of America in 2019. It has the highest mutational load, many of them occurring in key driver pathways, most commonly the BRAFV600E in the Mitogen Activated Protein Kinase (MAPK) pathway. This driver mutation is targeted clinically with FDA approved therapies using small molecule inhibitors of oncogenic BRAFV600E and MEK. Though highly immunogenic, melanoma is notorious for inducing an immunosuppressive microenvironment; relieved by checkpoint inhibitor therapy. The two molecules currently approved clinically are ipilimumab and nivolumab targeting the molecules Cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and PD-1 respectively. A plethora of immunomodulatory molecules exist, many with redundant functions. Additionally, these molecules are not only expressed by immune cells, but also by tumor cells within the tumor microenvironment. Tumor profiling of these cell surface checkpoint molecules is necessary to optimize a clinical response. In this study we identified the presence of immunomodulatory molecules in melanoma using data from The Cancer Genome Atlas, and validated this expression in two model systems: human melanoma tissues and patient derived melanoma cells. The level of expression of B and T Lymphocyte Attenuator (BTLA), TIM1, and CD226 concurrently with the BRAFV600E mutation status significantly dictated overall survival in melanoma patients. These molecules, along with Herpes Virus Entry mediator (HVEM) and CD160, two molecules that are a part of the HVEM/BTLA/CD160 axis, had a higher expression in human melanoma tissues compared to normal skin xxvi melanocytes, and have unique roles to play in T cell activation. In patient derived melanoma cell lines, MEL2, MELV, KFM, and GLM2 (BRAFV600E positive), and 3MM (BRAF negative) we found a differential expression of immunomodulatory molecules compared to normal adult melanocytes. Inhibition of BRAFV600E by treatment with vemurafenib led to a significant upregulation of BTLA, HVEM, CD160, TIM1, and CD226 expression in patient derived cell lines positive for the BRAFV600E mutation. BRAFV600E inhibition additionally led to the upregulation of Microphthalmia Induced Transcription Factor (MITF), that has binding sites in the genes of BTLA, HVEM, CD160, TIM1, and CD226 and is suppressed by a constitutively active MAPK pathway. These molecules were expressed at the protein level, as evidenced by western blots and were localized to the cell membrane, as evidenced by immunofluorescence and flow cytometry. We uncovered a link between the expression of immunomodulatory molecules and the BRAFV600E genetic lesion in melanoma. Using stringent criteria, we identified the presence of BTLA on the surface of melanoma cells making it a feasible immuno-oncology target. A combined inhibition of BRAFV600E and MEK by vemurafenib and trametinib, lowered the surface expression of BTLA in patient derived melanoma cells that harbor the BRAFV600E mutation. We conclude that small molecule inhibitors of the MAPK pathway regulate the surface expression of this multifaceted molecule making BTLA a promising target for immuno-oncology to be targeted in combination with small molecule inhibitors, potentially alleviating T regulatory cell activation and improving patient prognosis.

Download

Share

COinS