Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Eric Lau, Ph.D.

Committee Member

Vince Luca, Ph.D.

Committee Member

Shari A. Pilon-Thomas, Ph.D.

Committee Member

Keiran Smalley, Ph.D.

Committee Member

Chrystal M. Paulos, Ph.D.

Keywords

anti-melanoma immunity, CD4+ T cells, fucosylation, HLA-DRB1 MHC Class II, Immunotherapy, L-fucose

Abstract

Melanoma is the one of the most lethal skin malignancies due to its ability to rapidly metastasize and evade the immune system. One factor that influences melanoma’s ability to metastasize and evade the immune system is the tumor microenvironment. The tumor microenvironment is a complex ecosystem that consists of melanoma cells interacting with different proteins and cell types such as cytokines, extra cellular matrix proteins, and various immune cells. While different immune cells can have various implications in the tumor microenvironment, some tumor infiltrating lymphocytes (TIL) have the potential to suppress these tumors. Recent therapeutic strategies aim to enhance TILs in melanoma, either by increasing the total TIL in melanoma (i.e., adoptive cell therapy) or by inhibiting exhaustion of the tumor suppressive TILs (i.e., immune checkpoint blockade), known as immunotherapy. Although these therapies have produced long-term response in some patients with melanoma, there is a significant population of patients (~60% of melanoma patients) that do not respond to immunotherapies. Preliminaries studies have reported that poor response to immune checkpoints have correlated with poor infiltration of TILs. Additionally, there are currently suboptimal biomarkers to determine who would respond to this therapy. Our study investigates how a dietary sugar called L-fucose can suppress melanomas by increasing TIL in melanoma, which can potentially serve as an adjuvant to immunotherapies or serve as a biomarker of response to immunotherapy.

L-fucose is a dietary sugar found in abundance in red and brown seaweed. It has been used as an experimental therapeutic in leukocyte adhesion deficiency II (LAD II), a genetic disorder in the de novo L-fucose synthesis pathway which leukocytes are unable extravasate out of capillaries. L-fucose can enter the cell and processed by the L-fucose salvage pathway to be post-translationally attached to proteins and/or glycans. Thus, fucosylation can affect different aspects of the protein’s biology, such as protein stability, trafficking, or cell surface abundance.

In melanoma, levels of fucosylation decrease with melanoma progression. Additionally, melanoma patients with higher tumor-specific fucosylation levels have a higher survival probability than those with lower levels. A preliminary in vivo murine model found that either increasing fucosylation through dietary supplementation or genetic overexpression in the L-fucose salvage pathway can reduce tumor growth and decrease metastasis. Preliminary studies showed that L-fucose-treated tumors had increased abundance of TILs. My project focuses on determining the underlying immunoregulatory mechanism affected by tumor fucosylation that is driving melanoma suppression.

To determine how the immune system is affected by L-fucose supplementation, we profiled TILs from control or L-fucose-treated mice. We observed TILs increased ~10-50 fold in melanomas from L-fucose-treated mice. Of the immune subpopulations analyzed, CD3+ T cells were most increased, with both CD4+ and CD8+ T cells increasing. Immunodeficient murine models determine that the immune system is necessary for L-fucose-mediated tumor suppression. Immunodepletion of CD4+ and CD8+ T cells identified CD4+ T cells as the crucial immune population for L-fucose-triggered tumor suppression, with additional models showing the requirement of trafficking immune cells to the tumor. Further, we observed that stem central and central memory CD4+ T cells were increased in L-fucose-treated tumors.

From our observation that increasing L-fucose on the melanoma’s themselves is sufficient to decrease tumor volume, we were interested in identifying proteins that were fucosylated on melanomas. We implemented a click chemistry approach to identify all fucosylated proteins on melanomas. Of the proteins that were identified, HLA-A and HLA-DRB1 were the only immune-related and cell surface proteins in our screen. We further confirmed their expression and fucosylation in a panel of melanoma cell lines. To determine their importance in L-fucose-mediated tumor suppression, we knocked down mouse orthologs of HLA-A and HLA-DRB1, H2K1 and EB1 respectively, and observed that HLA-DRB1 is necessary of L-fucose-triggered tumor suppression in melanoma.

In order to determine how fucosylation affects HLA-DRB1’s function, we identified asparagine 48 and threonine 129 as potential fucosylation sites using glycosylation prediction software. We mutated both amino acids and identified asparagine 48 as the amino acid containing a fucosylated glycan. To determine how the fucosylation of HLA-DRB1 affects its function in vitro, we observed that cell surface HLA-DRB1 decreased with inhibition of fucosylation and increased with L-fucose supplementation. Importantly, the glycosylation-fucosylation site is necessary for in vivo L-fucose-triggered melanoma suppression, as L-fucose associated tumor suppression was abrogated in fucosylation deficient mutants, similar to total loss of murine HLA-DRB1.

Due to immune checkpoint inhibitor’s failure being associated with poor infiltration of TIL, we sought to test if L-fucose could be combined with the immune checkpoint inhibitor anti-PD-1 to further suppress tumor growth. We observed that L-fucose either suppressed melanomas as well as anti-PD-1 or further suppressed melanomas in different mouse models. We were also interested in determining if fucosylated HLA-DRB1 could be used as a prognostic biomarker for anti-PD-1 immunotherapy, a current insufficiency in immunotherapy. To test this, we adapted a proximity ligation assay to visualize fucosylated HLA-DRB1 in immunotherapy treated melanoma patient samples. We observed a trend across 3 independent cancer centers in increased fucosylation and fucosylated HLA-DRB1 in patients that responded to immunotherapy. Taken together, L-fucose and fucosylated proteins could potentially enhanced immunotherapies.

Our data supports the notion of using L-fucose therapeutically to treated melanomas. Further studies on how L-fucose affect different immune subpopulations, such as dendritic cells, are expected to help increase our understanding of how L-fucose could be applied to immunotherapies. We also observed that intratumoral fucosylation levels were lower in male patients in comparison to female patients. Subsequent studies are expected to elucidate how fucosylation is regulated by sex. Additionally, further studies will investigate how utilizing L-fucose as an adjuvant in different cancer types to enhance immunotherapies.

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