Plasma glycoproteomic biomarkers identify metastatic melanoma patients with reduced clinical benefit from immune checkpoint inhibitor therapy

Abstract

The clinical success of immune-checkpoint inhibitors (ICI) in both resected and metastatic melanoma has confirmed the validity of therapeutic strategies that boost the immune system to counteract cancer. However, half of patients with metastatic disease treated with even the most aggressive regimen do not derive durable clinical benefit. Thus, there is a critical need for predictive biomarkers that can identify individuals who are unlikely to benefit with high accuracy so that these patients may be spared the toxicity of treatment without the likely benefit of response. Ideally, such an assay would have a fast turnaround time and minimal invasiveness. Here, we utilize a novel platform that combines mass spectrometry with an artificial intelligence-based data processing engine to interrogate the blood glycoproteome in melanoma patients before receiving ICI therapy. We identify 143 biomarkers that demonstrate a difference in expression between the patients who died within six months of starting ICI treatment and those who remained progression-free for three years. We then develop a glycoproteomic classifier that predicts benefit of immunotherapy (HR=2.7; p=0.026) and achieves a significant separation of patients in an independent cohort (HR=5.6; p=0.027). To understand how circulating glycoproteins may affect efficacy of treatment, we analyze the differences in glycosylation structure and discover a fucosylation signature in patients with shorter overall survival (OS). We then develop a fucosylation-based model that effectively stratifies patients (HR=3.5; p=0.0066). Together, our data demonstrate the utility of plasma glycoproteomics for biomarker discovery and prediction of ICI benefit in patients with metastatic melanoma and suggest that protein fucosylation may be a determinant of anti-tumor immunity.

Variable PD-1 glycosylation modulates the activity of immune checkpoint inhibitors

“…Although currently underexplored, glycoprofiling of new or validated drug targets represents a valuable opportunity for the development of next-generation therapeutics that may achieve exquisite selectivity by binding to distinct glycosylation variants. In addition, the recent discovery of fucosylated biomarkers in the blood of melanoma patients that failed to achieve extended survival after treatment with immune checkpoint inhibitors points to a role of glycosylation not only in the tumor but also in the periphery (26,27). Therefore, glycosylation analysis of circulating glycoproteins may offer crucial information for both drug design and biomarker discovery.…”

“…Here, we observed that camrelizumab and cemiplimab specifically interact with a fucose moiety within the N58 glycan of PD-1. As fucosylation is increased in cancer and fucosylated biomarkers in blood have been associated with a lack of benefit of immune checkpoint inhibitor therapy (26,27), we investigated the influence of fucosylation on PD-1 in antibody activity by a combination of protein-and cell-based assays. Furthermore, we characterized fucosylation of sPD-1 in the serum of individuals with non-smallcell lung cancer (NSCLC).…”

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells

“…Glycosylation is a common post-translational modification of proteins, and it impacts their polarity, solubility, structure, cellular localization, and interactions with other molecules. , Thus, it is unsurprising that changes in glycosylation coincide with many diseases and aberrant glycosylation is implicated in cancer development and progression. − In addition, glycoproteins have a long-standing recognition as cancer biomarkers. − Poly- N -acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N -acetylglucosamine and galactose residues that is found mainly on tri- and tetra-antennary N -glycans and preferentially on the branch initiated by the β1–6 N -acetylglucosaminyltransferase-V encoded by the Mgat5 gene. , PolyLacNAc contributes to cancer resistance against T cell killing and increases the stability of the PD-L1 protein at the cell surface, leading to an enhanced interaction with the PD-1 receptor and reduced cytotoxic T cell responses in cancer . PolyLacNAc may also exhibit context-dependent roles in cancer cells: the presence of sialyl Lewis X on polyLacNAc-elongated glycans may lead to metastasis, while the same epitope on truncated N -glycans results in the death of tumor cells in lung blood vessels .…”

“… 3 − 6 In addition, glycoproteins have a long-standing recognition as cancer biomarkers. 7 − 9 Poly- N -acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N -acetylglucosamine and galactose residues that is found mainly on tri- and tetra-antennary N -glycans and preferentially on the branch initiated by the β1–6 N -acetylglucosaminyltransferase-V encoded by the Mgat5 gene. 10 , 11 PolyLacNAc contributes to cancer resistance against T cell killing 12 and increases the stability of the PD-L1 protein at the cell surface, leading to an enhanced interaction with the PD-1 receptor and reduced cytotoxic T cell responses in cancer.…”

Dataset: Impact of β-Galactosylceramidase Overexpression on the Protein Profile of Braf(V600E) Mutated Melanoma Cells

“…However, the mechanisms by which GALC exerts its pro-tumorigenic functions in human melanoma remain poorly understood. Analysis of the proteome may provide valuable information for the characterization of the biological pathways leading to melanoma progression [12][13][14][15][16] and for the identification of diagnostic and prognostic biomarkers [17][18][19][20]. As stated above, GALC may exert a pro-oncogenic role in Braf wild-type murine melanoma cells [5].…”