Melanoma is one of the most lethal skin malignancies due to its high propensity to metastasize. Emerging immunotherapies have increased therapeutic options, but durable responses have been limited to as yet poorly defined subsets of patients. Further mechanistic insight is needed to improve stratification and therapeutic efficacy and to reveal newer, potentially more effective therapeutic approaches. In this regard, we report compelling immunotherapeutic implications for protein fucosylation, an intriguing subtype of glycosylation that is increasingly reported to play roles in cancer pathogenesis 1 and that can be modulated by dietary L-fucose.
Previously, we discovered that during melanoma progression, global substrate control and downstream structural subtypes of fucosylation are downregulated and altered, respectively, correlating with poor survival outcomes 2. In vitro experiments in that early study suggested that melanoma motility is restrained when we increased fucosylation by overexpressing the upstream substrate regulator fucokinase (FUK) or by supplementing melanoma-bearing mice with dietary L-fucose. Whereas either approach reduced primary tumor volume and metastasis to the lung, we unexpectedly observed significant increases in intratumoral immune cells, suggesting fucosylation-triggered anti-tumor immune responses. The lack of clear underlying immunological mechanism prompted the initiation of this study, in which we tested our hypothesis that L-fucose and fucosylation trigger anti-tumor immune responses that can suppress melanoma. Long-term, this line of study aims to delineate how L-fucose/tumoral fucosylation could be therapeutically leveraged.
In a systematic series of genetically manipulated and oral L-fucose supplementation mouse models of melanoma, we show here that—independent of oncogenic driver mutation or sex—boosting tumor fucosylation increases a range of intratumoral immune cells that suppress tumor growth. We identified CD4+T cells as crucial mediators of L-fucose-triggered tumor suppression and induction of other intratumoral cell populations. Based on our observation that ectopic FUK expression alone in melanoma cells drove similar immune induction and tumor suppression, we reasoned that fucosylated melanoma-expressed protein(s) likely play crucial role(s) in the effects induced by oral L-fucose.
An initial click-chemistry-based proteomic screen, followed by biochemical validation and knockdown/reconstitution tumor modeling, identified the Class II MHC protein HLA-DRB1 as a crucial fucosylated melanoma protein necessary for oral L-fucose-triggered intratumoral immune cell induction and tumor suppression. In studying how glycosylation-fucosylation impacts HLA-DRB1 protein behavior, we mapped the fucosylation of HLA-DRB1 onto the predicted glycan HexNAc(4)Hex(3)Fuc(1) conjugated on Asparagine 48 (Asn 48) and determined that this site-specific glycosylation-fucosylation promotes the cell surface accumulation of this protein. Loss of glycosylation-fucosylation at Asn 48 reduces the cell surface presence of HLA-DRB1 and completely abrogates L-fucose-triggered tumor suppression and immune induction (see Schematic).
We next sought to assess the translational potential of our findings, in terms of therapeutic and biomarker applications.
As Class II MHC expression and CD4+T cell populations have been reported to correlate with responsiveness to immune checkpoint blockade therapy in melanoma 3-5, our data suggested that administration L-fucose might augment the efficacy of immune checkpoint blockade agents. We tested if L-fucose enhances anti-PD1 efficacy and found that oral L-fucose could suppress tumor progression as effectively as anti-PD1 or could augment anti-PD1 efficacy in different mouse models—effects that were associated with altered tumor immunological profiles induced by L-fucose. The biological and mechanistic underpinnings of the differential enhancement of immunotherapy efficacy remain unknown; subsequent studies are expected to provide key insights that will inform the future implementation of L-fucose to enhance immunotherapies.
As we observed that L-fucose supplementation resulted in variable enhancement of anti-PD1 efficacy, we reasoned that tumor fucosylation or fucosylated HLA-DRB1 levels might exhibit potential prognostic utility. Thus, we immunofluorescently analyzed pre-treatment tumor biopsies from anti-PD1 monotherapy-treated melanoma patients from Moffitt Cancer Center, MD Anderson Cancer Center, and Harvard Massachusetts General Hospital. Although specific types of fucosylated glycans can be visualized using a previously reported fluorescently-conjugated lectin staining technique 6, the retrospective immunofluorescent visualization of the specific glycosylated/fucosylated species of a specific protein of interest has until now been a long-standing technical hurdle. To overcome this obstacle, we developed a modified fucose-binding lectin- and antibody-based proximity ligation assay technique (L-PLA) that allows for the visualization of glycosylated/fucosylated forms of specific proteins of interest in cells grown in vitro as well as in fixed tissues.
Using L-PLA and traditional immunofluorescent staining, we assessed levels of total and fucosylated HLA-DRB1, total tumor core fucosylation, and numbers of CD4+T cells in the patient specimens. We performed single-cell segmented measurements of immunofluorescent signal intensities per tumor and non-tumor cell in each biopsy. These analyses revealed trends of increased populations of cells with increased levels of fucosylated HLA-DRB1 and tumoral fucosylation in clinically defined anti-PD1 responders compared with non-responders. Despite the remarkably consistent trends across patient cohorts from the 3 independent cancer centers, the trends did not reach statistical significance, an issue that we believe is attributable to the small patient cohort sizes per institute and significant potential confounding factors. The acquisition of patient samples for this study proved to be a challenge due to extremely limited availability of anti-PD1 monotherapy pre-treatment specimens, and further, to difficulty in identifying and acquiring specimens from patients who fit clinical responder vs. non-responder inclusion criteria. Other confounding factors included heterogeneity in time from pre-treatment biopsy to initiation of therapy as well as the type of biopsy. Regarding the latter, we found that discrepant staining intensities appeared to correspond with different core (containing limited/no stroma) vs. whole tumor biopsies (containing significant amounts of stroma). Our findings highlight the importance of streamlining of (i) the pre-treatment specimen harvest/acquisition process to ensure for specimens reflecting comparable time to treatment initiation, and (ii), biopsy type (i.e., specimens with comparable proportions of tumor vs. stromal interface that would likely result in statistically significant prognostic correlations). Subsequent prospective tissue collection that is more uniformly designed in these aspects are expected to mitigate these issues and facilitate more statistically robust analyses.
Together, our findings highlight how fucosylation regulates melanoma:immune signal transduction and support the potential clinical implementation of L-fucose to improve anti-tumor immune responses and immunotherapies, with particular implications for immune checkpoint blockade and adoptive cell transfer therapies. Further studies are expected to clarify the extent of clinical utility of L-fucose and fucosylated-biomarkers.
1 Adhikari, E. et al. L-fucose, a sugary regulator of antitumor immunity and immunotherapies. Mol Carcinog 61, 439-453 (2022). https://doi.org:10.1002/mc.23394
2 Lau, E. et al. The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation. Sci Signal 8, ra124 (2015). https://doi.org:10.1126/scisignal.aac6479
3 Rodig, S. J. et al. MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma. Sci Transl Med 10 (2018). https://doi.org:10.1126/scitranslmed.aar3342
4 Johnson, D. B. et al. Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy. Nat Commun 7, 10582 (2016). https://doi.org:10.1038/ncomms10582
5 Ribas, A. et al. PD-1 Blockade Expands Intratumoral Memory T Cells. Cancer Immunol Res 4, 194-203 (2016). https://doi.org:10.1158/2326-6066.CIR-15-0210
6 Norton, P. et al. Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma. Proteomics 16, 3126-3136 (2016). https://doi.org:10.1002/pmic.201600064