Editing the immunopeptidome of melanoma cells using a potent inhibitor of endoplasmic reticulum aminopeptidase 1 (ERAP1)

Abstract

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, often is limited by ineffective presentation of antigenic peptides that elicit T-cell-mediated anti-tumor cytotoxic responses. Manipulation of antigen presentation pathways is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides as part of the system that generates peptides for binding to MHC class I molecules (MHC-I). We hypothesized that pharmacological inhibition of ERAP1 in cells could regulate the cellular immunopeptidome. To test this hypothesis, we treated A375 melanoma cells with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting bound peptides, and identifying them using capillary chromatography and tandem mass spectrometry (LC-MS/MS). Although the inhibitor did not reduce cell-surface MHC-I expression, it induced qualitative and quantitative changes in the presented peptidomes. Specifically, inhibitor treatment altered presentation of about half of the total 3204 identified peptides, including about one third of the peptides predicted to bind tightly to MHC-I. Inhibitor treatment altered the length distribution of eluted peptides without change in the basic binding motifs. Surprisingly, inhibitor treatment enhanced the average predicted MHC-I binding affinity, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9-12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 may be a viable approach for manipulating the immunopeptidome of cancer.

Analysis of Secondary Structure Biases in Naturally Presented HLA-I Ligands

“…Additional studies could be useful to verify this hypothesis, by investigating experimentally the role of proteasome and other proteases like ERAP1 on the generation of the peptidome, following for example the work of Admon et al (10, 49, 74). A very recent publication on ERAP1 inhibition showed that the average predicted affinity of MHC-I binding peptides was enhanced, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9–12-mers, suggesting that baseline ERAP1 activity in this cell line (A375, melanoma cells) is destructive for many potential epitopes (74).…”

“…Additional studies could be useful to verify this hypothesis, by investigating experimentally the role of proteasome and other proteases like ERAP1 on the generation of the peptidome, following for example the work of Admon et al (10, 49, 74). A very recent publication on ERAP1 inhibition showed that the average predicted affinity of MHC-I binding peptides was enhanced, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9–12-mers, suggesting that baseline ERAP1 activity in this cell line (A375, melanoma cells) is destructive for many potential epitopes (74). Based on the published results we hypothesize that the edited immunopeptidome in ERAP1 inhibited melanoma cells could still present a helix enrichment because: (1) ERAP1 inhibition increased the presentation of 9–12-mers peptides which were found in our study to show higher helical content when compared to longer peptides; (2) ERAP1 inhibition increased the frequency of N-terminal AA such as ALA, LEU, TYR, and MET which are all known to have a high helical propensity; (3) the inhibition does not affect the basic sequence motifs of the presented peptides.…”

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1

“…Several studies have already demonstrated significant effects of ERAP1 down-regulation on the immunopeptidome of cells and have associated these changes with alterations of adaptive immune responses in autoimmunity and cancer (13). Furthermore, pharmacological regulation of ERAP1 activity is an emerging approach for cancer immunotherapy and thus a deep mechanistic knowledge on how ERAP1 regulates the immunopeptidome is crucial for optimizing potential therapies (46) (47).…”

“…Although ERAP1 has been extensively studied for its ability to generate antigenic peptides from N-terminally elongated precursors, it can also over-trim antigenic peptides to lengths not suitable for binding onto MHCI, essentially destroying the epitope (3). Recent analysis on the effect of ERAP1 on the immunopeptidome of cells have suggested that the destructive properties of ERAP1 may have been underestimated and could actually be the dominant function of the enzyme (47,52,53). The data presented herein highlight the ability of MHCI to protect peptides from ERAP1mediated degradation and are thus consistent with the hypothesis that ERAP1 acts to limit available peptides for MHCI.…”

IRAP Inhibitors: M1-Aminopeptidase Family Inspiration

“…The broader activity and/or selectivity of these ligands needs to be considered (Talma et al, 2019). It is an interesting feature that these peptidomimetic transition state analogs possess intracellular activity also (Koumantou et al, 2019).…”