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. 2016 Apr 8;6(4):e411.
doi: 10.1038/bcj.2016.14.

Carfilzomib Alters the HLA-presented Peptidome of Myeloma Cells and Impairs Presentation of Peptides With Aromatic C-termini

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Carfilzomib Alters the HLA-presented Peptidome of Myeloma Cells and Impairs Presentation of Peptides With Aromatic C-termini

D J Kowalewski et al. Blood Cancer J. .
Free PMC article

Abstract

Recent studies suggest that multiple myeloma is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell immunotherapy. As standard of care in myeloma includes proteasome inhibitor therapy, it is of great importance to characterize the effects of this treatment on HLA-restricted antigen presentation and implement only robustly presented targets for immunotherapeutic intervention. Here, we present a study that longitudinally and semi-quantitatively maps the effects of the proteasome inhibitor carfilzomib on HLA-restricted antigen presentation. The relative presentation levels of 4780 different HLA ligands were quantified in an in vitro model employing carfilzomib treatment of MM.1S and U266 myeloma cells, which revealed significant modulation of a substantial fraction of the HLA-presented peptidome. Strikingly, we detected selective down-modulation of HLA ligands with aromatic C-terminal anchor amino acids. This particularly manifested as a marked reduction in the presentation of HLA ligands through the HLA allotypes A*23:01 and A*24:02 on MM.1S cells. These findings implicate that carfilzomib mediates a direct, peptide motif-specific inhibitory effect on HLA ligand processing and presentation. As a substantial proportion of HLA allotypes present peptides with aromatic C-termini, our results may have broad implications for the implementation of antigen-specific treatment approaches in patients undergoing carfilzomib treatment.

Figures

Figure 1
Figure 1
Effects of carfilzomib on myeloma cell viability and HLA class I surface expression. Quantification of HLA surface expression was performed using a bead-based flow-cytometric assay using the pan-HLA class I-specific monoclonal antibody W6/32. (a) Viability of MM.1S cells before in vitro treatment and 24 h/48 h after a 1-h pulse with 100 nm carfilzomib (Carfilzomib) or 5% glucose (MOCK). Cell viability was determined using the trypan blue exclusion test. (b) Absolute counts of HLA class I surface molecules on MM.1S cells under in vitro treatment. (c) Longitudinal analysis of relative, mock-normalized changes in HLA class I surface expression on four different MCLs under in vitro treatment. (d) Longitudinal analysis of relative, mock-normalized changes in HLA class I surface expression on primary CD38+CD138+ myeloma cells from seven different patients (UPN1-7) under in vitro treatment. (e) Absolute counts of HLA class I surface molecules on primary CD38+CD138+ myeloma cells from bone marrow aspirate of two different patients before commencement of therapy and after 4 weeks of treatment with carfilzomib.
Figure 2
Figure 2
Mass spectrometric analysis of the HLA-presented peptidome of MM.1S cell under carfilzomib treatment. (a) HLA class I ligand extracts of MM.1S cells before in vitro treatment and 24 h/48 h (t24h, t48h) after a 1-h pulse with 100 nm carfilzomib (Carfilzomib) or 5% glucose (Mock) were analyzed in biological triplicates using sample shares of 20% in dose-finding mass spectrometry runs. The raw number of HLA ligand identifications and the summed area under the curves (AUC) of their extracted ion chromatograms are indicated in gray and black bars, respectively. (b) Numbers of peptide identifications after adjustment of the injected sample amounts. The sample amounts were adjusted according to their ratios of summed AUC and each condition was analyzed in five technical replicates, allowing for LFQ of the relative peptide abundances. (c) Overlap of the 1908 different HLA class I ligands identified on MM.1S cells compared with a set of 197 different myeloma-associated peptides described in an earlier study.
Figure 3
Figure 3
Carfilzomib induces substantial qualitative and quantitative changes in the HLA ligandome of MM.1S cells. (a) Overlap analysis of HLA class I ligands identified on carfilzomib-treated versus untreated (mock controls and pre-therapy) cells. (b) Frequency-based analysis of peptide presentation on treated versus untreated MM.1S cells. HLA ligands are indicated on the x axis and the numbers of samples on which they were detected on the y axis. The box on the right highlights HLA ligands showing significant treatment-associated presentation (exclusive presentation on ⩾4/6 treated samples, FDR=3.0%), whereas the box on the left indicates peptides lost after carfilzomib therapy (exclusive presentation on ⩾5/9 untreated samples, FDR=4.9%). (cf) Volcano plots of modulations in the relative abundances of HLA ligands on MM.1S cells comparing the conditions indicated. Each dot represents a specific HLA ligand. Log2-fold changes of their abundance are indicated on the x axis and the corresponding significance levels after Benjamini–Hochberg correction on the y axis. HLA ligands showing significant up- or down-modulation (>4-fold change in abundance with P<0.01) are highlighted in red and blue, respectively. The numbers and percentages of these significantly modulated ligands are specified in the corresponding quadrants. (c, d) Volcano plots comparing HLA ligand abundances on carfilzomib versus mock-treated cells at t24h and t48h, respectively. (e, f) Control volcano plots comparing HLA ligand abundances on mock-treated cells at t24h and t48h to baseline levels before therapy.
Figure 4
Figure 4
Kinetics of HLA class I ligand presentation on MM.1S cells after treatment with carfilzomib. (a) Longitudinal analysis of the presentation of 14 highly specific myeloma-associated peptides on MM.1S cells after treatment with carfilzomib. Modulations in peptide abundance are indicated on the y axis as fold-change compared with mock-treated controls. (b) Clustering of 1068 different HLA class I ligands according to their presentation kinetics upon treatment with carfilzomib. Modulations in relative peptide abundance are indicated on the y axis as fold-change compared with mock-treated controls. (c) Distribution of HLA restrictions of peptides represented in the down-modulated cluster 8 compared with the distribution of all 1068 tracked peptides.
Figure 5
Figure 5
Carfilzomib alters HLA class I restricted peptide presentation in an HLA allotype-specific manner. (a) Distribution of HLA restrictions among peptides identified on carfilzomib-treated (n=1575 peptides) versus untreated MM.1S cells (n=1,304 peptides). (b) Longitudinal analysis of HLA ligand abundances after carfilzomib treatment grouped according to HLA restrictions. HLA allotype-specific fold-change values were calculated as the mean fold-change of all peptides restricted by the respective allotype. Data points represent mean fold-change values of three biological replicates±s.d. (c) Radar plots of the distribution of HLA restrictions among peptides showing significant down-modulation (blue lines) or up-modulation (red lines) compared with the distribution among all HLA ligands (gray dashed lines). Radar plots consist of overlays of three biological replicates. (d) Longitudinal analysis of HLA ligand abundances after carfilzomib treatment dichotomized according to their C-terminal anchor amino-acid groups. Data points represent mean fold-change values of three biological replicates±s.d.

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