Mass Spectrometry-Based Immunopeptidome Analysis of Acute Myeloid Leukemia Cells Under Decitabine Treatment Delineates Induced Presentation of Cancer/Testis Antigens on HLA Class I Molecules
Authors: Bauer Jens , Zieger Nora , Nelde Annika , Bichmann Leon , Salih Helmut R. , Kanz Lothar , Rammensee Hans-Georg , Stevanovic Stefan , Walz Juliane S. 
Published: October 2019

Abstract
In recent years, therapeutic approaches for acute myeloid leukemia (AML) have been improved, however the disease is still characterized by high relapse rates and a poor overall survival mainly in elderly patients aged 60 years and older. The standard therapy for these AML patients involves hypomethylating agents (HMAs) such as decitabine. With this, treatment remission can be achieved in some patients, however effective post-remission therapies are still overdue. Recent data suggests that HMAs induce gene expression of various cancer/testis antigens (CTAs), which could lead to the presentation of cancer/testis antigen-derived peptides on human leukocyte antigen (HLA) molecules. These CTA-derived peptides might serve as prime targets for tailored T cell-based immunotherapy approaches, which could represent an effective post-remission combination therapy.
Here we present a mass spectrometry-based study, which longitudinally maps the HLA-presented immunopeptidome and in particular cancer/testis antigens of AML cells under in vitro decitabine treatment. To analyze the impact of decitabine on the presentation of HLA ligands we treated the AML cell lines U937 and MONO-MAC-6 as well as primary AML cells (n = 1) with decitabine for 48 h (t48) and 72 h (t72) in vitro. Upon flow cytometry-based quantification of HLA class I and II surface expression levels, no significant changes of HLA surface molecule levels under decitabine treatment compared to untreated controls were observed. Implementing label-free quantitation mass spectrometry, we then quantitatively assessed HLA class I ligand presentation under decitabine treatment. Only minor effects of decitabine on the whole HLA class I-restricted peptidome were observed: We detected a significant upregulation of 2.6 ± 0.9% of HLA class I ligands (fold change (FC) ≥ 4, p ≤ 0.01) after 48 h of decitabine treatment, whereas 9.6 ± 5.7% of the ligands were altered in their abundance over time without treatment. At t72 similar proportions of decitabine modulation were observed with 4.2 ± 2.7% of up-regulated HLA ligands.
A total of 69 HLA class I ligands derived from 31 different CTAs were identified by mass spectrometric analysis, 9 of these ligands were presented exclusively under decitabine treatment. Furthermore, we showed that decitabine exposure caused a significantly increased presentation of 7/69 CTA-derived HLA ligands at least at one time point in the cell lines and the primary AML cells (FC ≥ 4, p ≤ 0.01). From the CTA cyclin A1, two HLA class I-presented peptides were significantly upregulated in U937 cells at t48 (FC 79.0 and 8.2) and t72 (FC 14.1 and 12.4). In primary AML cells, two peptides derived from JARID1B and KIAA0100 were significantly upregulated at either t48 (FC 21.8) or t72 (FC 6.6). In addition, we screened our dataset for HLA ligands derived from previously described decitabine-regulated genes and identified a HLA class I-presented peptide from DAZL, which was significantly upregulated in U937 cells at t72 under decitabine treatment (FC 57.2).
Taken together, our results demonstrate a modulatory effect of the hypomethylating agent decitabine on the HLA ligandome of AML cells, enhancing the presentation of CTA-derived peptides on HLA class I molecules. The latter will be further evaluated for their eligibility as targets for tailored peptide-based immunotherapeutic approaches in AML patients undergoing HMA treatment.

Disclosures
Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1.