Induction of Cancer Testis Antigen Expression in Circulating Acute Myeloid Leukemia Blasts Following Hypomethylating Agent Monotherapy
Authors: Srivastava Pragya , Paluch Benjamin E. , Matsuzaki Junko , James Smitha R. , Collmat-Lai Golda , Blagitko-Dorfs Nadja , Ford Laurie Ann , Naqash Rafeh , Luebbert Michael , Karpf Adam R. , Nemeth Michael J. , Griffiths Elizabeth A. 
Published: October 2019

Abstract
Background: The development of immunotherapeutic strategies for the treatment of leukemia has shown considerable promise but targeting suitable myeloid leukemia antigens remains a significant challenge. Cancer testis antigens (CTA) have been identified as promising targets for immunotherapy in solid tumors, but dense promoter methylation silences their expression in acute myeloid leukemia, limiting their potential as targets. Patients with acute myeloid leukemia are frequently treated with hypomethylating agents (HMAs) and previous studies have established that exposure of leukemia cell lines to HMAs induces expression of CTAs. In this study, we tested the hypothesis that patients receiving HMAs exhibit increased expression of CTAs.
Methods: AML patients receiving decitabine were enrolled under an IRB-approved protocol (Roswell Park) or with approval of the Ethics Committee (University of Freiburg). Peripheral blood samples were serially collected prior to decitabine treatment and two to four times per week during their first cycle of decitabine therapy (20 mg/m2 per day for 10 days).
Results: We analyzed expression and demethylation of CTA genes in peripheral blood samples serially isolated from AML patients (n = 5) during and following their first cycle of decitabine therapy (20 mg/m2 per day for 10 days). These patients demonstrated induction of MAGEA1 (1/5 patients), XAGE1 (3/5), MAGEA3/A6 (3/5), and NY-ESO-1 (5/5). Western blot analysis demonstrated increased expression of NY-ESO-1 protein following decitabine treatment. The induction of NY-ESO1 mRNA was confirmed in an independent group of AML patients (5/7, treated at University of Freiburg) receiving decitabine. Since NY-ESO-1 is an established cancer immunotherapy target with clinically translatable vaccines in development, we further examined induced NY-ESO-1 expression in a larger cohort of AML patients. We performed sodium bisulfite pyrosequencing to quantify changes in NY-ESO-1 promoter methylation pre-decitabine to the post-decitabine nadir time point for each patient (n = 22). There was a statistically significant decrease in NY-ESO-1 promoter methylation (p < 0.001). Prior to decitabine treatment, 18% (4/22) of samples exhibited detectable levels of NY-ESO-1 mRNA as measured by quantitative PCR. Following decitabine therapy, 78% (17/22) of samples had detectable levels of NY-ESO-1 mRNA. Treatment with decitabine was associated with a significant increase in NY-ESO-1 expression when comparing pre-treatment expression to the maximum expression at any time interval post decitabine (p < 0.0001). We then tested whether levels of NY-ESO-1 induction were different in patients who demonstrated a clinical response compared with those who did not. Overall, 7/22 patients (32%) demonstrated a clinical response to decitabine. 6/7 patients who clinically responded to decitabine demonstrated a significant increase in NY-ESO-1 mRNA (p < 0.03). Crucially, NY-ESO-1 mRNA levels were also significantly increased in 11 out of the 15 patients that did not demonstrate a response to decitabine (p = 0.001). To test whether AML blasts expressing NY-ESO-1 could induce a T-cell response, we stimulated HLA compatible NY-ESO-1- specific CD8+ T cells with AML blasts isolated from HLA-A*0201+ AML patients before and after decitabine treatment. T-cell responses were determined by intracellular cytokine staining (IFN-γ, TNF-α and IL-2) and the expression of CD107a/b, a marker for T-cell degranulation. Co-culture of AML blasts harvested post-decitabine, resulted in increased levels of IFN-γ, TNF-α, IL-2, and CD107a/b in HLA-A*0201/NY-ESO-1157-165 tetramer+ CD8+ T-cells in three of the four patients studied, compared to T-cells co-cultured with AML blasts from the same four patients obtained prior to decitabine exposure.
Conclusion: We observed enhanced expression of NY-ESO-1 in AML patients receiving decitabine and this induction was sufficient to produce a cytotoxic response in HLA-compatible antigen specific T-cells. A majority of patients who did not respond to decitabine still exhibited an increase NY-ESO-1 mRNA, suggesting that immunotherapies that target NY-ESO-1 have the potential to be effective even in patients who have not demonstrated a prior response to decitabine. These data support the combination of decitabine with immunotherapeutic approaches targeting NY-ESO-1 and other CTAs in myeloid cancer.

Disclosures
Griffiths: Alexion Pharmaceuticals: Honoraria; Astex: Research Funding; Celgene: Honoraria. Off Label Use: Decitabine is in routine clinical use in the United States for the management of unfit/elderly patients with AML. Patients received decitabine as standard of care off label in the hospital per Bloom et al PNAS 2010.