Conventional Dose Hypomethylating Agents Induce CG Antigen Genes In Vivo

Abstract

Abstract 2441 Background:Cancer testis or cancer germline (CG) antigens are a group of genes whose usual expression is limited to embryonic or germ cell tissues and which can also be aberrantly expressed in a variety of malignancies (Fratta E et al. Mol Oncol. 2011;5:164). The CG antigen genes MAGE-A1, XAGE-1, NY-ESO-1, and PRAME (among others) are epigenetically regulated. Most adult non-germline tissues demonstrate dense CpG island promoter methylation, with no gene expression. Endogenous expression of CG genes in epithelial malignancy can be immunogenic and a number of vaccine strategies are currently underway, however studies in hematologic malignancy have shown limited baseline expression, and thus there has been limited interest in them as a target in AML. Recently, several groups have shown that treatment with the hypomethylating agents, azacitidine(Aza) and decitabine(Dac) are sufficient to re-induce gene expression in myeloid cell lines, prompting interest in these genes as a therapeutic target in myeloid malignancy (Almstedt M et al. Leuk Res 2010;34:899). Re-expression of these genes has also been observed in MDS/AML patients treated with Dac, albeit at higher doses (15–270mg/m2) than those currently in standard use (Sigalotti L et al. Blood 2003;101:4644). Hypothesis:We hypothesized that conventional therapy with Aza(75mg/m2) and Dac(20mg/m2) induces CG promoter hypomethylation and gene expression, and that this may be sufficient to induce anti-CG antigen directed antibody responses. Methods:We obtained samples every 2–4 days from 2 AML patients during the first cycle of Aza or Dac treatment, and analyzed baseline and post-treatment samples for methylation (by pyrosequencing) and CG gene expression (by RT-PCR) of the above genes. We further tested serum from 10 patients for the presence of baseline and post treatment (following 1–10 cycles of Aza or Dac) anti-CG antibodies using an ELISA-based method. Results:Both AML patients treated with hypomethylating agents demonstrated a time dependent decrease in global methylation (by LINE-1 pyrosequencing), and CG antigen promoter methylation which nadired at day 6 post treatment. Baseline methylation of MAGE-A1 (90%), XAGE-1 (85%), NY-ESO-1 (92%) and PRAME (50–60%) was similar and high in both patients pre-treatment. Both Dac and Aza induced hypomethylation of all the CG genes studies, and this hypomethylation correlated with changes in LINE-1 methylation. Low level expression of MAGE-A1, XAGE-1, and PRAME were induced in a time dependent fashion following treatment with both Aza and Dac, however only limited re-expression of NY-ESO-1 was observed. ELISA testing for antibodies against a panel of 25 CG antigens (including MAGE-A1, XAGE-1 and NY-ESO-1) was performed on 10 patients with either MDS or AML. Following hypomethylating agents there was evidence for increased antibody levels, most notably against MAGE proteins, compared to baseline. Conclusion:Conventional hypomethylating agent therapy can induce CG gene expression, which may be sufficient to induce anti-CG antigen antibody responses. Augmentation of anti-tumor immune responses may be possible in myeloid malignancies using a combination of hypomethylating agents and CG antigen-directed vaccines. Disclosures:Odunsi:Ludwig Institute for Cancer Reserach: Membership on an entity's Board of Directors or advisory committees, Research Funding.