LINE1 and Alu repetitive element DNA methylation in tumors and white blood cells from epithelial ovarian cancer patients.

OBJECTIVE: We determined whether DNA methylation of repetitive elements (RE) is altered in epithelial ovarian cancer (EOC) patient tumors and white blood cells (WBC), compared to normal tissue controls. METHODS: Two different quantitative measures of RE methylation (LINE1 and Alu bisulfite pyrosequencing) were used in normal and tumor tissues from EOC cases and controls. Tissues analyzed included: i) EOC, ii) normal ovarian surface epithelia (OSE), iii) normal fallopian tube surface epithelia (FTE), iv) WBC from EOC patients, obtained before and after treatment, and v) WBC from demographically-matched controls. RESULTS: REs were significantly hypomethylated in EOC compared to OSE and FTE, and LINE1 and Alu methylation showed a significant direct association in these tissues. In contrast, WBC RE methylation was significantly higher in EOC cases compared to controls. RE methylation in patient-matched EOC tumors and pre-treatment WBC did not correlate. CONCLUSIONS: EOC shows robust RE hypomethylation compared to normal tissues from which the disease arises. In contrast, RE are generally hypermethylated in EOC patient WBC compared to controls. EOC tumor and WBC methylation did not correlate in matched patients, suggesting that RE methylation is independently controlled in tumor and normal tissues. Despite the significant differences observed over the population, the range of RE methylation in patient and control WBC overlapped, limiting their specific utility as an EOC biomarker. However, our data demonstrate that DNA methylation is deranged in normal tissues from EOC patients, supporting further investigation of WBC DNA methylation biomarkers suitable for EOC risk assessment.

Induction of cancer testis antigen expression in circulating acute myeloid leukemia blasts following hypomethylating agent monotherapy.

Cancer testis antigens (CTAs) are promising cancer associated antigens in solid tumors, but in acute myeloid leukemia, dense promoter methylation silences their expression. Leukemia cell lines exposed to HMAs induce expression of CTAs. We hypothesized that AML patients treated with standard of care decitabine (20mg/m2 per day for 10 days) would demonstrate induced expression of CTAs. Peripheral blood blasts serially isolated from AML patients treated with decitabine were evaluated for CTA gene expression and demethylation. Induction of NY-ESO-1 and MAGEA3/A6, were observed following decitabine. Re-expression of NY-ESO-1 and MAGEA3/A6 was associated with both promoter specific and global (LINE-1) hypomethylation. NY-ESO-1 and MAGEA3/A6 mRNA levels were increased irrespective of clinical response, suggesting that these antigens might be applicable even in patients who are not responsive to HMA therapy. Circulating blasts harvested after decitabine demonstrate induced NY-ESO-1 expression sufficient to activate NY-ESO-1 specific CD8+ T-cells. Induction of CTA expression sufficient for recognition by T-cells occurs in AML patients receiving decitabine. Vaccination against NY-ESO-1 in this patient population is feasible.

Immunomodulatory action of the DNA methyltransferase inhibitor SGI-110 in epithelial ovarian cancer cells and xenografts.

We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.

Immunomodulatory action of SGI-110, a hypomethylating agent, in acute myeloid leukemia cells and xenografts.

The mechanism of clinical action for the FDA approved hypomethylating drugs azacitidine and decitabine remains unresolved and in this context the potential immunomodulatory effect of these agents on leukemic cells is an area of active investigation. Induced expression of methylated Cancer Testis Antigen (CTA) genes has been demonstrated in leukemic cell lines following exposure to hypomethylating drugs in vitro. SGI-110 is a novel hypomethylating dinucleotide with prolonged in vivo exposure and clinical activity in patients with MDS and AML. We demonstrate that this agent, like decitabine, produces robust re-expression of the CTAs NY-ESO-1 and MAGE-A, both in vitro and in leukemia-bearing AML xenografts. Upregulation of these genes in vitro was sufficient to induce cytotoxicity by HLA-compatible CD8+ T-cells specific for NY-ESO-1, a well-recognized and immunogenic CTA. Additionally, exposure to SGI-110 enhances MHC class I and co-stimulatory molecule expression, potentially contributing to recognition of CTAs. SGI-110, like the parent compound decitabine, induces expression of CTAs and might modulate immune recognition of myeloid malignancy.