Adam R. Karpf

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.

DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene MAGEA11.

MAGEA11 is a cancer germline (CG) antigen and androgen receptor co-activator. Its expression in cancers other than prostate, and its mechanism of activation, has not been reported. In silico analyses reveal that MAGEA11 is frequently expressed in human cancers, is increased during tumor progression, and correlates with poor prognosis and survival. In prostate and epithelial ovarian cancers (EOC), MAGEA11 expression was associated with promoter and global DNA hypomethylation, and with activation of other CG genes. Pharmacological or genetic inhibition of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs) activated MAGEA11 in a cell line specific manner. MAGEA11 promoter activity was directly repressed by DNA methylation, and partially depended on Sp1, as pharmacological or genetic targeting of Sp1 reduced MAGEA11 promoter activity and endogenous gene expression. Importantly, DNA methylation regulated nucleosome occupancy specifically at the -1 positioned nucleosome of MAGEA11. Methylation of a single Ets site near the transcriptional start site (TSS) correlated with -1 nucleosome occupancy and, by itself, strongly repressed MAGEA11 promoter activity. Thus, DNA methylation regulates nucleosome occupancy at MAGEA11, and this appears to function cooperatively with sequence-specific transcription factors to regulate gene expression. MAGEA11 regulation is highly instructive for understanding mechanisms regulating CG antigen genes in human cancer.

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.

DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.

NY-ESO-1 Vaccination in Combination with Decitabine Induces Antigen-Specific T-lymphocyte Responses in Patients with Myelodysplastic Syndrome

Purpose: Treatment options are limited for patients with high-risk myelodysplastic syndrome (MDS). The azanucleosides, azacitidine and decitabine, are first-line therapy for MDS that induce promoter demethylation and gene expression of the highly immunogenic tumor antigen NY-ESO-1. We demonstrated that patients with acute myeloid leukemia (AML) receiving decitabine exhibit induction of NY-ESO-1 expression in circulating blasts. We hypothesized that vaccinating against NY-ESO-1 in patients with MDS receiving decitabine would capitalize upon induced NY-ESO-1 expression in malignant myeloid cells to provoke an NY-ESO-1–specific MDS-directed cytotoxic T-cell immune response. Experimental Design: In a phase I study, 9 patients with MDS received an HLA-unrestricted NY-ESO-1 vaccine (CDX-1401 + poly-ICLC) in a nonoverlapping schedule every four weeks with standard-dose decitabine. Results: Analysis of samples serially obtained from the 7 patients who reached the end of the study demonstrated induction of NY-ESO-1 expression in 7 of 7 patients and NY-ESO-1–specific CD4+ and CD8+ T-lymphocyte responses in 6 of 7 and 4 of 7 of the vaccinated patients, respectively. Myeloid cells expressing NY-ESO-1, isolated from a patient at different time points during decitabine therapy, were capable of activating a cytotoxic response from autologous NY-ESO-1–specific T lymphocytes. Vaccine responses were associated with a detectable population of CD141Hi conventional dendritic cells, which are critical for the uptake of NY-ESO-1 vaccine and have a recognized role in antitumor immune responses. Conclusions: These data indicate that vaccination against induced NY-ESO-1 expression can produce an antigen-specific immune response in a relatively nonimmunogenic myeloid cancer and highlight the potential for induced antigen-directed immunotherapy in a group of patients with limited options. Clin Cancer Res; 24(5); 1019–29. ©2017 AACR. See related commentary by Fuchs, p. 991

Functional characterization of a panel of high-grade serous ovarian cancer cell lines as representative experimental models of the disease

Genomic analysis of ovarian cancer cell lines has revealed a panel that best represents the most common ovarian cancer subtype, high-grade serous ovarian cancer (HGSOC). However, these HGSOC-like cell lines have not been extensively applied by ovarian cancer researchers to date, and the most commonly used cell lines in the ovarian cancer field do not genetically resemble the major clinical type of the disease. For the HGSOC-like lines to serve as suitable models, they need to be characterized for common functional assays. To achieve that objective, we systematically studied a panel of HGSOC cells CAOV3, COV362, Kuramochi, OVCAR4, OVCAR5, OVCAR8, OVSAHO and SNU119 for migration, invasion, proliferation, clonogenicity, EMT phenotype and cisplatin resistance. They exhibited a range of efficacies and OVCAR5, OVCAR8 and Kuramochi were the most aggressive. SNU119 and OVSAHO cells demonstrated the lowest functional activities. Wide differences in expression of EMT markers were observed between cell lines. SNU119 were the most epithelial and OVCAR8 had the most mesenchymal phenotype. COV362 was the most resistant to cisplatin while CAOV3 was the most sensitive. Taken together, our systematic characterization represents a valuable resource to help guide the application of HGSOC cells by the cancer research community.

DNA methylome analyses implicate fallopian tube epithelia as the origin for high-grade serous ovarian cancer

High-grade serous ovarian cancer (HGSC) is the most common and lethal form of epithelial ovarian cancer (EOC). Two distinct tissues have been suggested as the tissue of origin: ovarian surface epithelia (OSE) and fallopian tube epithelia (FTE). We hypothesized that the DNA methylome of HGSC should more closely resemble the methylome of its tissue of origin. To this end, we profiled HGSC (n = 10), and patient-matched OSE and FTE (n = 5) primary fresh-frozen tissues, and analyzed the DNA methylome using Illumina 450K arrays (n = 20) and Agilent Sure Select methyl-seq (n = 7). Methylomes were compared using statistical analyses of differentially methylated CpG sites (DMC) and differentially methylated regions (DMR). In addition, methylation was evaluated within a variety of different genomic contexts, including CpG island shores and Homeobox (HOX) genes, due to their roles in tissue specification. Publicly available HGSC methylome data (n = 628) were interrogated to provide additional comparisons with FTE and OSE for validation. These analyses revealed that HGSC and FTE methylomes are significantly and consistently more highly conserved than are HGSC and OSE. Pearson correlations and hierarchal clustering of genes, promoters, CpG islands, CpG island shores, and HOX genes all revealed increased relatedness of HGSC and FTE methylomes. Thus, these findings reveal that the landscape of FTE more closely resembles HGSC, the most common and deadly EOC subtype. Implications: DNA methylome analyses support the hypothesis that HGSC arise from the fallopian tube and that due to its tissue-specificity and biochemical stability, interrogation of the methylome may be a valuable approach to examine cell/tissue lineage in cancer. Mol Cancer Res; 14(9); 787–94. ©2016 AACR.

G-1 Inhibits breast cancer cell growth via targeting colchicine-binding site of tubulin to interfere with microtubule assembly

G-protein–coupled estrogen receptor 1 (GPER1) has been reported to play a significant role in mediating the rapid estrogen actions in a wide range of normal and cancer cells. G-1 was initially developed as a selective agonist for GPER. However, the molecular mechanisms underlying the actions of G-1 are unknown, and recent studies report inconsistent effects of G-1 on the growth of breast cancer cells. By employing high-resolution laser scanning confocal microscopy and time-lapse imaging technology, as well as biochemical analyses, in the current study, we provide convincing in vitro and in vivo evidence that G-1 is able to suppress the growth of breast cancer cells independent of the expression status of GPERs and classic estrogen receptors. Interestingly, we found that triple-negative breast cancer cells (TNBC) are very sensitive to G-1 treatment. We found that G-1 arrested the cell cycle in the prophase of mitosis, leading to caspase activation and apoptosis of breast cancer cells. Our mechanistic studies indicated that G-1, similar to colchicine and 2-methoxyestradiol, binds to colchicine binding site on tubulin, inhibiting tubulin polymerization and subsequent assembly of normal mitotic spindle apparatus during breast cancer cell mitosis. Therefore, G-1 is a novel microtubule-targeting agent and could be a promising anti-microtubule drug for breast cancer treatment, especially for TNBC treatment. Mol Cancer Ther; 16(6); 1080–91. ©2017 AACR.

White blood cell DNA methylation and risk of breast cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)

BackgroundSeveral studies have suggested that global DNA methylation in circulating white blood cells (WBC) is associated with breast cancer risk.MethodsTo address conflicting results and concerns that the findings for WBC DNA methylation in some prior studies may reflect disease effects, we evaluated the relationship between global levels of WBC DNA methylation in white blood cells and breast cancer risk in a case-control study nested within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) cohort. A total of 428 invasive breast cancer cases and 419 controls, frequency matched on age at entry (55–59, 60–64, 65–69, ≥70 years), year of entry (on/before September 30, 1997, on/after October 1, 1997) and period of DNA extraction (previously extracted, newly extracted) were included. The ratio of 5-methyl-2’ deoxycytidine [5-mdC] to 2’-deoxyguanine [dG], assuming [dG] = [5-mdC] + [2’-deoxycytidine [dC]] (%5-mdC), was determined by liquid chromatography-electrospray ionization-tandem mass spectrometry, an especially accurate method for assessing total genomic DNA methylation.ResultsOdds ratio (OR) estimates and 95% confidence intervals (CI) for breast cancer risk adjusted for age at entry, year of entry, and period of DNA extraction, were 1.0 (referent), 0.89 (95% CI, 0.6–1.3), 0.88 (95% CI, 0.6–1.3), and 0.84 (95% CI, 0.6–1.2) for women in the highest compared to lowest quartile levels of %5md-C (p for trend = .39). Effects did not meaningfully vary by time elapsed from WBC collection to diagnosis.DiscussionThese results do not support the hypothesis that global DNA hypomethylation in WBC DNA is associated with increased breast cancer risk prior to the appearance of clinical disease.

Genome-Wide Hypomethylation and Cancer Risk—Letter

Brennan and Flanagans recent publication in Cancer Prevention Research describing a meta-analysis of blood methylation and cancer risk is an outstanding contribution to this burgeoning field ([1][1]). The authors discuss a wide-ranging set of important issues and make numerous salient points