Daniela Matei

Abstract 3439: Tissue tranglutaminase (TG2) targeting by multifunctional field responsive gold nanoparticles

Background: Ovarian cancer (OC) spreads by direct exfoliation into the peritoneal cavity, bypassing vascular mechanisms. Our previous studies demonstrated that intraperitoneal (IP) metastasis is critically dependent on TG2, an enzyme that catalyzes protein post-translational modifications and crosslinking. In this study we assessed TG2 expression in OC, measured effects of TG2 overexpression on OC cell proliferation and tumorigenicity, and designed a novel method for cancer-specific TG2 targeting. As in-vivo delivery of siRNAs is inefficient and nonspecific, we engineered folate-coated gold nanorods (GNR) that deliver and release TG2 siRNAs into tumor cells by photothermal activation, and used small animal models to evaluate the efficacy of siRNA delivery. Materials & Methods: Immunohistochemistry (IHC) evaluated TG2 expression in 58 OC specimens. Correlation with histological subtype, grade, and stage was performed by Kruskal-Wallis test. Human full length TG2 and mutants lacking enzymatic activity (C277S), GTPase activity (R580A), or both (TG1-140) were stably expressed in OV90 cells. Cell proliferation was measured by MTT and clonogenic assays. Tumorigenicity and IP metastasis were assessed in orthotopic xenografts. GNRs were synthesized by seed mediated growth, cleansed by treatment with polystyrenesulfonate, then functionalized with a mixture of thiolated siRNA and folate-conjugated PEG for targeted TG2 knockdown in tumor cells overexpressing the folate receptor (FR). Localization of FR-targeted GNRs in orthotopic ovarian tumors and metastatic colonies in the peritoneum was assessed ex vivo by confocal microscopy, two-photon luminescence (TPL), and fluorescence imaging. Results: TG2 expression was noted in 84% of OC specimens, of which 71% had > 2+ expression. TG2 expression levels correlated with histological subtype (p=0.005), and with high grade (p=0.005), but not with surgical stage. Forced expression of TG2 or of one of the three mutants did not affect OV90 cell proliferation in vitro or tumor volume in vivo. Mice injected with TG2-OV90 cells developed milliary peritoneal metastases (8/8) compared to mice implanted with control cells (2/9, p=0.002). TG2 knockdown via siRNA yielded >70% reduction in TG2 expression level in vitro. The uptake of folate-labeled GNRs into SKOV3 cells was confirmed in vitro by confocal microscopy and TPL. FR-targeted GNR delivery in vivo was most notable in peritoneal metastases, primary ovarian tumor, and liver. Little or no uptake was noted in other normal tissues, suggesting that this modality achieves cancer-specific delivery. Conclusions: TG2 is overexpressed in OC and its overexpression increases peritoneal metastasis. GNR uptake and delivery to ovarian tumors is facilitated by FR targeting. Ongoing studies are assessing the effects of GNRs carrying TG2 siRNA on OC metastasis in an orthotopic xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3439. doi:1538-7445.AM2012-3439

Abstract A2: Exploratory textural CT evaluation of the combination of TRC105 (anti-endoglin monoclonal antibody; MAb) and bevacizumab (BEV) indicates partial response by Choi criteria in BEV refractory advanced cancer patients (pts) and identifies candidate markers of response.

Background: Endoglin (CD105) is an endothelial cell membrane receptor, highly expressed on angiogenic tumor vessels, that is essential for angiogenesis and upregulated by hypoxia and VEGF inhibition. TRC105 is an anti-endoglin MAb that potentiates VEGF inhibitors in preclinical models. TRC105 10 mg/kg weekly was well tolerated with BEV 10 mg/kg q2wk and the combination demonstrated activity in BEV and VEGF TKI refractory pts. This study assessed radiographic responses to TRC105 + BEV using Choi criteria and tumor morphology by applying novel quantitative textural analysis (QTA: TexRad -University of Sussex, UK) in pts with durable stable disease by RECIST, to determine predictive markers of response.nnMethods: Contrast enhanced CT scans from 5 pts with advanced solid tumors who demonstrated stable disease by RECIST in a trial of escalating doses of TRC105 (3, 6, 8 or 10 mg/kg/wk) plus BEV were reviewed. Seventeen target lesions were selected from baseline scans and assessed for target lesion diameter, whole lesion density, and tumor volume at baseline and follow-up. QTA analysis was assessed on the same target lesions using six different filter levels at baseline and on follow-up scans. The results were correlated to anatomic tumor response using non-parametric evaluation and regression analysis. Statistical significance was defined as a two-tailed p < 0.05.nnResults: Scans from 5 patients (median age 56; M:F 2:3; median 4 prior regimens; 3 metastatic colorectal and 2 ovarian cancer) 4 of whom progressed following VEGF inhibitor treatment were selected and demonstrated stable disease by RECIST for at least 4 months (range: 4-14 months) of treatment with TRC105 + BEV. Four of five patients (80%) had partial responses by Choi criteria. Predictive markers of tumor response on baseline scans included 1) elevated mean pixel density (median values of responder (R) vs non responder (NR): 27.2 vs -4.3) that correlated with subsequent tumor size reduction, 2) elevated entropy (a measure of tumor heterogeneity; median R vs NR: 5.1 vs 4.7) that correlated with subsequent decrease in mean tumor volume, and 3) low kurtosis (a measure of tumor heterogeneity; median R vs NR: 0.2 vs 1.1) that correlated with subsequent reduction in lesion density (p<0.01). Mean positive pixel values (an indicator of hypoxia) on follow-up scans correlated with decreased tumor density.nnConclusions: Assessment of radiographic response using Choi criteria identified VEGF inhibitor refractory patients who demonstrated partial response to the combination of TRC105 + BEV. Using novel QTA measures, markers of tumor heterogeneity and hypoxia correlated with individual lesion responses and are worthy of prospective evaluation as predictive imaging biomarkers.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):A2.nnCitation Format: Ron L. Korn, Michael S. Gordon, Lee S. Rosen, Francisco Robert, Daniela Matei, Jonathan W. Goldman, David S. Mendelson, E. Gabriela Chiorean, Robert Matthew Strother, Ben K. Seon, Delia Alvarez, Bonne J. Adams, Charles P. Theuer. Exploratory textural CT evaluation of the combination of TRC105 (anti-endoglin monoclonal antibody; MAb) and bevacizumab (BEV) indicates partial response by Choi criteria in BEV refractory advanced cancer patients (pts) and identifies candidate markers of response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A2.

Abstract A70: Epigenome and genome alterations in platinum resistant ovarian tumors.

Purpose: Epigenetic changes, particularly in DNA methylation, have been implicated in acquired resistance to platinum in ovarian cancer (OC). The goal of the current study was to analyze and integrate global RNA expression and DNA methylation profiles of platinum resistant tumors compared to untreated, platinum-sensitive ovarian tumors, as well as to measure genomic and epigenomic changes induced by guadecitabine (SGI-110) in tumors. Methods: An ongoing phase I/II multi-institutional clinical trial uses the novel DNA methyltransferase (DNMT) inhibitor guadecitabine to re-sensitize recurrent platinum resistant OC to carboplatin. Patients enrolled in this trial had recurrent platinum resistant OC. Tumor biopsies were collected at baseline and after two cycles of guadecitabine administered daily for 5 days at a low (30mg/m2) dose (28 days per cycle). RNA and DNA were extracted from 48 and 57 baseline tumors and analyzed for next generation sequencing approaches to interrogate transcriptomes (RNA-seq) and methylomes (Infinium Human Methylation450 (HM450) arrays), respectively. Differential gene expression and DNA methylation profiles were generated and used for Ingenuity Pathway Analysis (IPA) to identify the top altered pathways in response to guadecitabine. Expression of DNMTs was examined by real-time RT-PCR and immunohistochemistry. LINE1 methylation and promoter methylation of selected genes (MAGE-A2, MAGE-A3, MAGE-A11, NY-ESO, RASSF1, MLH1, and HOXA11) were quantified by pyrosequencing before and after guadecitabine treatment (n=12 paired samples). Results: Analysis of a limited number of paired samples before and after treatment (n=8) revealed significant changes in global gene expression profiles induced by guadecitabine, with 960 altered genes representing immunopathway enrichment including cytokine production in macrophages and T helper cells by IL-17A and IL-17F, granulocyte /agranulocyte adhesion and inflammation, IL-8 signaling, p38 MAPK signaling, cAMP-mediated signaling, and innate immunity. Epigenetic profiling using HM450 revealed extensive methylation changes when comparing recurrent platinum resistant ovarian tumors (n=42) to primary, untreated ovarian cancer specimens analyzed as part of the TCGA project (n=10). Six hundred and four promoters were significantly differentially methylated (adjusted p 0.2), among which, 498 and 106 were hypermethylated or hypomethylated respectively in recurrent platinum resistant ovarian tumors. IPA analysis of baseline tumor transcriptome and methylome demonstrated significant enrichment in a wide range of pathways associated with cancer, stem cells, inflammation and the immune system. DNMT1, 3A, and 3B mRNA levels in the tumors were highly variable (n=19). Analysis of a limited number of paired samples (n=7) revealed no significant changes in global methylation or in DNMT expression levels induced by treatment with guadecitabine (adjusted p>0.05). However, the DNMT inhibitor induced significant methylome alterations in selected patients. Significant hypomethylation of MAGE-A3 and MAGE–A11 promoters (p Conclusions: These data suggest that treatment with the DNMT inhibitor guadecitabine induces a reactivation of immune responses in human OC. Correlations between methylation changes and expression profiles are being explored. Citation Format: Fang Fang, Horacio Cardenas, Dave Miller, Aaron Buechlein, Qing Yu, Yunlong Liu, Guanglong Jiang, Pietro Taverna, Harold Keer, Doug Rusch, Daniela Matei, Kenneth P. Nephew. Epigenome and genome alterations in platinum resistant ovarian tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A70.

Abstract 4024: Epigenetic priming potentiates immune checkpoints inhibitors in ovarian cancer

Background: Ovarian cancer (OC) progression is accompanied by the establishment of stable and transcriptionally repressive epigenetic modifications. An important mechanism of immune evasion is represented by epigenetic silencing of tumor antigens (NY-ESO-1, Muc16 and MAGE). We hypothesize that by reversing DNA methylation, DNA methyl transferase inhibitors (DNMTIs) restore the expression of such antigens, potentiating anti-tumor immune response. The targeting of immune checkpoints regulated by programmed cell death protein-1(PD-1) signaling represents a novel therapeutic strategy in cancer, including in OC. Here we set out to measure the anti-tumor effects of epigenetic priming in combination with PD-1/PDL-1 blockade in OC preclinical models. Methods: The ID8 intraperitoneal (ip) immunocompetent syngeneic mouse model was used to measure the effects of the novel DNMTI guadecitabine (SGI-110, Astex Pharmaceuticals Inc) and PDL1 blockade. The experimental groups consisted of non-specific IgG (control), guadecitabine 2mg/m2 sq bi-weekly, murine anti-PDL1 inhibitory antibody (10mg/kg) bi-weekly and combination of guadecitabine with anti-PDL1 antibody (n = 6 mice/group, 3 week treatment). Immune cells collected from the ascites and spleens of tumor bearing mice were either directly processed or co-cultured with ID8 cells for 48 hours. Cells were immuno-phenotyped by flow cytometry. In OC cells treated with guadecitabine, changes in gene expression were analyzed by real time-PCR. Results: In ID8 tumors bearing mice, the combination of PDL1 blockade with guadecitabine significantly decreased primary tumor formation (P Conclusions: Guadecitabine in combination with anti-PDL1 antibody induced striking anti-tumor effects in an immunocompetent OC syngeneic model by activating cytotoxic T-cells. These data support clinical strategies utilizing epigenetic priming using DNMTI in combination with immune checkpoints inhibitors. Citation Format: Yu Qing, Salvatore Condello, Katie J. Meyer, Andrea Caperell-Grant, Kenneth P. Nephew, Daniela Matei. Epigenetic priming potentiates immune checkpoints inhibitors in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4024.

Abstract CT222: Differences in pharmacokinetics of TRC105 (anti-endoglin antibody) when administered as a single agent versus in combination with bevacizumab (Bev)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnBackground: TRC105 is an anti-endoglin chimeric monoclonal antibody that inhibits angiogenesis and tumor growth and is being studied in randomized Phase 2 trials with Bev. TRC105 is cleared through binding to endoglin expressed on proliferating endothelium when given as a single agent to cancer patients (Spencer et al, ASCO 2012). Preclinical data indicate that endoglin expression is increased in response to VEGF targeted treatment, and increased endoglin expression in response to Bev may increase the clearance of TRC105 when administered in combination to cancer patients.nnMethods: Patients (pts) with solid tumors (ST) or ovarian cancer (OC) received 10 mg/kg/wk TRC105 as a single agent, and additional patients with ST (primarily ovarian and colorectal) received 10 mg/kg/wk TRC105 with Bev. Peak and trough levels were assessed by ELISA in 39 patients who received TRC105 alone and compared to that of 35 patients (largely Bev refractory) who received TRC105 and Bev. Pts administered TRC105 and Bev were considered a population sub-group and treated as a covariate. A population pharmacokinetic model of TRC105 disposition was built using rich sampling from the ST trial, with sparse data from OC and TRC105+Bev included in the base model. A two-compartment model with nonlinear elimination best fit the data, utilizing Michaelis-Menten parameters for saturable clearance.nnResults: TRC105 peak and trough concentrations exceeded target serum concentrations of TRC105 known to saturate endoglin receptors in all pts dosed with 10 mg/kg/wk of TRC105 with and without Bev. The PK of TRC105 given with Bev had mean predicted (following 10,000 simulations) parameters of volume of distribution in the central compartment (VC), VMAX, and KM that were increased compared to population estimated parameters of TRC105 given as a single agent [VC= 44.5±2 (SE) (mL/kg) Pop mean estimate vs. 68.8±4 (mL/kg) Bev predicted; VMAX = 92.6±16 (μg/hr) vs. 297.5±40 (μg/hr) predicted, and KM= 5.91±2 (μg/mL) vs. 61.6±8.8 (μg/mL)]. All differences were significant (p<0.001). Observable data from patients administered the combination yielded PK parameters that were consistent with increased target-mediated clearance of TRC105 when given with Bev.nnConclusions: Peak and trough TRC105 serum levels exceed target serum concentrations when given at 10 mg/kg/wk as a single agent or with Bev. Central compartment distribution of TRC105 increased when given with Bev, which is consistent with increased endoglin expression on proliferating endothelium following Bev treatment. The maximum rate of elimination (Vmax) also increased, consistent with increased turnover; however the intrinsic clearance ratio of Vmax/Km remained the same, suggesting no change in endoglin turnover efficiency. Future studies will assess whether PK parameters correlate with responses to the combination of TRC105 and Bev in Bev refractory patients.nnCitation Format: Shawn D. Spencer, Lee S. Rosen, Michael S. Gordon, Francisco Robert, Daniela Matei, Cody J. Peer, Bonne Adams, Delia Alvarez, Ben K. Seon, Charles P. Theuer, W. Douglas Figg. Differences in pharmacokinetics of TRC105 (anti-endoglin antibody) when administered as a single agent versus in combination with bevacizumab (Bev). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT222. doi:10.1158/1538-7445.AM2014-CT222

Abstract 2317: SGI-110 alters ovarian cancer stem cells to prevent recurrent and chemoresistant ovarian cancer

Ovarian cancer stem cells (OCSCs) are associated with drug resistance and tumor relapse. Epigenetic aberrations, especially DNA methylation, result in silencing of tumor suppressor and differentiation-associated genes and regulate OCSCs9 survival. To test the hypothesis that DNA hypomethylating agents can “reset” OCSCs towards differentiation, we investigated the effect of the DNA methytransferase inhibitor SGI-110 on OCSCs, defined by aldehyde dehydrogenase 1 (ALDH)(+) cells. We treated ALDH(+) cells from platinum-sensitive A2780 and platinum-resistant A2780-cp OC cells with SGI-110 (100nM, 72 hr) or with cisplatin (CDDP; 1.67M, 24hr). After a 4 day recovery period, %ALDH+ was analyzed using FACS, cell viability was determined, and ALDH(+) cells were grown as spheroids in culture or as xenografts in mice. The overall %ALDH+ cells was higher (P Citation Format: Yinu Wang, Horacio Cardenas, Fang Fang, Salvatore Condello, Pietro Taverna, Gavin Choy, Mohammad Azab, Kenneth Nephew, Daniela Matei. SGI-110 alters ovarian cancer stem cells to prevent recurrent and chemoresistant ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2317. doi:10.1158/1538-7445.AM2014-2317

887PTRC105 (ANTI-ENDOGLIN ANTIBODY) IN COMBINATION WITH BEVACIZUMAB (BEV) AND AS A SINGLE AGENT FOR PLATINUM RESISTANT OVARIAN CANCER

ABSTRACT Aim: Endoglin (CD105) is an endothelial cell membrane receptor densely expressed on tumor vessels that is essential for angiogenesis and upregulated by hypoxia and VEGF inhibition. TRC105 is an anti-endoglin monoclonal antibody that potentiates BEV in preclinical models. Methods: Patients with advanced metastatic ovarian cancer, ECOG PS 0-1, and normal organ function were treated with escalating doses of IV TRC105 (3, 6, 8 or 10u2003mg/kg/wk) plus BEV at 15u2003mg/kg q3wk or 10u2003mg/kg q2wk (n = 11) or with 10u2003mg/kg/wk TRC105 as a single agent (n = 23). Patients were assessed for safety, pharmacokinetics and response. Results: TRC105 was well tolerated as a single agent in 23 patients with advanced platinum resistant ovarian cancer (median age = 63; median 2 prior therapies; range 1-5). The combination of TRC105 and BEV was well tolerated at the recommended single agent doses (10u2003mg/kg) when the initial dose of TRC105 was divided over two days to limit the frequency of headache. The concurrent administration of BEV and TRC105 did not otherwise potentiate known toxicities of TRC105 or BEV. Target TRC105 serum concentrations were achieved in all patients who received the combination or single agent TRC105 at 10u2003mg/kg. Mucocutaneous telangiectasia, a marker of TRC105 target modulation, was observed at higher rates with the combination of the two drugs (64% vs 35%; p = 0.15). Reductions in CA-125 were noted in 7 of 20 patients treated with TRC105 as a single agent and 7 of 11 treated with the combination, including 5 of 8 patients who received prior BEV or VEGF receptor tyrosine kinase inhibitor (VEGFR TKI) therapy. Radiographic reductions in tumor volume were observed in 7 of 11 patients (64%) treated with the combination including 4 of 8 patients who received prior BEV or VEGFR TKI, including two patients with PFS > 6 months; one of whom had a partial response by RECIST 1.1. Conclusions: TRC105 10u2003mg/kg wkly was well tolerated with and without BEV 10u2003mg/kg q2wk in patients with advanced platinum resistant ovarian cancer. The combination demonstrated activity in BEV and VEGFR TKI refractory patients. Disclosure: A.A. Garcia: The institution received funding for participating in the study; E.A. Alvarez: Dr. Alvarez receives compensation from TRACON Pharmaceuticals, Inc for service on the Safety Review Board Committee; D.S. Mendelson: Institution received funding to conduct the study; B.K. Seon: I am an inventor of TRC105. I do not own stock of TRACON Pharma. I am not a member on the advisory board or board of directors of TRACON Pharma. I had a corporate-sponsored research agreement with TRACON Pharma; D. Alvarez, B.J. Adams and C.P. Theuer: I am employed by TRACON Pharma and own stock in the company. M. Gordon: Institution received funding to conduct the study. All other authors have declared no conflicts of interest.

Abstract IA17: Targeting the methylome for epigenetic resensitization of ovarian cancer

Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. Although most patients respond to platinum-based chemotherapy, relapses are common, leading to platinum-resistant OC, which is uniformly fatal. OC progression is associated with accumulation of epigenetic alterations. In particular, deoxycytosine methylation of CpG islands in promoter regions of tumor suppressor genes (TSGs) plays a prominent role in the development and progression of drug-resistant epithelial OC. We recently demonstrated for the first time in a clinical trial that therapeutic interventions targeting the OC methylome reverse drug resistance and induce meaningful clinical responses. We showed that repetitive low-dose decitabine reactivated silenced genes and restored sensitivity to carboplatin, providing strong clinical and biological support for further study of hypomethylating agents in heavily pre-treated, platinum-resistant ovarian cancer patients. While the FDA-approved demethylating agent decitabine is prone to deamination by cytidine deaminase, SGI-110 (Astex Pharmaceuticals, Inc.), a dinucleotide analogue of decitabine, is more stable, less toxic, and a promising alternative to restoring silenced TSG expression in cancer cells by reversal of DNA methylation. Our preclinical evaluation demonstrated that SGI-110 resensitized platinum-resistant OC cell lines to cisplatin (CDDP) (3-fold reduction in IC50) and reduced the CDDP IC50. SGI-110 treatment induced significant demethylation and subsequent transcriptional derepression of tumor suppressors and differentiation-associated genes in OC cells. SGI-110 alone or in combination with CDDP was well tolerated in non-tumor bearing mice. Significant antitumor activity was observed in mice harboring subcutaneous OC tumors and treated with single SGI-110 and SGI-110 + CDDP treatment in both a biweekly and daily (QD5) regimen. In addition to reducing tumor growth in xenografts, SGI therapy was effective in causing global as well as TSG demethylation and gene reexpression in vivo. Furthermore, the antitumor activity of SGI-110 was associated with reduced chromatin compaction, allowing greater CDDP intercalation into DNA, as assessed by increased DNA platinum adducts in SGI-treated OC cells. The results of our preclinical study support our recently activated clinical trial NCT01696032 using SGI-110 in combination with carboplatin in patients with recurrent, platinum-resistant OC. Clinical specimens (tumor and plasma samples) will be analyzed for epigenetic biomarker changes. We seek to bring forward the new concept of epigenetic targeting in platinum resistant OC by priming the tumors with SGI-110 and set the stage for interventions targeting the OC epigenome, as well as guide and impact the design of future clinical investigations in OC. Citation Format: Kenneth P. Nephew, Daniela Matei, Pietro Taverna, Fang Fang, Jessica Tang, Gavin Choy, John Lyons, Mohammad Azab, Jay Pilrose, John Turchi. Targeting the methylome for epigenetic resensitization of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA17.

Abstract 2978: DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnEpithelial-to-mesenchymal transition (EMT) is a required step in the process of metastasis and has been linked to cancer cell stemness. We hypothesized that aside from defined genetic changes associated with EMT, epigenetic mechanisms are also involved. We determined DNA methylation changes in SKOV3 ovarian cancer (OC) cells undergoing TGFβ-induced EMT by using the Infinium HumanMethylation450 array. Methylation of sites was expressed as s-values ranging from 0 (completely unmethylated) to 1 (completely methylated). Methylation of 468 and 390 sites changed significantly (P 1.25-fold. The majority of changes (∼66%) reflected TGFβ-induced demethylation. Methylation of 160 sites changed similarly at 48 and 120h indicating that approximately 40% of changes were sustained. PCA analysis demonstrated that the removal of TGFβ was associated with reversal of DNA methylation changes at most sites and restitution of the baseline methylome. Pathway analysis identified DNA methylated genes involved in functional networks relevant to EMT and cancer progression, including: 1) cell morphology and development (39 genes), 2) cell growth and proliferation, and cell death and survival (34 genes), 3) cellular assembly and organization (22 genes), 4) DNA replication, recombination and repair (17 genes), and 5) gene expression, cell signaling, and cellular movement (15 genes). Validation of the relevant genes is ongoing. Treatment of OC cells with the DNA methyl transferase inhibitor SGI-110 prevented TGFβ-induced EMT and reduced by two-fold the number of cells with stem cell characteristics (ALDH1+). These results demonstrate that DNA methylation is a dynamic process involved in regulation of genes implicated in EMT and metastasis.nnCitation Format: Horacio Cardenas, Chirayu Goswami, Kenneth P. Nephew, Daniela Matei. DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2978. doi:10.1158/1538-7445.AM2013-2978

Abstract 4081: In vivo effects of decitabine on global gene expression profiles in tumors from patients with platinum-resistant ovarian cancer

Deoxycytosine methylation of CpG islands within promoter regions of tumor suppressor genes (TSGs) plays a prominent role in the development and progression of drug-resistant epithelial ovarian cancer (OC). Recently, in a phase I/II trial of the DNA methylation inhibitor decitabine in combination with carboplatin in platinum-resistant OC patients (NCT00477386), we demonstrated that decitabine-altered DNA methylation resulted in platinum resensitization and significant clinical activity, with a response rate of 35% and median PFS of 10 months (ASCO abstract #79158). The objective of the current study was to investigate the molecular basis underlying the clinical response observed. For this, we compared differential gene expression in paired tumor biopsies and ascitic fluid collected before and one week after decitabine treatment. Of the 17 patients enrolled in NCT00477386, samples evaluable from 14 patients were subjected to global gene expression profiling using GeneChip Human Gene 1.0 St arrays, and selected genes were validated by quantitative RT-PCR. Data analysis was performed using significance of microarray analysis (SAM), clustering, functional pathway prediction using DAVID, gene ontology (GO), and gene set enrichment analysis (GSEA). In the patients with progression free survival (PFS) greater than six months (n=6), 274 genes were uniquely upregulated by decitabine. Enriched signaling pathways included Hedgehog, focal adhesion, and gap junction. Specifically, genes upregulated (fold change >1.2, P 6 months compared to patients with PFS 6 months and decreased in patients with PFS 6 months, including 33 genes of the “stemness” and chemoresistance-associated Wnt pathway. Notably, underexpression of those 33 genes has been correlated with prolonged survival in previous ovarian cancer studies. In summary, these findings show that repetitive low-dose decitabine reactivates silenced genes and restores sensitivity to carboplatin, providing strong clinical and biological support for further study of hypomethylating agents in heavily pre-treated, platinum-resistant ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4081. doi:1538-7445.AM2012-4081