HDAC Inhibitor Entinostat Research Articles

Histone Deacetylase Inhibitor Entinostat Inhibits Tumor-Initiating Cells in Triple-Negative Breast Cancer Cells.

Mortality following breast cancer diagnosis is mainly due to the development of distant metastasis. To escape from the primary site, tumor cells undergo the epithelial-to-mesenchymal transition (EMT), which helps them acquire a more motile and invasive phenotype. In our previous study, we showed that class I selective HDAC inhibitor entinostat reverses the EMT phenotype through reversal of epigenetic repression of E-cadherin. Recent evidence suggests that a subset of cells within a breast tumor may drive the metastatic outgrowth following escape from the primary site. These cells, termed tumor-initiating cells (TIC), represent a great threat to overall prognosis. They are critical in terms of drug resistance and tumor initiation at metastatic sites. Acquisition of EMT traits has also been shown to impart TIC phenotype to the cells, making EMT a "dual-threat" for prognosis. In the current study, we show that entinostat treatment can reduce the percentage of TIC cells from triple-negative breast cancer (TNBC) cells. Entinostat treatment was able to reduce the CD44(high)/CD24(low) cell population, ALDH-1 activity, as well as protein and mRNA expression of known TIC markers such as Bmi-1, Nanog, and Oct-4. Next, we inoculated MDA-MB-231 cells transfected with firefly luciferase (231/Luc) in mammary fat pad of NSG mice. The mice were then treated with entinostat (2.5 mg/kg/d), and tumor development and formation of metastasis were assessed by bioluminescence imaging. Treatment with entinostat significantly reduced tumor formation at the primary site as well as lung metastasis. As such, entinostat may help prevent development of distant metastasis.

Bcl-xl and Mcl-1 are the major determinants of the apoptotic response to dual PI3K and MEK blockage

The dual targeting of PI3K-AKT-mTOR and Ras-Raf-MEK-ERK pathways is a potential anticancer therapy, but unfortunately, the response rate has been low in early phase clinical trials. Pre-clinical models have suggested that an apoptotic response to dual PI3K and MEK targeting is relatively rare and understanding apoptotic avoidance could lead to increased clinical efficiency. This study investigated solid cancer cell lines, which are known to be sensitive to dual PI3K and MEK inhibition but to have a limited apoptotic response. The cells were exposed to dual PI3K and MEK blockage in combination with a panel of additional pharmacological agents and cytotoxicity and apoptosis were analyzed. Our results indicated that the BH3 mimetic ABT-263, the HDAC inhibitor entinostat and the multikinase inhibitor dasatinib increased the cytotoxicity and apoptotic response of dual PI3K and MEK targeting. Furthermore, ABT-263 and entinostat was able to induce apoptosis in combination with single agent PI3K and MEK inhibitors. Protein expression, immunoprecipitation and siRNA knockdown models suggested that Bcl-xl and Mcl-1 were the most important factors circumventing PI3K and/or MEK inhibition-mediated apoptosis. The results suggest that the cytotoxicity of PI3K and/or MEK inhibitor treatments can be augmented by combinatory approaches targeting anti-apoptotic mediators Bcl-xl and Mcl-1.

Abstract 5384: Systematic high-throughput drug sensitivity and resistance testing (DSRT) of ovarian cancer cell lines indicates novel therapeutic possibilities with existing and emerging drugs

Abstract There is an urgent need to develop better understanding of the drug responsivenes in ovarian cancer (OC), particularly in the clear cell and mucinous subtypes, which tend to be resistant to the commonly used platinum-based regimens. This would help to improve survival and facilitate precision medicine approaches to therapy. In order to discover previously unsuspected anti-cancer effects with approved and emerging drugs, we applied Drug Sensitivity and Resistance Testing (DSRT) technology (Pemovska et al., Cancer Discovery, 2013) to determine detailed dose-response effects of 300 drugs in 25 established OC cell lines (10 serous, 10 clear cell and 5 mucinous ovarian cancer cell lines). The panel covered 160 approved drugs as well as 162 emerging, investigational and pre-clinical oncology compounds such as kinase and non-kinase inhibitors. All drugs were tested in 5 different concentrations covering a 10,000-fold concentration range using Labcyte nano-dispensing technology, in order to generate detailed dose-response curves for each drug in each cell line. The area under the curve was estimated from the dose-response curve to obtain the drug sensitivity score. Bioinformatic processing of the drug response data from OC cell lines resulted in several key observations. First, the DSRT data made it possible to classify OC cell lines into four functional taxonomic subtypes based on comprehensive drug responses. Interestingly, these clusters did not depend on the histological origin of the OC cell lines. Second, the 322 drugs were clustered into functional subsets based on the response data across the 25 OC cell lines. Most of this clustering followed the expected chemical similarity and target space of the drugs, such as topoisomerase II inhibitors, MEK inhibitors, mTOR inhibitors and Taxanes each clustering in their own subgroups. Third, we found that many emerging, currently not-yet-approved drugs were active in subgroups of OC cell lines, including both kinase as well as non-kinase drugs, such as HDAC inhibitors Entinostat and Belinostat. DSRT technology provides a powerful strategy for assessing drug response in OC cell models, and it could also help to asses drug responses in patient-derived ex vivo model systems of OC. Analysis of correlations of DSRT data with genomic data of the cell lines is underway and will yield new translational and pharmacogenomic opportunities for OC. Citation Format: Akira Hirasawa, Astrid Murumägi, Mariliina Arjama, Bhagwan Yadav, John Patrick Mpindi, Krister Wennerberg, Tero Aittokallio, Daisuke Aoki, Olli Kallioniemi. Systematic high-throughput drug sensitivity and resistance testing (DSRT) of ovarian cancer cell lines indicates novel therapeutic possibilities with existing and emerging drugs. [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 5384. doi:10.1158/1538-7445.AM2014-5384

HDAC inhibitors restore the capacity of aged mice to respond to haloperidol through modulation of histone acetylation.

Antipsychotic drugs are widely prescribed to elderly patients for the treatment of a variety of psychopathological conditions, including psychosis and the behavioral disturbances associated with dementia. However, clinical experience suggests that these drugs may be less efficacious in the elderly individuals than in the young. Recent studies suggest that aging may be associated with epigenetic changes and that valproic acid (VPA), a histone deacetylase inhibitor, may reverse such changes. However, it is not yet known whether HDAC inhibitors can modulate age-related epigenetic changes that may impact antipsychotic drug action. In this study, we analyzed conditioned avoidance response (CAR) and c-Fos expression patterns to elucidate the effect of HDAC inhibitors VPA and entinostat (MS-275) on behavioral and molecular markers of the effects of haloperidol (HAL) in aged mice. Our results showed that HAL administration failed to suppress the avoidance response during the CAR test, suggesting an age-related decrease in drug efficacy. In addition, HAL-induced c-Fos expression in the nucleus accumbens shell and prefrontal cortex was significantly lower in aged mice as compared with young mice. Pretreatment with VPA and MS-275 significantly improved HAL effects on the CAR test in aged mice. Also, VPA and MS-275 pretreatment restored HAL-induced increases in c-Fos expression in the nucleus accumbens shell and prefrontal cortex of aged mice to levels comparable with those observed in young mice. Lastly, but most importantly, increases in c-Fos expression and HAL efficacy in the CAR test of the HAL+VPA and HAL+MS-275 groups were correlated with elevated histone acetylation at the c-fos promoter region in aged mice. These findings suggest that pretreatment with VPA or MS-275 increases the behavioral and molecular effects of HAL in aged mice and that these effects occur via modulation of age-related histone hypoacetylation in the nucleus accumbens shell and prefrontal cortex.

Abstract YR01: New treatment options in metastatic breast cancer

Abstract During 2013 there have been significant new data from various clinical trials of systemic therapies for metastatic breast cancer (MBC) – the issue is to what extent has this changed clinical practice. The most significant results continue to be in HER2+ MBC where novel HER2 targeted drugs given either alone or in combination with trastuzumab have re-defined the optimal treatment strategy. It would appear that dual combination therapy is superior to trastuzumab alone. In the CLEOPATRA trial the combination of the two monoclonal antibodies pertuzumab & trastuzmab with docetaxel showed a very significant improvement in progression-free survival (18.5 vs 12.4 months) compared with trastuzumab-docetaxel therapy, and in 2013 this became a new standard of care in the first-line setting. Following the results of the EMILIA trial which showed a substantial improvement in PFS (9.6 vs 6.4 months) for the antibody conjugate T-DM1 compared with lapatinib and capecitabine in the second-line setting, in the USA this option has become an approved second-line therapy. In addition, compelling data were published this year showing superiority of T-DM1 compared with docetaxel-tratsuzumab in the first-line setting. The role of lapatinib remains more uncertain, although the combination with trastuzumab recently received approval in hormone receptor negative HER2+ MBC previously treated with trastuzmab and chemotherapy. In ER+ breast cancer attempts are continuing to prevent or reverse endocrine resistance. It is clear that different endocrine therapies, including the combination of fulvestrant and anastrozole provide only 3-4 months benefit following progression on a non-steroidal aromatase inhibitor as shown in the SoFEA trial. This is in contrast to the very positive data reported for the mTOR inhibitor everolimus combined either with exemestane (BOLERO-2) or tamoxifen (TAMRAD), an approach that has become a new standard of care for endocrine resistant breast cancer. Whether benefit exists in the untreated 1st line setting for this approach remains unclear, as demonstrated by negative results from the HORIZON study. Attempts to see whether biomarkers of the PI3-kinase pathway might predict those who benefit from mTOR inhibition were presented at ASCO 2013, but failed to identify any single predictive profile. For other signalling therapies in ER+ MBC, positive results were published this year for the combination of the HDAC inhibitor entinostat with exemestane, while negative results were reported for the combination of the IGF-1R mAb therapy ganitumab with endocrine therapy. While progress with systemic therapies has been rapid with novel therapeutics in both ER+ and HER2+ breast cancer, for other subtypes (ie. triple negative MBC) no new therapeutics have emerged during the last 12 months. Instead, prospective studies (SAFIR0l) are providing personalised targeted treatment options based on molecular characterisation of key mutations / amplifications in metastatic biopsy samples, in the hope that this will ultimately provide a more efficient clinical development pathway for novel therapeutics in MBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr YR01.

Entinostat Prevents Leukemia Maintenance in a Collaborating Oncogene-Dependent Model of Cytogenetically Normal Acute Myeloid Leukemia

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis, and therapeutic intervention based on improved patient stratification. Relevant preclinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML), and a conditional transplantation mouse model was developed that demonstrated oncogene dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity map analysis identified Entinostat as a drug with the potential to alter the leukemic condition toward the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation, and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML.

Cross-species synthetic lethal interaction screening as a strategy for the identification of novel therapeutic targets in cancer.

11105 Background: Mutation, deletion, or epigenetic silencing of tumor suppressor genes is a near universal feature of malignant cells. However, therapeutic strategies for restoring the function of mutated or deleted genes have proven difficult. Synthetic lethality, an event in which the simultaneous perturbation of two genes results in cellular death, has been proposed as a method to selectively target cancer cells. Identifying and pharmacologically inhibiting proteins encoded by genes that are synthetic lethal with known tumor suppressor mutations should result in selective toxicity to tumor cells. Methods: To identify candidate target proteins we measured all pair-wise genetic interactions between all known orthologs of human tumor suppressor genes (162 genes) and all orthologs of druggable human proteins (~400 genes) in the model organism S. Cerevisiae. Analysis of the data uncovered 2,087 distinct synthetic lethal interactions between a tumor suppressor and druggable gene. A computational algorithm was then developed to identify those interactions which were likely to be conserved in humans based on conservation of the synthetic lethal relationship in the distant fission yeast S. pombe. Results: Our bioinformatic analysis suggested a high probability of conservation of the synthetic lethal interactions between the yeast RAD51 (ortholog of BRCA1) and RAD57 (ortholog of XRCC3) with HDA1 (a histone deacetylase; HDAC). We confirmed this by treating LN428 cells with stable lentiviral knockdown of BRCA1 or XRCC3 with the HDAC inhibitors vorinostat (SAHA) and entinostat (MS-275). Both the BRCA1 and XRCC3 knockdown cell lines were significantly more sensitive to HDAC inhibition relative to wild-type (non-silencing lentiviral control) cell line (Table). Conclusions: These results demonstrate that high-throughput approaches for screening synthetic lethal interactions in model organisms such as S. cerevisiae and S. pombecan serve as a valuable resource in helping to identify novel therapeutic targets in human cancer. [Table: see text]

Abstract 679: SGI-110 and entinostat therapy reduces lung tumor burden and reprograms the epigenome .

Abstract The DNA methyltransferase inhibitors (DNMTi) Vidaza (5-azacytidine) and Dacogen (5-aza-2′-deoxycytidine, decitabine) are approved by the FDA for chemotherapy against myelodysplastic syndrome, a heterogeneous bone marrow disorder. Vidaza in combination with the HDAC inhibitor entinostat has shown some promise in treating lung cancer and this has been replicated in our orthotopic lung cancer model. However, the effectiveness of these DNMTi against solid tumors is likely impacted in part, by their stability and rapid inactivation by cytidine deaminase in the liver. These studies were initiated to test the efficacy of SGI-110, a prodrug for decitabine. SGI-110, a dinucleotide consisting of 5-aza-2′-deoxycytidine followed by a deoxyguanosine, has been shown to be effective in vitro as a DNA methylation inhibitor and less prone to deamination by cytidine deaminase. In this study, we used our in vivo lung cancer model to explore the potential therapeutic effect of SGI-110 alone and in combination with entinostat. In vivo plasma concentrations and pharmacokinetic properties of SGI-110 and its metabolite decitabine were evaluated in nude rats after a single SC dose of SGI-110. Upon SC administration, SGI-110 is rapidly converted to decitabine. Dose proportional pharmacokinetics showed no significant difference in accumulation of SGI-110 or decitabine, while the half life of decitabine was increased to 4hrs. SGI-110 alone or in combination with entinostat significantly reduced tumor burden by 56% of Calu-6, a K-ras/p53 mutant lung adenocarcinoma cell line engrafted orthotopically in nude rats. On a molecular level, global changes in gene profiles were associated with a reduction in tumor burden. Microarray analysis revealed that a 212 and 592 genes were differentially expressed in SGI-110 alone or with combination therapy. Highly significant gene expression changes were seen in key cancer regulatory pathways. In addition, SGI-110 treatment activated p21 and apoptotic genes BIK and BAK in these tumors. SGI-110 caused widespread epigenetic reprogramming that induced demethylation of an average of 291 genes; in particular 208 genes were polycomb target genes. The polycomb targets were enriched for transcription factors. These preclinical in vivo findings demonstrate that SGI-110 alone shows strong demethylating activity and when combined with entinostat more effectively retards tumor growth. (Supported by Astex Pharmaceutical and SU2C) Citation Format: Carmen S. Tellez, Marcie J. Grimes, Maria A. Picchi, Yushi Liu, Tom H. March, Aram Oganesian, Pietro Taverna, Steven A. Belinsky. SGI-110 and entinostat therapy reduces lung tumor burden and reprograms the epigenome . [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 679. doi:10.1158/1538-7445.AM2013-679

Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells

We reported that the class I HDAC inhibitor entinostat induced apoptosis in erbB2-overexpressing breast cancer cells via downregulation of erbB2 and erbB3. Here, we study the molecular mechanism by which entinostat dual-targets erbB2/erbB3. Treatment with entinostat had no effect on erbB2/erbB3 mRNA, suggesting a transcription-independent mechanism. Entinostat decreased endogenous but not exogenous erbB2/erbB3, indicating it did not alter their protein stability. We hypothesized that entinostat might inhibit erbB2/erbB3 protein translation via specific miRNAs. Indeed, entinostat significantly upregulated miR-125a, miR-125b, and miR-205, that have been reported to target erbB2 and/or erbB3. Specific inhibitors were then used to determine whether these miRNAs had a causal role in entinostat-induced downregulation of erbB2/erbB3 and apoptosis. Transfection with a single inhibitor dramatically abrogated entinostat induction of miR-125a, miR-125b, or miR-205; however, none of the inhibitors blocked entinostat action on erbB2/erbB3. In contrast, co-transfection with two inhibitors not only reduced their corresponding miRNAs, but also significantly abrogated entinostat-mediated reduction of erbB2/erbB3. Moreover, simultaneous inhibition of two, but not one miRNA significantly attenuated entinostat-induced apoptosis. Interestingly, although the other HDAC inhibitors, such as SAHA and panobinostat, exhibited activity as potent as entinostat to induce growth inhibition and apoptosis in erbB2-overexpressing breast cancer cells, they had no significant effects on the three miRNAs. Instead, both SAHA- and panobinostat-decreased erbB2/erbB3 expression correlated with the reduction of their mRNA levels. Collectively, we demonstrate that entinostat specifically induces expression of miR-125a, miR-125b, and miR-205, which act in concert to downregulate erbB2/erbB3 in breast cancer cells. Our data suggest that epigenetic regulation via miRNA-dependent or -independent mechanisms may represent a novel approach to treat breast cancer patients with erbB2-overexpressing tumors.

Abstract LB-473: The HDAC inhibitor entinostat targets erbB2/erbB3 receptors via functional cooperation of miR-125a, miR-125b, and miR-205 in breast cancer cells

Abstract Breast cancer is highly heterogeneous and has been classified into five distinct subtypes, among which erbB2-overexpressing (erbB2+) and basal breast cancers have the least favorable prognosis and express erbB receptors that are excellent targets for anti-cancer therapies. We previously reported that the class I HDAC inhibitor, entinostat (also known as MS-275, SNDX-275, Syndax Pharmaceuticals, Inc., Waltham, MA) selectively inhibited proliferation and induced apoptosis in erbB2+ breast cancer cells. Entinostat preferably targeted erbB2 and erbB3 receptors rather than the epidermal growth factor receptor (EGFR). Here, we study the molecular mechanism through which entinostat downregulates erbB2/erbB3 and promotes the breast cancer cells undergoing apoptosis. We showed that entinostat dramatically reduced the protein levels of erbB2/erbB3, whereas it had no significant effect on their mRNA expression in MDA-MB-453 and BT474 breast cancer cells. In addition, entinostat specifically decreased the endogenous, but not the exogenous erbB2/erbB3, suggesting that it did not promote erbB2/erbB3 protein degradation. Therefore, we hypothesizes that entinostat reduces erbB2/erbB3 receptors through transcription-independent mechanism; it may inhibit erbB2/erbB3 protein translation via induction of specific miRNA. Quantitative real-time (qRT) PCR revealed that entinostat significantly upregulated the expression of three erbB2/erbB3-targeting miRNAs, miR-125a, miR-125b, and miR-205 in erbB2+ breast cancer cells. Specific miRNA inhibitors were used to determine whether these miRNAs played a causal role in entinostat-induced downregulation of erbB2/erbB3 and apoptosis. While inhibition of any one of the three miRNAs failed to block entinostat action, simultaneous inhibition of any two of them significantly abrogated entinostat-induced erbB2/erbB3 reduction and apoptosis. Collectively, these data demonstrate that entinostat targets erbB2/erbB3 receptors via functional cooperation of miR-125a, miR-125b, and miR-205. Our findings suggest that miRNA-mediated epigenetic regulation may represent a novel strategy targeting erbB2/erbB3 to treat the aggressive erbB2+ breast cancer. The combinations of entinostat and erbB2-targeted therapy may be more efficacious for breast cancer patients whose tumors overexpress erbB2. This hypothesis will be directly tested by a recently initiated clinical study determining the activity of entinostat in combination with lapatinib in breast cancer patients that are erbB2+ and progressed on trastuzumab. 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 LB-473. doi:1538-7445.AM2012-LB-473

Functional Activation of the Estrogen Receptor-α and Aromatase by the HDAC Inhibitor Entinostat Sensitizes ER-Negative Tumors to Letrozole

Approximately 25% of breast cancers do not express the estrogen receptor-α (ERα) and consequently do not respond to endocrine therapy. In these tumors, ERα repression is often due to epigenetic modifications such as methylation and histone deacetylation. For this reason, we investigated the ability of the histone deacetylase inhibitor entinostat (ENT) to trigger reexpression of ERα and aromatase in breast cancer cells, with the notion that this treatment would restore sensitivity to the aromatase inhibitor (AI) letrozole. ENT treatment of tumor cells increased expression of ERα and aromatase, along with the enzymatic activity of aromatase, in a dose-dependent manner both in vitro and in vivo. Notably, ERα and aromatase upregulation resulted in sensitization of breast cancer cells to estrogen and letrozole. Tumor growth rate was significantly lower in tumor xenografts following treatment with ENT alone and in combination with letrozole than in control tumors (P > 0.001). ENT plus letrozole also prevented lung colonization and growth of tumor cells, with a significant reduction (P > 0.03) in both visible and microscopic foci. Our results show that ENT treatment can be used to restore the letrozole responsiveness of ER-negative tumors. More generally, they provide a strong rationale for immediate clinical evaluation of combinations of histone deacetylase and aromatase inhibitors to treat ER-negative and endocrine-resistant breast cancers.

Abstract PD05-03: HDAC Inhibitor Entinostat Restores Responsiveness of Letrozole Resistant MCF-7Ca Xenografts to AIs through Modulation of Her-2

Abstract Development of aromatase inhibitors (AIs) has significantly improved the treatment outcome of hormone responsive post-menopausal breast cancer. However, not all tumors respond and some eventually acquire resistance. We have developed a xenograft model that mimics post-menopausal hormone responsive breast cancer (MCF-7Ca). Results obtained using this model have been confirmed by numerous clinical trials. Using this model, we have established that single agent AI is better than tamoxifen in controlling tumor growth. We also observed that although, AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. A cell line was isolated from these Long-Term Letrozole Treated tumors and designated as LTLT-Ca. These cells and the tumors had lower expression of ER and aromatase compared to parental MCF-7Ca cells. On the other hand, growth factor receptor Her-2 and downstream kinases such as MAPK, Akt were upregulated. Inhibition of Her-2 using trastuzumab resulted in reversal of resistance and restoration of sensitivity to estrogens, antiestrogens and AIs. In order to restore sensitivity to AIs, we treated letrozole resistant MCF-7Ca tumors to HDAC inhibitor entinostat (ENT). In studies performed with ER negative MDA-MB-231 cells, ENT has upregulated ERα and restored sensitivity to AIs. To examine the effect of this combination on tumors that have acquired resistance to letrozole, we utilized MCF-7Ca xenografts model. We inoculated ovariectomized athymic nude mice MCF-7Ca cells and then tumors were allowed to form in the presence of androstenedione (≥4A), aromatizable substrate for estrogen. When the tumors reached ∼300mm3, the mice were randomized such that the mean tumor volume was not significantly different across the groups (p=0.13). 50 mice were treated with letrozole for total 16 weeks, during this time the tumors regressed, but eventually began to grow. When the tumors had reached double the initial volume, they were randomized again into 5 groups; letrozole continued, exemestane (250µg/day), ENT (50µg/day) or the combination of ENT with letrozole or exemestane. The mice were treated till week 26. The growth rates of tumors of mice treated with the combination of ENT with letrozole or exemestane was significantly better than single agent alone (P<0.05). Tumors were collected at autopsy and analyzed. ENT was able to increase ERα expression and aromatase activity in the letrozole resistant tumors. In addition, ENT also downregulated Her-2, p-Her-2, p-MAPK and p-Akt. We have shown that upregulation of Her-2 in LTLT-Ca cells is a result of longer half-life of Her-2 protein and not gene amplification. To determine the mechanism of effect of ENT on Her-2 expression, we examined the interaction of Her-2 with HSP-90 in LTLT-Ca cells and effect of ENT on this interaction. We observed that ENT reduced the association of Her-2 protein with HSP-90, possibly reducing the stability of Her-2 protein the LTLT-Ca cells. This reduced interaction was also seen the tumors treated with the combination of ENT plus letrozole or exemestane. Our results suggest that HDAC inhibitor entinostat may modulate Her-2 and result in reverse the acquired resistance of letrozole resistant cells and tumors. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-03.

Expression of ERα and Aromatase in MDA-MB-231 Tumors by HDAC Inhibitor Entinostat Leads to Growth Inhibition by Aromatase Inhibitor Letrozole.

Abstract The treatment for hormone receptor-positive breast cancer has improved significantly since the development of aromatase inhibitors (AIs). Nevertheless, AIs are ineffective in estrogen receptor-negative (ER-) tumors, which comprise of approximately 25% of breast cancers and tend to be more aggressive. Studies have shown that repression of ER in these hormone receptor-negative tumors may be due to epigenetic modifications. The discovery of recruitment of histone deacetylase enzymes in gene silencing provides a rationale for inhibition of HDAC activity to release transcriptional repression as a potential therapeutic strategy. The objective of the present study was to express ERα and aromatase with HDACI treatment and thereby sensitize tumors to growth inhibition with aromatase inhibitors. In this study we used ER negative, hormone refractory MDA-MB-231 human breast cancer cells. Treatment with HDAC inhibitor entinostat led to upregulation of ERα, aromatase and its activity in a dose dependent manner in cells and xenografts. MDA-MB-231 xenografts were grown in ovariectomized female nude mice. Mice were inoculated with 2.5 X 106 cells per site subcutaneously. When the tumors reached 150 mm3, the mice were grouped into 4 groups (n=10), so that the mean tumor volume was not statistically different across groups (p=0.88). Tumor volumes were measured twice weekly. The mice in the letrozole group had a mean tumor growth rate (β = 0.023 ± 0.014) that was not statistically different (p=0.76) from that of the control group (β = 0.038 ± 0.007). Also, the growth rate of entinostat group (β = 0.034 ± 0.011) was not significantly lower than that of the control (p=0.33). However, the growth rate of entinostat plus letrozole group ((β = -0.003 ± 0.013) was significantly lower than that of the control (p=0.01), entinostat (p=0.03) and letrozole (p=0.049) groups. The combined treatment of entinostat plus letrozole was significantly more effective than either agent alone. In addition, the ability of this combination to inhibit migration in vitro was examined by wound healing assay. The combination of entinostat plus letrozole provides superior inhibition of migration (p<0.001) compared to control, entinostat and letrozole alone. To test the efficacy of this combination in preventing the outgrowth of tumor foci in the lung, mice received an inoculation of 3 X 106 cells intravenously via the tail vein. They were treated three weeks later with entinostat alone, letrozole alone, or the combination. Mice were treated for six weeks, and then euthanized. Treatment with entinostat alone or letrozole was not significantly effective, but the combination resulted in a significantly reduced number of both visible and micrometastases (p=0.03) compared to no treatment (control) and the entinostat alone group. Thus, up-regulation of ERα and aromatase resulted in sensitization of tumors to significant inhibition of growth, cell migration and formation of micro-metastases by the aromatase inhibitor letrozole.Our results provide the basis for possible use of AIs in combination with HDAC inhibitors for the treatment of hormone refractory ERα negative breast cancer. This could open a new avenue for the management of ER- breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 401.

Involvement of TET2 in Regulation of Epigenetic Silencing in MDS and Related Myeloid Malignancies.

Abstract 2908Poster Board II-884Genomic (chromosomal gain losses and UPD) and genetic damage (mutations) are hallmarks of MDS and related myeloid malignancies. In addition, aberrant hypermethylation of CpG islands, leading to epigenetic silencing of tumor suppressor and differentiation genes, appears to play an important pathogenic role in evolution to AML. While various mechanisms leading to chromosomal instability have been identified, the pathogenesis of epigenetic instability leading to aberrant methylation remains unexplored. The recent discovery of mutations in TET2 gene in MDS/MPN, sAML and especially CMML provided an intriguing possibility that dysfunction of this bona fide tumor suppressor gene may result in increased promoter methylation. This hypothesis is based on the description of the function of the highly related TET1 gene: the corresponding protein mediates hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which prevents the DNMT1-catalyzed maintenance methylation during cell division. Consequently, TET2 mutations could lead to epigenetic instability and accumulation of aberrantly hypermethylated CpG sites.Both homozygous and heterozygous TET2 mutations were found suggesting that the presence of WT allele or residual activity is not protective. A large proportion of these mutations results in introduction of frame shifts or stop codons resulting in loss of function whilst missense mutations may lead to decreased function. To clarify the function of TET2 and consequences of the mutations we transduced M5 THP-1 AML cell line with lentiviral vectors, containing partial TET2 cDNA (TET2+) and shRNA (TET2-), to achieve overexpression or knockdown of TET2 gene. Following selection, TET2+ cell line showed 3-fold TET2 mRNA overexpression while TET2- cells demonstrated 85% knockdown of TET2 gene as compared to control cells. Based on the putative function of TET2, we performed thin layer chromatography (TLC) comparing 5hmC DNA content in THP1, TET2+, TET2- and appropriate control cells. Parental THP1 cells revealed abundant level of 5hmC. Whilst a 1.5-fold increase of 5hmC content was found in TET2+ cells, it remained unchanged in TET2- cells. However, early results showed a 20% decrease in 5hmC in bone marrow of patients with myeloid malignancies and homozygous TET2 mutations. Illumina 27k methylation arrays were used to assess the effects of altered TET2 expression on methylation patterns. Although global methylation levels, as expressed by averaged β-values, did not significantly differ, the number of hypermethylated CpG sites, defined by individual β-values >0.9, was decreased by 25% (3756 vs. 2839) in TET2+ cells. No difference was seen in TET2- cells vs. controls. However, a number of promoter CpG in TET2- displayed >2-fold change in methylation level compared to TET2+, including MGMT, PCGF2, PTPRO and NOX1. Among differentially silenced promoter CpG we selected CDKN2B (p15) promoter as a marker for methylation activity. When p15 promoter controlling a luciferase reporter was transfected into TET2+ and control cells, a 2.4-fold increase of luciferase activity occurred, consistent with increased demethylating activity in cells expressing higher levels of TET2. Additionally, qPCR showed a 2.5-fold increase in p15 expression in TET2+ cells. Based on this result we also studied methylation status of p15 promoter in patients with WT (N=20) and mutant TET2 (N=27); patients with MDS and MDS/MPN who carried TET2 mutations presented consistent methylation of various promoters including p15 in 2/3 mutant cases vs. 1/3 WT cases.Whereas proliferation kinetics was not significantly altered by changes in TET2 levels, 72h treatment with decitabine (0.5 μM) resulted in a differential effect on TET2+ and TET2- cells. TET2+ cells were not significantly affected however survival of TET2- cells decreased by 30%. In addition, TET2+ cells showed a relative resistance to HDAC inhibitor Entinostat (1μM) with 26% inhibition of proliferation vs. 60% seen in TET2- cells measured after 72h by cell proliferation assay (MTS).In sum, our results suggest that TET2 gene may be involved in epigenetic regulation of promoter CpG that are relevant to proliferation/differentiation of myeloid cells and changes in the expression/activity of this gene may result in altered methylation and thereby epigenetic silencing patterns. Disclosures:No relevant conflicts of interest to declare.