#6128 It is well established that approximately 75% of human breast cancers are ER+ and therefore treated with endocrine therapy. In a past few decades endocrine therapy has made significant advancements. However, the application of these agents is limited to ER+ cancers since ER- patients are unresponsive to endocrine therapy primarily due to lack of ER expression in the tumor. The main purpose of this project is to determine whether ER- breast cancer tumors that display poor anti-proliferative response to aromatase inhibitor (AI) letrozole, can be sensitized by co-treatment with HDACi entinostat. Based on preliminary studies, we hypothesize that by inhibiting HDAC ER is re-expressed making the cells sensitive to the anti-proliferative effects of AIs. In our previous studies we have shown that HDACi entinostat can revert the ERα repression and upregulate ERα and aromatase in vitro and in vivo. In this study we are showing that this activation of aromatase and re-expression of ERα renders ER- breast cancer tumors (xenografts of MDA-MB-231 cells) responsive to letrozole.
 MDA-MB-231 xenografts were grown in ovariectomized female nude mice. Mice were inoculated with 2.5X 106 cells per site subcutaneously. When the tumors reached a measurable size ∼150 mm3, the mice were grouped into 6 groups (n=10), such that the mean tumor volumes across the groups was not statistically different (p=0.99). The mice were injected with Δ4A (100 μg/day), Δ4A plus letrozole (10 μg/day), entinostat (2.5 mg/kg/day), entinostat plus Δ4A, entinostat plus Δ4A plus letrozole or vehicle. The mice were injected 5 times a week. The tumors were measured every week with calipers and the tumor volumes were calculated using formula, 4/3 π r12r2. The mice in the entinostat plus Δ4A plus letrozole group had the least tumor growth rate (0.004+0.081), which was lower than entinostat plus Δ4A (0.115+0.079) and Δ4A plus letrozole (0.096+0.080). This data suggests a trend towards improved inhibition of tumor growth with combination of entinostat plus letrozole. The mice were sacrificed on week 9 due to large tumor volumes. The tumors and uteri were excised, cleaned, weighed and stored for additional analysis.
 In addition, 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. This suggests that the combination of entinostat plus letrozole has potential of inhibiting metastatic spread along with tumor growth.
 These findings indicate that ERα in ER- breast cancer cells is silenced along with aromatase but can be restored with HDACi. Thus activation of silenced ER and intratumoral aromatase by HDACi could open a new avenue for management of ER- advanced breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6128.

#2133 Background: All-trans-retinoic acid (ATRA) and other retinoids play key roles in prevention and therapy of many proliferative diseases including cancers. VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], which is a new generation novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of ATRA. The inhibitory effects of VN/14-1 on the growth of human breast cancer cells and human breast tumors in the nude mouse model have been previously demonstrated. The purpose of this study was to evaluate the effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, as well as on the uterus in immature ovariectomized (OVX) rats.
 Methods: (1) VN/14-1 (5, 10, and 20 mg/kg/d) was given by oral gavage to grouped female Sprague Dawley (SD) rats bearing MNU-induced mammary carcinoma for 8 weeks, after which tumor weight and volume, as well as histology were measured. (2) VN/14-1 (10 and 20 mg/kg/d) and b-estradiol (10 mg/kg/d) were given alone or in combination, by oral gavage (VN/14-1) and subcutaneous injection (b-estradiol), to immature OVX SD rats for 3 days, after which uterine weight and histology were measured.
 Results: (1) At the end of the treatment period, the administration of 5, 10 and 20 mg/kg/d VN/14-1 caused significant reductions of 19.1, 34.4 and 44.3%, respectively, in mean tumor weight compared with the control animals (all p < 0.05). The cumulative tumor growth was also significantly slower in groups receiving 5, 10 and 20 mg/kg/d compared to the control group in a dose-dependent manner. (2) Immature OVX rats given VN/14-1 at doses of 10 and 20 mg/kg, had reduction in uterine wet weight of up to 56% compared to OVX controls (P < 0.001). OVX rats given VN/14-1 of 10 and 20 mg/kg in combination with β-estradiol had reduction in uterine wet weight of up to 58% compared to the OVX rats given β-estradiol alone (P < 0.001). The adverse toxic effects such as fatigue and anorexia were occurred in the groups at high dose of 20 mg/kg.
 Conclusions: RAMBA VN/14-1 was able to inhibit the growth of tumors in the MNU-induced ER positive rat mammary tumor model and antagonized the stimulatory effect of β-estradiol on the uterus. The studies suggest VN/14-1 might be an effective novel therapy for ER positive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2133.

Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC), an aggressive and highly metastatic disease. Activation of eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (Mnk1/2) play a critical role in the development, progression and metastasis of TNBC. Herein, we undertook a comprehensive study to evaluate the activity of a first-in-class Mnk1/2 protein degraders, in clinically relevant models of TNBC. These studies enabled us to identify racemic VNLG-152R as the most efficacious Mnk1/2 degrader. By targeting Mnk1/2 protein degradation (activity), VNLG-152R potently inhibited both Mnk-eIF4E and mTORC1 signaling pathways and strongly regulated downstream factors involved in cell cycle regulation, apoptosis, pro-inflammatory cytokines/chemokines secretion, epithelial-mesenchymal transition (EMT) and metastasis. Most importantly, orally bioavailable VNLG-152R exhibited remarkable antitumor and antimetastatic activities against cell line and patient-derived TNBC xenograft models, with no apparent host toxicity. Collectively, these studies demonstrate that targeting Mnk-eIF4E/mTORC1 signaling with a potent Mnk1/2 degrader, VNLG-152R, is a novel therapeutic strategy that can be developed as monotherapy for effective treatment of patients with primary/metastatic TNBC. Keywords:Breast cancer / metastasis / Mnk1/2 degraders / Mnk/eIF4E/mTORC1 / TNBC.