Abstract PS2-13-02: Daxx is a novel predictive biomarker of response to anti-notch plus endocrine therapy to target cancer stem cells in ER-positive breast cancer

Abstract Background: Notch promotes cancer stem cells (CSC) and resistance to endocrine therapy (ET) in ER+ breast cancer (BC). A Pre-surgical window study using ET plus a γ-secretase inhibitor (MK-0752) in ER+ BC identified novel Notch-regulated genes. Expression profiling showed more genes were downregulated by ET + GSI than ET alone. Of the few genes increased by ET plus the GSI, DAXX was the only gene required for GSI-mediated blockade of CSC survival in two distinct ER+ BC cell lines (MCF-7 and BT474) as measured by mammosphere-forming efficiency (MFE). Goals of the current research were to determine if DAXX is a 1) novel target gene of Notch1 or Notch4 and repressed by Notch signaling, 2) bona fide CSC suppressor, 3) predictive biomarker of anti-tumor efficacy of the GSI MK-0752 in a xenograft model, and 4) marker of response to ET in the neoadjuvant setting to inform if MK-0752 should be added to ET. Methods: Enrichment of Notch1 on CSL elements for validated genes was performed using ChIP-PCR. CSC survival was assessed by MFE when DAXX was knocked down. RT-PCR and western blotting measured DAXX RNA and protein expression under GSI treatment or Notch1 or Notch4 knockdown. RNA-sequencing was conducted from MCF-7 cells-expressing DAXX and depleted for DAXX. Tumor xenograft studies were performed using MCF-7 cells both expressing DAXX (DAXX+) and depleted for DAXX (DAXX-). Mice were treated with estrogen, estrogen deprivation, to mimic the use of an aromatase inhibitor, 75mg/kg GSI MK-0752, or estrogen deprivation plus MK-0752 for 80 days. Tumor volume was measured weekly. Survival of mice was monitored using Kaplan-Meier analysis. On day 80, tumors were excised and their masses weighed. To inform which patients would benefit from addition of MK-0752 to ET, DAXX and Ki67 protein expressions were measured by IHC and blindly scored in 35 untreated and then either ET- or chemotherapy-treated human ER+ tumors. Statistical calculations were performed using linear regression analysis, a T-test for two groups, or an ANOVA for multiple groups. Results: Notch1 enrichment on CSL elements increased with ET versus estradiol by 2-fold and attenuated with GSI treatment for most of the genes except for NOXA and PGR. The GSI eliminated CSCs from DAXX-expressing MCF-7 (P=9.4e-15) and BT474 cells (P