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<0.0001). DAXX-depleted cells were enriched for CSCs (MCF-7:P=0.004; BT474:P=0.0003). DAXX knockdown partially rescued the GSI-mediated inhibition of CSCs (MCF-7:P=0.003; BT474:P=0.0003). GSI or Notch1/Notch4 knockdown increased DAXX RNA and protein expression. RNA-sequencing of DAXX+ and DAXX- MCF-7 cells showed DAXX knockdown increased pluripotent stem cell genes (NANOG, SOX2, KLF4, and ALDH1A1) and decreased luminal genes (GATA3, CDH1, ESR2, and FOXA1). In mice, ET (estrogen deprivation) combined with MK-0752 GSI reduced tumor growth of MCF-7 xenografts compared to ET alone (P<0.05). DAXX was required for the anti-tumor efficacy of ET plus MK-0752 GSI (P=0.0084). Out of 35 patient tumors scored for DAXX and Ki67, 10/35 expressed less DAXX post-treatment (P=0.0052) and 25/35 retained expression. Lower DAXX was associated with less change in Ki67 levels (P=0.0097). High DAXX correlates with better response to ET as measured by Ki67. Tumors with decreased DAXX may potentially benefit from addition of MK-0752. Conclusions: This research in both cell lines and xenografts uniquely demonstrated that GSI-induced DAXX was necessary for the anti-CSC and anti-tumor efficacy of the GSI. DAXX is a potent CSC inhibitor and possible predictive biomarker of response to ET or ET plus the GSI MK-0752. In summary, ET plus a GSI modulated Notch and other CSC genes, in particular DAXX, a potential predictive biomarker of ET plus GSI efficacy in ER+ BC. Testing of anti-Notch or DAXX-promoting agents should be a clinical trial priority. Funding: BCRF Citation Format: C. Osipo, D. Wyatt, P. Tang, L. Miele, K. S. Albain. 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]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-13-02.