Abstract S4-03: Identification of a notch-driven breast cancer stem cell gene signature for anti-notch therapy in an ER+ presurgical window model

Abstract

Background: Resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) for ER+ breast cancer is a major cause of mortality and new treatment paradigms are needed. Cancer stem cells drive breast cancer growth and are resistant to standard therapy. Notch signaling aids survival of these resistant stem cells and is inhibited by gamma-secretase inhibitors (GSI). We showed combining GSI with ET in mice caused shrinkage of breast cancer tumors. A presurgical window biomarker modulation model was used to confirm this discovery in humans. Methods: The GSI MK-0752 was added to ET in patients before definitive surgery (ClinTrials.gov NCT00756717). There were 3 biopsies: day 0 (prestudy), day 14 (after ET alone), and day 25 at definitive surgery (after continued ET plus MK-0752, 350 mg orally 3d on, 4d off, 3d on). Biopsies were analyzed for genes increased or decreased by GSI, to confirm that Notch and cancer stem cell pathways were inhibited. Real-time PCR was used to validate expression of genes identified in pathway analyses of microarray datasets generated from the biopsies. Mammosphere-forming assays were performed to confirm that ET+GSI impacts breast cancer stem cells. The qRT-PCR data were evaluated using ANOVA with repeated measures and ANOVA was performed on mammosphere results. Results: The accrual goal was met and therapy well-tolerated in 20 evaluable women (PSABCS 2011, abs# S1-5). Of 33 genes identified by analysis of expression microarrays, 19 genes (FDR<8%) were impacted significantly by GSI+ ET (3 increased, 16 decreased) compared to initial biopsy and/or ET alone. Genes with increased expression were DAXX, NOXA (both pro-apoptotic) and LNFG (tumor suppressor). Six of 16 genes that decreased (NOTCH1, NOTCH4, HEYL, HES1, HES5, and HEY2) are Notch pathway-associated genes. The GSI decreased expression of 3 genes from cell cycle and proliferative pathways (Ki67, CCND1, CCNA2) and inhibited 2 genes expressed in cancer stem cells (RUNX1 and ALDH1A1). Five genes directly/indirectly regulated by Notch were decreased by GSI (RICTOR, RPTOR, MMP7, ADAM19, and PRH). Estrogen deprivation for 3 days, mimicking short exposure to an AI, increased mammosphere-forming ability of ER+ breast cancer cells more than 2 fold. The GSI MRK-003 blocked this mammosphere formation by 95%-98%. Conclusions: A 7-day course of the GSI MK-0752 added to ET in the presurgical window had significant biomarker responses: decrease in Notch signaling, cancer stem cell genes, proliferation-associated genes, the mTORC1 and 2 complex genes RICTOR and RPTOR, metalloproteinases that promote metastasis, and PRH; as well as increase in 3 key genes that promote apoptosis and tumor suppression. These results suggest that 1) GSI inhibited the intended Notch pathway, 2) putative breast cancer stems cells can be targeted by this strategy, and 3) the biomarkers identified create a gene signature for anti-Notch therapy in ER+ breast cancer. Validation of efficacy of the GSI+ET therapy combination and this gene signature in a clinical trial is planned. Support: Breast Cancer Research Foundation (research grant), Merck Oncology (drug/arrays), Swim Across America (clinical trial costs), and DOD BC073237 (KRC). Citation Format: Kathy S Albain, Andrei Y Zlobin, Kyle R Covington, Brian T Gallahger, Susan G Hilsenbeck, Cheryl M Czerlanis, Shelly Lo, Patricia A Robinson, Ellen R Gaynor, Constantine Godellas, Davide Bova, Kathy Czaplicki, Barbara Busby, Patrick J Stiff, Suzanne AW Fuqua, Lucio Miele, Clodia Osipo. Identification of a notch-driven breast cancer stem cell gene signature for anti-notch therapy in an ER+ presurgical window model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S4-03.

DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

“…Thus, there is a critical need for new therapeutic targets to reduce TICs in ER þ breast cancer to prevent resistance, tumor recurrence, tumor, and severe side effects. Through a biomarker clinical trial (ClinTrials.gov Identifier: NCT00756717), we identified DAXX as a novel gene whose transcript is upregulated after short exposure to a GSI during endocrine therapy preceding definitive surgery in a window biomarker trial (19). From publicly available expression data (kmplot.com), high DAXX RNA correlates with a longer RFS in women with ER þ breast cancer following endocrine therapy (Fig.…”

“…To identify new therapeutic NOTCH biomarkers and targets, Albain and colleagues completed a biomarker presurgical window trial (ClinTrials.gov Identifier: NCT00756717; ref. 19). Death domain-associated protein 6 (DAXX) was identified as a novel NOTCH target with potential clinical significance in ER þ breast cancer (manuscript in preparation).…”

Notch Signaling in Breast Cancer: A Role in Drug Resistance

“…A promising approach is identification Notch downstream targets that are specifically expressed in breast tumors as biomarkers for prognosis or predictive markers of sensitivity to treatment. The identification of breast tumor specific Notch targets was conducted by using a pre-surgical window trial in women with early stage ER+ breast cancer [ 132 ]. In this trial, 22 women received neo-adjuvant letrozole (an aromatase inhibitor) or tamoxifen for 14 days, followed by addition of MK-0572 GSI for 11 days.…”

“…Furthermore, many novel Notch target transcripts were also regulated by the GSI. One of these was death-domain-associated protein 6 (DAXX), which was upregulated with GSI treatment [ 132 ]. Specifically, DAXX was identified to be a novel Notch1 target gene, and high expression of Notch1 repressed DAXX (unpublished data).…”

A Phase Ib Dose Escalation Trial of RO4929097 (a γ-secretase inhibitor) in Combination with Exemestane in Patients with ER + Metastatic Breast Cancer (MBC)

“…More recently, Gelsomino et al showed that mutations in the ESR1 gene encoding ERα associated with endocrine resistance promote a CSC phenotype through activation of Notch4 14 . A pilot clinical trial of short term GSI MK0752 in combination with letrozole or tamoxifen in early stage ERα+ breast cancer patients showed that this regimen was safe and produced molecular changes in tumors consistent with the inhibition of proliferation and "stemness" and the induction of apoptosis 15 . Based on these findings, we designed a phase 1b study of GSI-exemestane combination in the metastatic setting.…”