Abstract A020: Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting

Abstract

Abstract Triple negative breast cancer (TNBC) is a heterogeneous molecular breast cancer (BC) subtype that still has a very poor prognosis. Genetic, molecular and clinical tumor heterogeneity represent major pathobiological factors involved in poor clinical outcome. The sequence specific transcriptional repressor and proto-oncogene B cell lymphoma protein 6 (BCL6) has emerged as a suitable therapeutic target as it plays broad and critical roles in many hematological and solid cancers. BCL6 exerts control over cellular proliferation and survival by binding to specific responsive elements located within the regulatory regions of target genes and recruiting of chromatin modifying co-repressor complexes such as silencing mediator for retinoid and thyroid receptors (SMRT), BCL6 corepressor (BCoR) and histone deacetylases (HDACs). Moreover, in hematological malignancies, BCL6 transcriptional activity is also associated to the recruitment of the chromatin remodeling factor, Enhancer of Zeste 2 (EZH2). The role of BCL6 in BC is still not well defined; however emerging studies report its involvement in tumorigenesis, disease progression and poor survival. We recently showed that BCL6 is expressed at significantly higher levels in TNBC mammospheres, 3D multicellular structures enriched in cancer stem cells (CSCs), than in their differentiated bulk cell counterparts. Additionally, we found that the functional role of BCL6 is crucial for mammosphere formation, as its loss of function decreased this process. Intriguingly, we uncovered a functional interplay occurring between BCL6 and EZH2, which triggers the BCL6/Notch stemness signaling axis by inhibiting Numb transcription in both SUM149 and SUM159 TNBC cell lines. We exploited the molecular mechanisms responsible for this specific interplay, taking advantage of two molecules, FX1, a small molecule inhibitor able to block BCL6 recruiting activity and EED226, a potent Polycomb Repressive Complex 2 (PRC2) inhibitor. In order to define the role of both BCL6, as epigenetic regulator, and NUMB, as inhibitor of NOTCH stemness pathway in TNBC, SUM149 and SUM159 cell lines were stably transduced with lentiviral shRNA to derive stable NUMB-silenced matched cells. The expression of Notch downstream target genes, such as HES1/2 and HEY1/2, was assessed by qRT PCR analysis in the engineered TNBC cell models and resulted only slightly increased following NUMB downmodulation. Upon FX1 or EED226 administration, a strongest effect was observed, indicating that NUMB is not the only player in regulating the Notch pathway. Further experiments are currently ongoing aiming at identify additional players and molecular pathway(s). Collectively, our preliminary results provide additional preclinical evidences on the role exerted by BCL6 as epigenetic regulator of TNBC and underlie its interplay with EZH2thus paving the way for a novel potential therapeutic strategy targeting BCL6/Notch signaling axis. Citation Format: Francesca De Santis, Francesca Putti, Giorgia Bravin, Carla Portulano, Federica Zonca, Lorenzo Castagnoli, Michele Magni, Filippo de Braud, Serenella M Pupa, Massimo Di Nicola. Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A020.