Abstract PD3-10: Dual epigenetic/autophagy inhibition as a novel strategy to tackle triple negative breast cancer
Authors: Tauro Marilena , Li Tao , Lynch Conor C 
Published: March 2022

Abstract
Clinical Significance: Despite initial response to therapies, patients with triple negative breast cancer (TNBC) have the highest risk of metastatic relapse within 5 years of diagnosis. There is an urgent need for better treatments for this deadly disease. To this end, understanding the molecular drivers can reveal novel therapeutic targets. Background: The TNBC oncogenic program is known to be driven by MYC. MYC expression is regulated by bromodomain proteins and several bromodomain inhibitors such as JQ1 have proven effective at inhibiting MYC expression. However, resistance can occur rapidly upon activation of alternative cell survival mechanisms, such as autophagy. TNBC is noted for its elevated basal autophagy. Interestingly, publicly available datasets show that ULK3, an initial effector of the autophagy program, is upregulated in TNBC patients while the opposite is observed for the classical autophagy initiator ULK1. Moreover, TNBC patients with low ULK3 expression levels tend to achieve longer relapse free survival (RFS) than higher expression cohort, in opposition to ULK1. Importantly, ULK3 has not been investigated to date in regulating TNBC cell intrinsic autophagy and no drugs exist that can inhibit ULK3. Methods and Results: Given the importance of MYC and ULK3 in TNBC progression, our team developed a novel class of small molecules, namely dual BRD4/ULK3 inhibitors, that are superior to JQ1 alone in limiting TNBC viability. In vitro data demonstrate that TNBC cells are more sensitive to the cytotoxic activity of the dual inhibitors compared to other breast cancer subtypes. Further investigation through western blot analysis confirms the effectiveness of these novel dual inhibitors in abrogating MYC expression overtime, and completely blocking the autophagy program (measured by downstream ULK3, ULK1, LC3B, p62 protein levels), resulting in cell death. Interestingly, the human cell line SUM159R, that is resistant to BRD4 inhibitor JQ1 and cross-resistant to standard chemotherapeutics (doxorubicin) is also highly sensitive to our dual inhibitors, implying their potential use for the treatment of refractory TNBC disease. Excitingly, and for the first time, using a genetic silencing approach (shULK3 in SUM159), we demonstrated that ULK3 is critical for TNBC autophagy and cell survival. As a proof of concept, we reproduced the same genetic silencing experiment in a different cell line, namely WM1366-LC3BmCherry-GFP, an LC3B conveniently engineered system used to visualize proper autophagosome formation and material degradation through autophagy. We verified that both genetic ablation and pharmacologic inhibition of ULK3 are strategies that lead to cancer cell death. Conclusions: In conclusion, we 1) characterized a novel class of dual BRD4/kinase compounds, capable of inhibiting BRD4 activity and autophagy, and superior to the BRD4 inhibitor, JQ1. 2) showed that ULK3 is over-expressed in TNBC. 3) demonstrated that silencing ULK3 blocks autophagy in TNBC cell lines and results in significantly lower cell viability. 4) found that our dual inhibitors present a double bind for TNBC cells that results in increased cytotoxicity and potentially will be useful for the treatment of resistant TNBC. Clinically, we also posit that our dual inhibitors could be easily administered as a single agent, avoiding the potential complication of pharmacokinetics/pharmacodynamics associated with administering multiple therapies.
Citation Format: Marilena Tauro, Tao Li, Conor C Lynch. Dual epigenetic/autophagy inhibition as a novel strategy to tackle triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-10.