Abstract 3890: Mitochondrial MCL1 maintains triple negative breast cancer stem cells and contributes to chemotherapy resistance

Cytotoxic chemotherapy is the standard of care for patients with triple negative breast cancer (TNBC). Most patients with advanced TNBC progress after chemotherapy and die from metastatic disease. MCL1 is an anti-apoptotic Bcl-2 family member known to sequester and inactivate pro-apoptotic Bcl-2 family proteins and, thus, contribute to chemotherapy resistance. We previously reported that ~45% of residual TNBCs that remain in the breast after neoadjuvant chemotherapy harbor MCL1 amplification, suggesting a causal role for MCL1 in drug resistance. A recent report (Goodwin et al. 2015) suggested that siRNA-mediated ablation of MCL1 does not induce apoptosis in claudin-low TNBC cells with a cancer stem cell (CSC) gene expression signature. CSCs comprise a rare population of cells with tumor-initiating properties and refractoriness to chemotherapy. In this study, we showed that MCL1 expression is elevated in claudin-low TNBC SUM159PT and MDA436 CSCs as measured by ALDH + by flow cytometry and ability to form mammospheres. RNA interference of MCL1 in SUM159PT cells reduced CSCs and attenuated tumor formation in vivo. Mitochondrial oxidative phosphorylation (mtOXPHOS) plays a crucial role in maintenance of CSCs. MCL1 has been shown to localize in the mitochondrial matrix and contribute to mitochondrial respiration. Thus, we hypothesized that MCL1 contributes to enrichment of TNBC CSCs and chemotherapy resistance via mitochondrial regulation. Stable transfection and overexpression of MCL1 in MDA468 cells increased oxygen consumption ratio, mitochondrial membrane potential, and production of reactive oxygen species (ROS), all features of activated mtOXPHOS. Conversely, RNAi-mediated ablation of MCL1 in SUM159PT and MDA436 cells repressed these markers of activated mtOXPHOS. A mutant of MCL1 lacking its mitochondrial target sequences (MTS) was unable to localize in mitochondria and, when transfected into MDA468 cells, reduced the CD44 high /CD24 low fraction and mammosphere formation. We next tested VU0659158, a BH3 mimetic in development at Vanderbilt that disrupts MCL1 interactions with BH3 domain-containing proteins, such as BID, BIM, NOXA and PUMA. Treatment of SUM159PT cells with VU0659158 increased caspase activity but did not attenuate mammosphere formation. Analysis of mRNA expression in TCGA revealed that genes induced by mtOXPHOS involved in the hypoxia pathway are significantly up-regulated in MCL1 amplified breast cancers. Finally, pharmacological inhibition of HIF-1α, a key regulator of hypoxia, with digoxin decreased CSCs and attenuated tumor formation in vivo. These data suggest that 1) MCL1 confers resistance to chemotherapy by expanding CSCs via mtOXPHOS independent of its BH3 domain-mediated, anti-apoptotic function, and 2) targeting mitochondrial respiration and the hypoxia pathway may delay or reverse chemotherapy resistance in MCL1 amplified TNBC. Citation Format: Kyung-min Lee, Jennifer Giltnane, Justin Balko, Luis Schwarz, Angel Guerrero, Katie Hutchinson, Mellissa Hicks, Violeta Sanchez, Melinda Sanders, Taekyu Lee, Edward Olejniczak, Stephen Fesik, Carlos Arteaga. Mitochondrial MCL1 maintains triple negative breast cancer stem cells and contributes to chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2017-3890