Abstract 6: Targeting eukaryotic elongation factor 2 kinase (eEF2K) to combat cancer stem cells and tumorigenesis in triple-negative breast cancer

Abstract Breast cancer remains the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among women. Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers, is a highly aggressive and lethal form of breast cancer (BC) and characterized by the lack of ER, PR, and HER2 receptor expression, early relapses, drug resistance, and the poorest survival outcomes among all BC subtypes. Currently, there are no effective targeted therapies for TNBC, highlighting the urgent need for novel treatment strategies. eEF2K is an atypical alpha kinase that has been shown to promote TNBC cell proliferation, migration, invasion, and tumor growth. We previously validated eEF2K for the first time as a potential molecular target in TNBC and demonstrated that genetic inhibition of eEF2K completely suppresses in vivo TNBC tumor growth in mice. Overall data suggests that eEF2K is a critical oncogenic driver of TNBC tumorigenesis. Cancer stem cells (CSCs), a small but specific group of cancer cells, play a significant role in relapses and resistance to therapeutics including chemo-drugs. CSCs initiate tumor formation, promote tumor growth and cause relapses with their self-renewal and self-proliferation properties. While TNBC is known for the high number of CSCs population, the role of eEF2K in CSCs is unknown. In this study, we report the critical role of eEF2K in TNBC CSCs survival and tumorigenesis and found that the inhibition of eEF2K by siRNA and a small molecule inhibitor that we developed dramatically suppressed CSCs growth, migration, invasion and spheroid formation as well as TNBC cell proliferation and tumor growth in mice multiple TNBC models. Conversely, eEF2K overexpression promoted sphere formation. Our Kaplan-Meier analysis of the Cancer Genome Atlas (TCGA) revealed that high expression of eEF2K and ALDH1a3, a stemness marker, is associated with shorter overall and relapse-free survival. We also found that eEF2K is highly overexpressed in the ALDH-positive TNBC stem cell population. Genetic knockdown and pharmacological inhibition of eEF2K significantly suppressed the expression of key stemness signaling molecules, including ALDH1a3, Notch1, and Notch2. Currently, we are targeting eEF2K in MDA-MB-231 and MDA-MB-436 TNBC orthotopic xenograft and 4T1 mouse mammary tumor models in syngeneic mice to further assess its impact on CSCs-driven tumorigenesis. Our findings suggest that eEF2K plays a pivotal role in promoting TNBC stemness through the modulation of key stemness pathways, including ALDH1a3, Notch1, and Notch2 and tumor growth. Targeting eEF2K may therefore be a promising therapeutic strategy to eradicate TNBC by targeting primary and CSCs populations. Citation Format: Ezgi Biltekin, Nermin Kahraman, Yasemin M.Akay, Gulperi Oktem, Metin Akay, Bulent Ozpolat. Targeting eukaryotic elongation factor 2 kinase (eEF2K) to combat cancer stem cells and tumorigenesis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6.