DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer

Purpose: Although chemotherapies kill most cancer cells, stem cell–enriched survivors seed metastasis, particularly in triple-negative breast cancers (TNBC). TNBCs arise from and are enriched for tumor stem cells. Here, we tested if inhibition of DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations, would target TNBC stem cells. Experimental Design: Effects of DOT1L inhibition by EPZ-5676 on stem cell properties were tested in three TNBC lines and four patient-derived xenograft (PDX) models and in isolated cancer stem cell (CSC)-enriched ALDH1⁺ and ALDH1^- populations. RNA sequencing compared DOT1L regulated pathways in ALDH1⁺ and ALDH1^- cells. To test if EPZ-5676 decreases CSC in vivo, limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1⁺ and ALDH1^- cells were performed. Tumor latency, growth, and metastasis were evaluated. Antitumor activity was also tested in TNBC PDX and PDX-derived organoids. Results: ALDH1⁺ TNBC cells exhibit higher DOT1L and H3K79me2 than ALDH1^-. DOT1L maintains MYC expression and self-renewal in ALDH1⁺ cells. Global profiling revealed that DOT1L governs oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1⁺ but not in ALDH1^- cells. EPZ-5676 reduced tumorspheres and ALDH1⁺ cells in vitro and decreased tumor-initiating stem cells and metastasis in xenografts generated from ALDH1⁺ but not ALDH1^- populations in vivo. EPZ-5676 significantly reduced growth in vivo of one of two TNBC PDX tested and decreased clonogenic 3D growth of two other PDX-derived organoid cultures. Conclusions: DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell–enriched TNBC.