For a study, researchers sought to see if blocking DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations would target TNBC stem cells. EPZ-5676 inhibition of DOT1L was tested on stem cell properties in 3 TNBC lines, 4 patient-derived xenografts (PDX) models, and isolated cancer stem cell (CSC)-enriched ALDH1+ and ALDH1− populations. RNA sequencing was used to compare the pathways regulated by DOT1L in ALDH1+ and ALDH1− cells. Limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1+ and ALDH1− cells were performed to see if EPZ-5676 reduces CSC in vivo. Tumor growth, latency, and metastasis were all studied. TNBC PDX and PDX-derived organoids were also tested for antitumor activity. ALDH1+ TNBC cells had higher DOT1L and H3K79me2 levels than ALDH1−. DOT1L maintained MYC expression and self-renewal in ALDH1+ cells. DOT1L regulated oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1+ cells but not in ALDH1− cells, according to global profiling. EPZ-5676 decreased tumorspheres, and ALDH1+ cells in vitro and tumor-initiating stem cells and metastasis in xenografts generated from ALDH1+ but not ALDH1− populations in vivo EPZ-5676 inhibited in vivo growth of 1 of 2 TNBC PDX tested and inhibited clonogenic 3D growth of 2 other PDX-derived organoid cultures. In TNBC, DOT1L emerged as a critical CSC regulator. The findings supported further clinical research into DOT1L inhibitors to target stem cell-enriched TNBC.