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Updated: Oct 4, 2025
Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples
Published on: October 2, 2020
DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer
Hetakshi Kurani1,2,3, Seyedeh Fatemeh Razavipour3, Kuzhuvelil B Harikumar1,4
1Braman Family Breast Cancer Institute at Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Florida.
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.
Insights
DOT1L inhibition targets cancer stem cells (CSCs) in triple-negative breast cancer (TNBC). This epigenetic approach reduces tumor initiation and metastasis, supporting DOT1L inhibitors for TNBC treatment.
Area of Science:
- Oncology
- Epigenetics
- Cancer Stem Cell Biology
Background:
- Triple-negative breast cancer (TNBC) is enriched for tumor stem cells that drive metastasis.
- Chemotherapies often spare these cancer stem cells (CSCs), leading to treatment resistance and recurrence.
- DOT1L is an epigenetic regulator known to affect normal tissue stem/progenitor cells.
Purpose of the Study:
- To investigate if DOT1L inhibition can specifically target CSCs in TNBC.
- To evaluate the effects of the DOT1L inhibitor EPZ-5676 on TNBC stem cell properties both in vitro and in vivo.
Main Methods:
- Tested EPZ-5676 on TNBC cell lines, patient-derived xenograft (PDX) models, and isolated ALDH1+ (CSC-enriched) and ALDH1- populations.
- Utilized RNA sequencing to compare DOT1L-regulated pathways in ALDH1+ versus ALDH1- cells.
- Assessed tumor initiation, growth, and metastasis using limiting dilution assays and xenograft models.
Main Results:
- ALDH1+ TNBC cells showed higher DOT1L expression and H3K79me2 levels compared to ALDH1- cells.
- DOT1L inhibition by EPZ-5676 reduced tumorsphere formation and ALDH1+ cells in vitro.
- In vivo, EPZ-5676 decreased tumor-initiating CSCs and metastasis originating from ALDH1+ populations.
Conclusions:
- DOT1L is identified as a critical regulator of CSCs in TNBC.
- These findings suggest that DOT1L inhibitors warrant further clinical investigation for targeting stem cell-enriched TNBC.
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