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Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells
Published on: September 1, 2019
C-terminal mutation of RUNX1 attenuates the DNA-damage repair response in hematopoietic stem cells
Y Satoh1, I Matsumura, H Tanaka
1Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan. ysatoh@bldon.med.osaka-u.ac.jp
Abstract:
Loss-of-function mutations of RUNX1 have been found in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs). Although several reports have suggested roles for RUNX1 as a tumor suppressor, its precise function remains unknown. Because gene alterations of RUNX1 by themselves do not lead to the development of leukemia in mouse models, additional mutation(s) would be required for leukemia development. Here, we report that the C-terminal deletion mutant of RUNX1, RUNX1dC, attenuates DNA-damage repair responses in hematopoietic stem/progenitor cells. γH2AX foci, which indicate the presence of DNA double-strand breaks, were more abundantly accumulated in RUNX1dC-transduced lineage(-)Sca1(+)c-kit(+) (LSK) cells than in mock-transduced LSK cells both in a steady state and after γ-ray treatment. Expression profiling by real-time -PCR array revealed RUNX1dC represses the expression of Gadd45a, a sensor of DNA stress. Furthermore, bone marrow cells from MDS/AML patients harboring the RUNX1-C-terminal mutation showed significantly lower levels of GADD45A expression compared with those from MDS/AML patients with wild-type RUNX1. As for this mechanism, we found that RUNX1 directly regulates the transcription of GADD45A and that RUNX1 and p53 synergistically activate the GADD45A transcription. Together, these results suggest Gadd45a dysfunction due to RUNX1 mutations can cause additional mutation(s) required for multi-step leukemogenesis.
Insights
RUNX1 mutations impair DNA repair in hematopoietic stem cells by repressing Gadd45a, a DNA stress sensor. This dysfunction may drive multi-step leukemogenesis in acute myeloid leukemia and myelodysplastic syndromes.
Area of Science:
- Hematology
- Molecular Biology
- Cancer Research
Background:
- Loss-of-function mutations in RUNX1 are implicated in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs).
- RUNX1 is suspected to act as a tumor suppressor, but its exact role in leukemogenesis is unclear.
- RUNX1 alterations alone do not induce leukemia in mouse models, suggesting additional genetic events are necessary.
Purpose of the Study:
- To investigate the function of a C-terminal deletion mutant of RUNX1 (RUNX1dC) in DNA damage repair.
- To elucidate the mechanism by which RUNX1 mutations affect leukemogenesis.
- To explore the relationship between RUNX1, Gadd45a, and p53 in DNA damage response and leukemia development.
Main Methods:
- Transduction of hematopoietic stem/progenitor cells (LSK cells) with RUNX1dC.
- Quantification of DNA double-strand breaks using γH2AX foci.
- Gene expression profiling using real-time PCR array to analyze Gadd45a expression.
- Analysis of bone marrow cells from MDS/AML patients with wild-type and mutated RUNX1.
- Investigation of RUNX1 and p53's role in GADD45A transcription.
Main Results:
- RUNX1dC attenuated DNA-damage repair responses in hematopoietic stem/progenitor cells, evidenced by increased γH2AX foci.
- RUNX1dC repressed the expression of Gadd45a, a DNA stress sensor.
- MDS/AML patients with RUNX1-C-terminal mutations exhibited significantly lower GADD45A expression compared to those with wild-type RUNX1.
- RUNX1 directly regulates GADD45A transcription, synergistically with p53.
Conclusions:
- RUNX1 mutations, specifically C-terminal deletions, impair DNA double-strand break repair by downregulating Gadd45a.
- Gadd45a dysfunction resulting from RUNX1 mutations may contribute to the accumulation of additional mutations required for multi-step leukemogenesis.
- RUNX1 and p53 play a synergistic role in regulating GADD45A transcription, highlighting a critical pathway in maintaining genomic stability.
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