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RUNX1 Mutations Cause a Myeloid Differentiation Block Leading to the Formation of a Long Term Expanding CD34+/CD33+/CD45RA+/CD123+ Cell Population
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| Zeitschriftentitel: | Blood |
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| Personen und Körperschaften: | , , , , , |
| In: | Blood, 128, 2016, 22, S. 1979-1979 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society of Hematology
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| Schlagwörter: |
| Zusammenfassung: | <jats:title>Abstract</jats:title> <jats:p>RUNX1 (AML1) is a transcription factor critically involved in normal haematopoiesis. Inactivating RUNX1 mutations have been frequently described in a variety of myeloid neoplasms, including high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Here, we aimed to functionally and molecularly define the actions of a dominant negative mutant by in vitro and in vivo experiments and RNA- and ChIP-sequencing approaches.</jats:p> <jats:p>Overexpression of the RUNX1 mutant S291fs300X in cord blood (CB) CD34+ cells caused a decline in erythroid colony formation (p= 0.01) while the CFU-GM colonies showed enhanced replating capacity compared to control (>3 times). It appeared that the replating potential was restricted to CD14-/CD15- progenitor cells. Long-term suspension cultures with myeloid growth factors (IL-3, SCF) of RUNX1 S291fs300X CB CD34+ cells provided a rather homogenous cell population after 10 weeks of culture. These cells are growth factor dependent and are phenotypically defined by CD34+/CD38+/CD33+/IL1-RAP+/CD45RA+/CD123+ resembling a GMP phenotype which can be propagated for approximately 20 weeks in suspension. Comparable results were obtained with normal bone marrow CD34+cells transduced with the RUNX1 S291fs300X. Karyotype analyses demonstrated no abnormalities while integration site analysis showed a variety of different integration sites and differences between individual samples, suggesting that the myeloid differentiation block is related to the RUNX1 S291fs300X mutation.Long-term MS5 stromal co-cultures of transduced RUNX1 S291fs300X CB CD34+ cells showed after 8-10 weeks a rather homogenous cell population with limited potential to expand and localized under the stromal layer. This cell population is phenotypically defined by CD34+/CD38-. The interactions with the stroma appear to prevent proliferation but retain quiescence, indicating that sufficient niche-cell interactions might be crucial for transformation. NSG mice experiments are performed to test the reproducibility of these findings in vivo. Q-PCR studies demonstrated reduced expression of C/EBPα in RUNX1 S291fs300X CB CD34+ cells, one of the key targets in myeloid differentiation. Therefore, week 10 RUNX1 S291fs300X CB CD34+ cells were transduced with a retroviral C/EBPα overexpression vector. The re-expression of C/EBPα resulted in a reduction in cell proliferation, decline of undifferentiated blasts and an increase in CD15 expression. RNA- and ChIP-sequencing data revealed a decreased expression of crucial RUNX1 target genes including C/EBPα and Cited2 and also a retained binding of mutant RUNX1 on these loci in conjunction with a decrease of H3K27ac. Further research into the molecular mechanisms by which this RUNX1 S291fs300Xderegulates gene-expression is in progress.</jats:p> <jats:p>Our results implicate that overexpression of RUNX1 S291fs300X mutant leads to impaired erythroid differentiation and a strong differentiation block of the myeloid lineage resulting in the expansion and maintenance of a GMP-like cell population.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec> |
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| Umfang: | 1979-1979 |
| ISSN: |
0006-4971
1528-0020 |
| DOI: | 10.1182/blood.v128.22.1979.1979 |