Increased expression of RUNX3 inhibits normal human myeloid development

RUNX3 is a transcription factor dysregulated in acute myeloid leukemia (AML). However, its role in normal myeloid development and leukemia is poorly understood. Here we investigate RUNX3 expression in both settings and the impact of its dysregulation on myelopoiesis. We found that RUNX3 mRNA expression was stable during hematopoiesis but decreased with granulocytic differentiation. In AML, RUNX3 mRNA was overexpressed in many disease subtypes, but downregulated in AML with core binding factor abnormalities, such as RUNX1::ETO. Overexpression of RUNX3 in human hematopoietic stem and progenitor cells (HSPC) inhibited myeloid differentiation, particularly of the granulocytic lineage. Proliferation and myeloid colony formation were also inhibited. Conversely, RUNX3 knockdown did not impact the myeloid growth and development of human HSPC. Overexpression of RUNX3 in the context of RUNX1::ETO did not rescue the RUNX1::ETO-mediated block in differentiation. RNA-sequencing showed that RUNX3 overexpression downregulates key developmental genes, such as KIT and RUNX1, while upregulating lymphoid genes, such as KLRB1 and TBX21. Overall, these data show that increased RUNX3 expression observed in AML could contribute to the developmental arrest characteristic of this disease, possibly by driving a competing transcriptional program favoring a lymphoid fate.


Isolation, infection, and culture of human HSPC
Human neonatal cord blood was obtained from healthy full-term pregnancies at the University Hospital Wales, Cardiff, UK. These were obtained with informed consent and with approval from the South East Wales Research Ethics Committee in accordance with the 1964 Declaration of Helsinki. Normal human HSPC were isolated, cultured and transduced with retro/lentivirus as previously described [2]. Briefly, HSPC were infected with retro-or lentivirus by centrifugation for 120 minutes at 2200 x g and room temperature (RT) in 24-well plates pre-coated with 30 µg/mL RetroNectin ® (Takara, Paris, France) [3]. Following centrifugation, the retroviral supernatant was removed and HSPC were added to the wells (70-140 000 cells/well). The infection procedure was repeated on the following day to improve the transduction efficiency.

Phenotypic and differentiation analysis by flow cytometry
Transduced cultures were analyzed by flow cytometry at different time points using a panel of cell surface markers (Supplemental Table S2) as previously described [2]. CD13-APC in combination with CD36-biotin were used for lineage discrimination; Streptavidin PerCP-Cy5.5 was used as a second-step detection reagent. In addition, cells were incubated with one of the following Pacific Blue (PB)-labelled differentiation markers: CD11b, CD14, CD15 and CD34 (BioLegend, London, UK). All incubations were performed at 4 º C, and reactions were controlled with the appropriate isotypematched irrelevant antibody. Reagent concentrations were as recommended by the manufacturer.

Western Blot
Cytosolic and nuclear proteins were extracted from 5x10 6 HSPC using the Biovision Nuclear/Cytosol Fractionation Kit (Cambridge Bioscience, Cambridge, UK).
Briefly, cells were pelleted and washed, followed by sequential incubations with extraction buffers. Cytosol fractions were separated, and nuclear proteins were extracted using triethylammonium bicarbonate buffer (TEAB). Bradford protein assay was performed by measuring the absorbance of Bradford's reagent solution (Sigma Aldrich, Dorset, UK) at 595 nm.
SDS-PAGE was performed as previously described [4] using the NuPAGE ® electrophoresis system (Fisher Scientific UK Ltd, Loughborough, UK). Detection of RUNX3 protein expression was determined using a primary rabbit monoclonal antibody normalized against control cells.
Initially, the transwell 24-well plate was pre-incubated with serum free growth medium  [6,7]    a Control and RUNX3 HSPC (5 independent experiments; samples #1 to #10) were analyzed by RNA-seq as in b. b CD34 + HSPC were isolated using the MiniMACS™ magnetic sorting system for CD34 positivity and subsequently infected with control (PINCO GFP) or RUNX3 GFP retrovirus. Following enrichment for GFP expression by FACS, total RNA was extracted using the RNeasy Plus Mini Kit and RNA integrity number (RIN) was assessed using Agilent 2100 Bioanalyzer. Samples with a RIN above 8 were used for RNA-seq. Representation of the human Hematopoietic Cell Lineage KEGG pathway map (hsa04640). Cellular stages are identified by the specific expression of genes, which are highlighted in red for upregulation and green for downregulation associated with RUNX3 overexpression in HSPC. The lymphoid arm of hematopoiesis was removed from this pathway. Relevant cytokines involved in hematopoiesis are represented in each developmental stage. Important receptors and cytokines involved in hematopoiesis are represented in boxes and summarized under each developmental pathway/lineage.