Page 69 - Haematologica April 2020

RUNX3 in aging and erythroid-myeloid balance
throblast line shifted the cells to a less mature phenotype, characterized by increased CD71 and diminished CD235a expression, and blocked induction of hemoglobinization (Figure 2G and Online Supplementary Figure S2G-I). Conversely, retroviral overexpression of RUNX3 in HUDEP-2 pro-erythroblasts enhanced their hemoglo- binization (Figure 2H and Online Supplementary Figure S2I).
Progenitor deficiency of RUNX3 alters the balance of lineage output
To analyze in greater detail the effects of RUNX3 defi- ciency on progenitor fates, mass cytometry (CyTOF) was employed for comprehensive single cell profiling of cells in HSPC expansion culture, and cells in erythroid, megakaryocyte, or granulocyte culture conditions for 48 h. Cells from each culture condition were clustered into populations defined by surface marker staining, followed by construction of minimum spanning tree (MST) plots describing average fold changes in population abundance associated with RUNX3 knockdown (Figure 3A and Online Supplementary Figure S4A and B). These populations segregated into two main branches: a lower erythro- megakaryocytic compartment (Ery/Mk: red oval) defined by CD36 and/or CD41 positivity, and an upper compart- ment (Myeloid: blue oval) lacking both markers (Online Supplementary Figure S4C and D). As expected from results in Figure 2, RUNX3 knockdown selectively diminished cell populations within the Ery/Mk compartment in line- age culture conditions, but not in HSPC expansion condi- tions (Figure 3A and Online Supplementary Figure S4A). This contraction was associated with impaired prolifera- tion, as reflected by decreased Ki-67 expression, but with no evidence of increased apoptosis (Figure 3B and C).
Notably, populations in the myeloid compartment (blue oval) were augmented in RUNX3-deficient progenitors grown in erythroid medium but not in other culture con- ditions. Analysis of these populations revealed a myeloid- skewed shift in HSPC distribution, similar to what has been described in aged bone marrow. These populations displayed a GMP (granulocyte-monocyte progenitor) phe- notype, based on expression of CD34, CD38, CD123, and CD45RA in various combinations (Online Supplementary Table S1). Strikingly, RUNX3-deficient populations in the Ery/Mk compartment (red oval) exhibited aberrant reten- tion of CD123, as well as global upregulation of the GMP marker CD45RA and the myeloid differentiation antigen CD11b (Figure 3D-F).
The CyTOF panel permitted assessment of the frequen- cies of cells with marker profiles of megakaryocyte-ery- throid progenitors (MEP), common myeloid progenitors (CMP), and granulocyte monocyte progenitors (GMP).34 This analysis showed RUNX3 deficiency to decrease MEP frequency and increase CMP and GMP frequencies (Figure 3G and Online Supplementary Figure S4E). Within the CMP and MEP compartments, knockdown of RUNX3 was associated with diminished expression of erythroid mark- ers CD36 and CD235a, but enhanced expression of the myeloid marker CD11b (Figure 3H and I and Online Supplementary Figure S4F).
CD34+ cells and cells in erythroid culture for 24 h, before cell viability was impacted by RUNX3 deficiency. In line with our other data, few changes were found between control and RUNX3-deficient undifferentiated cells (<70 genes with differential expression). However, among the down-regulated genes were key erythroid transcription factors including KLF1, GATA1, and GFI1B (Figure 4A). Several globin- and erythroid blood group antigen-encod- ing genes were decreased as well (data not shown). Notably, Klf1, Gata1, and downstream erythroid target genes Gypa and Epor also underwent downregulation in aged versus young murine HSC (Figure 4B). When com- paring control and RUNX3-deficient progenitors in ery- throid culture, approximately 1,100 genes showed differ- ential expression. These included many of the same genes affected in the undifferentiated cells as well as additional erythroid genes such as CD36 and EPOR (Figure 4A). In addition, several granulocytic transcription factors were aberrantly up-regulated, including GFI1, JUN, and FOS (Figure 4A). Gene ontology (GO) analysis of genes differ- entially expressed in control versus RUNX3-deficient undifferentiated progenitors revealed only two significant functional categories, oxygen transport (i.e. erythroid; >100-fold enrichment; FDR 1.22E-6) and blood coagula- tion (i.e. megakaryocytic; 15.08-fold enrichment; FDR 1.91E-3), both of which showed downregulation. GO analysis of progenitors in erythroid culture yielded similar results but also included genes related to mitochondrial protein synthesis/transport and ribosomal biogenesis (Figure 4C).
Hematopoietic stem and progenitor cells RUNX3 deficiency occurs in human anemias associated with aging
Because RUNX3 expression levels strongly influence human erythroid differentiation, its downregulation could potentially contribute to anemias associated with aging. To address this possibility, we analyzed highly purified marrow progenitors from the following subjects: normal non-anemic young (20-35 years old), non-anemic aged (>65 years old), and aged (>65 years old) subjects with unexplained anemia of the elderly (UAE). The diagnosis of UAE was made by ruling out all other potential causes of anemia, as per the criteria of Goodnough and Schrier.35 Gene expression profiling by microarray confirmed RUNX3 downregulation in UAE versus aged HSC4 (GSE32719) (Figure 5A). Functional studies revealed intrinsic differences in lineage output between UAE and non-anemic old progenitors. UAE HSC yielded fewer ery- throid colonies (BFU-E) but similar numbers of myeloid colonies (CFU-GM) (Figure 5B). These findings resemble the effects of RUNX3 knockdown on colony formation by CD34+ progenitors (Figure 2B). Furthermore, UAE MEP also showed poor TGFβ responsiveness in erythroid colony (CFU-E) enhancement (Figure 5C), a notable find- ing given the known influences of HSC aging and RUNX3 expression on this pathway.3,36
Discussion
Hematopoietic stem cell alterations with aging are com- plex; they result from cell-autonomous and micro-envi- ronmental mechanisms, and involve transcriptional changes in numerous genes. Interestingly, several of the
RUNX3 deficiency causes perturbations in erythroid transcriptional program
To identify the genes affected by RUNX3 knockdown, we performed mRNA sequencing on undifferentiated
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