Runx1 loss minimally impacts long-term hematopoietic stem cells

Abstract

RUNX1 encodes a DNA binding subunit of the core-binding transcription factors and is frequently mutated in acute leukemia, therapy-related leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia. Mutations in RUNX1 are thought to confer upon hematopoietic stem cells (HSCs) a pre-leukemic state, but the fundamental properties of Runx1 deficient pre-leukemic HSCs are not well defined. Here we show that Runx1 deficiency decreases both apoptosis and proliferation, but only minimally impacts the frequency of long term repopulating HSCs (LT-HSCs). It has been variously reported that Runx1 loss increases LT-HSC numbers, decreases LT-HSC numbers, or causes age-related HSC exhaustion. We attempt to resolve these discrepancies by showing that Runx1 deficiency alters the expression of several key HSC markers, and that the number of functional LT-HSCs varies depending on the criteria used to score them. Finally, we identify genes and pathways, including the cell cycle and p53 pathways that are dysregulated in Runx1 deficient HSCs.

Figure 1. Runx1 deletion expands the phenotypic hematopoietic stem/progenitor pool in a cell-autonomous manner.

A. Schematic diagram of the experimental strategy. Mice stably engrafted with donor and competitor cells were injected with pIpC, and sacrificed for analyses. B. Representative FACS analysis of lineage depleted donor-derived bone marrow LSK cells and Flt3^- and Flt3⁺ fractions thereof 5 weeks post pIpC injection. C. Donor chimerism within the Lin^- fraction of recipient marrow. Upon deletion of Runx1 (∶/∶), there was no change in the percent donor contribution to the Lin^- fraction of cells (left panel), but within the donor-derived Lin^- fraction there was a significant increase in the percentage of Sca1⁺c-Kit⁺ cells (right panel). Dots show the distribution of donor chimerism, the horizontal lines reflect the mean (±95% CI). In animals whose bone marrow contained <30% Runx1-deficient Lin^- cells the difference was also significant (P<0.0001, not shown). D. Contribution of enriched donor LT-HSC/ST-HSCs (LSK Flt3^-) and MPP/LMPPs (LSK Flt3⁺) to recipient bone marrow (mean ± 95% CI). Left panel shows percent contribution, f/f n = 10, ∶/∶ n = 12. Right panel shows total numbers, f/f n = 14, ∶/∶ n = 18.

Figure 2. Effect of Runx1 deletion on fetal HSCs.

A. The percentage of phenotypic Runx1 deficient fetal liver HSCs (14.5 dpc) defined using SLAM markers. Runx1 was deleted in fetal HSCs using Vav1-Cre. The number of fetal liver cells was equivalent. Plots indicate the percentage of CD48^- CD150⁺ LSK cells in the fetal liver; error bars represent 95% CI. Data are compiled from 7 Runx1^f/f (f/f) and 6 Runx1^f/f; Vav1-Cre fetuses (Δ/Δ) in two experiments. B. Competitive limit dilution transplant. Dilutions of 14.5 dpc fetal liver cells (Ly5.2⁺) were injected along with 2×10⁵ Ly5.1⁺ adult bone marrow cells into irradiated Ly5.1/5.2 recipients. The contribution of Ly5.2⁺ cells to peripheral blood was assessed, and recipients with ≥5% donor-derived cells at ≥16 weeks were deemed reconstituted. Data are compiled from three different experiments with a total of 6 to 13 mice per data point. The frequency of LT-HSCs in the wild type fetal was 1 in 17,184 (95% CI range  =  1 in 22,355 to 1 in 13,208). The frequency of LT-HSCs in the Runx1^f/f;Vav1-Cre fetal liver was 1 in 68,413 (range  =  1 in 93,523 to 1 in 50,045).

Figure 3. Proliferation profile and CXCR4 expression on Runx1 deficient fetal liver HSCs.

A. Cell cycle analysis of sorted CD48^-CD150⁺ LSK cells stained with Hoechst and analyzed by FACS. Error bars represent 95% CI. Differences between f/f and ∶/∶ cells in G₀/G₁ and M/G₂ were significant (asterisks, P = 0.003 and 0.004, respectively). n = 6. B. Representative scatter plots for analysis of LSK and phenotypic LT-HSC, ST-HSC, and MPP populations are shown on left, and histograms for CXCR4 levels on the right. CD48^- CD150^- LSK cells are labeled ST-HSC based on analysis by Foudi et al. in adult marrow . C. Analysis of CXCR4 levels on Runx1 deficient fetal liver HSCs. The mean fluorescence intensity (MFI) of CXCR4 is plotted for LSK, phenotypic LT-HSC (CD48^-CD150⁺ LSK), ST-HSC (CD48^-CD150^- LSK), and MPP (CD48⁺CD150^- LSK) populations (n = 5 or 6 fetuses, error bars represent 95% CI). Significance was determined by unpaired, two-tailed Student's t-test.

Figure 4. LT-HSC marker expression is altered on Runx1 deficient HSCs.

A. Percentage of adult bone marrow LSK cells expressing SLAM markers. The gates were drawn based on fluorescence-minus-one controls using Runx1^f/f (f/f) and Runx1^f/f;Vav1-Cre (Δ/Δ) cells. The bar graph on the right represents the percentage of CD48^- CD150⁺ LSK cells in total bone marrow, averaged from 6-7 mice. Error bars indicate SEM. B. Real time PCR for mRNA encoding LT-HSC markers in donor derived LSKF^- cells sorted from recipients of Runx1^f/f and Runx1^f/f; Mx1-Cre bone marrow. n = 4–6 mice of each genotype. Error bars represent SEM. See Figure 1B for gating strategy. C. Mean fluorescence intensity of CD34, CD48, and CD150 on LSKF^- and LSKF⁺ cells (n = 6; error bars  =  SEM. D. Simultaneous analysis of all four LT-HSC markers on Runx1^f/f and Runx1^f/f;Vav1-Cre LSK cells. The LSK population (blue) and CD34^- Flt3^- LSK population (red) in the middle plots are analyzed for CD48 and CD150 expression in the right hand plots. Note the upward shift in CD48 levels, and rightward shift of C150 on Runx1 deficient HSCs. E. The percentage of phenotypic LT-HSCs and ST-HSCs in the bone marrow of 6-8 week old Runx1^f/f and Runx1^f/f; Vav1-Cre mice determined using CD34 and Flt3 markers. Data are averaged from 6 mice, error bars indicate 95% CI. F. Competitive limit dilution transplant. Bone marrow from 6-8 week old Runx1^f/f or Runx1^f/f;Vav1-Cre mice was injected along with 2×10⁵ Ly5.1⁺ adult bone marrow cells into irradiated Ly5.1/5.2 recipients. Contribution of Ly5.2⁺ cells to peripheral blood (top graph) and bone marrow LSK cells (bottom graph) were assessed. Recipients with ≥1% donor-derived cells at ≥16 weeks were deemed reconstituted. Data are compiled from three different experiments with a total of 5 to 13 mice per data point. G. Repopulating units (RU) determined by contribution to different blood cell populations. Competitor cells (2×10⁵, Ly5.1) were transplanted with either 1×10⁵ Runx1^f/f or 3×10⁵ Runx1^f/f; Vav1-Cre bone marrow cells. RUs were calculated according to Harrison et al. .

Figure 5. Alterations in cell cycle and apoptosis in Runx1 deficient adult HSCs.

A. Representative scatter plots for BrdU cell cycle analysis. Mice were exposed to BrdU for three days prior to analysis. CD34^- Flt3^- LSK and CD34⁺ Flt3^- LSK cells were analyzed for BrdU and DNA content in plots on the right. B. Summary of data from a total of 9 Runx1^f/f and Runx1^f/f;Vav1-Cre mice. Error bars indicate 95% CI. P values for differences between wild type and mutant cells in G₀/G₁ are indicated in the bars. C. Runx1^f/f and Runx1^f/f;Vav1-Cre CD34^- Flt3^- LSK cells were sorted from bone marrow by multicolor FACS, stained with Hoechst 33342 and Pyronin Y, and analyzed by flow cytometry. Scatter plots show representative cell cycle distributions. D. Summary of quiescence analysis (n = 6 to 8). Error bars represent 95% CI. E. Annexin V staining of Runx1^f/f and Runx1^f/f;Vav1-Cre CD34^- Flt3^- LSK and CD34⁺ Flt3^- LSK bone marrow cells (5-7 mice). Error bars represent SEM.

Figure 6. Runx1 deficient HSCs are not exhausted by proliferative stress.

A. Kaplan-Meier survival curve of mice following weekly injections of 5-FU (on days 1, 7, 14) is on the left. Runx1^f/f, n = 10; Runx1^f/f;Vav1-Cre, n = 9. The middle graph represents the percentage of c-Kit⁺ cells in peripheral blood before 5-FU injection (d0), and various days following weekly injection (6-12 mice per data point). Error bars are SEM. Right hand graph is percentage of LS cells in the bone marrow 7 days after the second 5-FU injection. Runx1^f/f, n = 7. Runx1^f/f;Vav1-Cre, n = 8. B. Serial transplantation of bone marrow cells. Numbers above bars indicate the number of transplant recipients, not including those that died within two weeks from radiation toxicity. The genotypes and doses represent the bone marrow cells used to engraft primary recipients. 2×10⁶ cells from primary recipients were transplanted into secondary recipients, and the process was repeated twice more. C. Homing assay. CD34^- Flt3^- LSK cells were isolated by FACS, labeled with CFSE, and 1000 cells transplanted into 4-5 recipients along with 2 x10⁵ unlabeled carrier cells. Bone marrow cells (>10⁷) were analyzed 16 hours later by FACS. Shown is the percentage of live CFSE⁺ Ter119^- cells. Data are representative of two experiments.

Figure 7. Microarray analysis of genes mis-expressed in Runx1 deficient HSCs.

A. Heat plot of 4707 probe sets, representing 3820 genes, differentially expressed between LSKF^- and LSKF⁺ cells isolated from mice transplanted with Runx1^f/f and Runx1^f/f; Mx1-Cre bone marrow, with a significance threshold of P = 0.005. Each column shows gene expression from donor cells from an independent transplant recipient. Red, > than the average, blue, < than the average. B. Quantitative real-time PCR (qRT-PCR) performed on several genes to independently validate the microarray data. Data shown are the mean fold change in expression (± SEM), normalized to Gapdh expression, between Runx1^Δ/Δ (n = 3) and Runx1^f/f (n = 3) LSKF^- samples. C. GSEA profiles illustrating the correlation between genes negatively regulated by Runx1 in LSK cells, and genes sets of cell cycle and p53 pathways. Enrichment score, p values and FDR q-values are shown under the enrichment plots. Number of permutations was set to 1000. D. Venn diagram illustrating overlap between dysregulated genes in Runx1 deficient LSKF^- and LSKF⁺ cells, and genes occupied by Runx1 in the HPC-7 line . For list of genes see Table S4.