Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation

Abstract

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

Figure 1.

Inhibition of the TRAF6-IRAK1/4 axis restores blood counts in Tifab^-/-;miR-146a^-/- mice. (A) Overview of del(5q) myelodysplastic syndromes (MDS) resulting in haploinsufficient expression of the 5q- genes miR-146a and TIFAB and a corresponding increase in expression of their targets, TRAF6 and IRAK1. (B) Immunoblotting of Tifab^-/-;miR-146a^-/- bone marrow (BM) cells treated with lipopolysaccharide (LPS) (1 mg/mL) and the IRAK1/4 (1 mM) or UBE2N (5 mM) inhibitor for 30 minutes. (C) Outline of BM transplantations using wild-type (WT) or Tifab^-/-;miR-146a^-/- BM cells. Peripheral blood (PB) analysis was performed monthly on recipient mice to monitor for cytopenias. At onset of cytopenias in the Tifab^-/-;miR-146a^-/- recipient mice, an IRAK1/4 inhibitor (NCGC-1481 at 30 mg/kg) or UBE2N inhibitor (UC-65 at 20 mg/kg) was administered daily (or phosphate-buffered saline [PBS], vehicle control). PB counts were performed weekly after the treatment was initiated. (D) PB counts of the recipient mice before (20 weeks post BM transplantation) and after treatment (post-Tx) with the IRAK1/4-inhibitor (N=4-5 per group). (E) PB counts of the recipient mice before (20 weeks post BM transplantation) and post-Tx with the UBE2N-inhibitor (N=4-5 per group). Significance for panels (D and E) was determined with a Student’s t test (*P<0.05) between treated and vehicle-treated groups.

Figure 2.

Low-dose lipoploysaccharide does not significantly impact the phenotype of Tifab^-/-;miR-146a^-/- mice. (A) Outline of bone marrow (BM) transplantations using wild-type (WT) or Tifab^-/-;miR-146a^-/- mice in the presence of low-dose lipopolysaccharide (LD-LPS). (B) Peripheral blood (PB) counts of the recipient mice at 4 months after transplantation (N=9-10 per group). (C) Summary of lymphoid (CD3⁺ and B220⁺), myeloid (CD11b⁺Gr1^-) proportions within the PB of the recipient mice (N=9-10 per group). (D) Overall survival of mice transplanted with WT or Tifab^-/-;miR-146a^-/- mice treated with either PBS or LPS (N=9-10). (E) PB counts of the moribund Tifab^-/-;miR-146a^-/- mice (N=4-5 per group) at 10-13 months after transplantation. (F) Proportion of the indicated hematopoietic stem and progenitor cells in the BM from moribund Tifab^-/-;miR-146a^-/- mice (N=5-7 per group). Significance for panels (B, C, E, and F) was determined with a Student’s t test (*P<0.05; **P<0.01; ***P<0.001).

Figure 3.

Tifab^-/-;miR-146a^-/- hematopoietic stem and progenitor cells are less quiescent following low-dose lipoploysaccharide. (A) Colony assay of wild-type (WT) or Tifab^-/-;miR-146a^-/- LSK treated with low-dose lipopolysaccharide (LD-LPS) (N=3 per group). (B) Outline of in vivo bromouridine (BrdU) incorporation assay using WT and Tifab^-/-;miR-146a^-/- mice in the presence of low-dose chronic inflammation. (C) Absolute number of hematopoietic stem cells (HSC) from WT and Tifab^-/-;miR-146a^-/- mice treated with LPS (N=6-10). Error bars represent the standard error of the mean (SEM). (D) Proportion of BrdU-positive cells within HSC from WT and Tifab^-/-;miR-146a^-/- mice treated with LPS (N=6-8). Error bars represent the SEM. (E) Proportion of 7AAD⁺ AnnexinV⁺ cells within HSC from WT and Tifab^-/-;miR-146a^-/- mice treated with LPS (N=3-5). Error bars represent the SEM. Significance for panels (C, D, and E) was determined with a Student’s t test (*P<0.05; **P<0.01; ***P<0.001).

Figure 4.

Tifab^-/-;miR-146a^-/- hematopoietic stem and progenitor cells are functionally defective following low-dose lipoploysaccharide. (A) Outline of competitive bone marrow (BM) transplantations using wild-type (WT) or Tifab^-/-;miR-146a^-/-mice in the presence of low-dose lipopolysaccharide (LD-LPS). (B) Summary of donor-derived peripheral blood (PB) proportions at the indicated time points (N=5-6 per group). Error bars represent the standard error of the mean (SEM). Statistical analysis was performed between Tifab^-/-;miR-146a^-/--phosphate-buffered saline (PBS) and -LPS. (C) Proportion of donor-derived PB cells from primary recipient mice (N=5-6 per group). Error bars represent the SEM. (D) Proportions of donor-derived BM cells from primary recipient mice is reported 3 months after transplantation. Error bars represent the SEM (N=5-6 per group). (E) Proportion of donor-derived PB cells from secondary recipient mice (N=6 per group). Error bars represent the SEM. (F) Proportions of donor-derived BM cells from secondary recipient mice is reported 3 months after transplantation. (G) Representative flow cytometric profiles of Tifab^-/-;miR-146a^-/- and WT BM hematopoietic stem cells (HSC) after the tertiary transplantation. Error bars represent the SEM (N=6 per group). LK: Lin^-cKit⁺; LSK: Lin^-cKit⁺Sca1⁺; MPP: multipotent progenitor (Lin^-cKit⁺Sca1⁺CD48⁺CD150^-); ST-HSC, Lin^-cKit⁺Sca1⁺CD48^-CD150^-; LT-HSC, Lin^-cKit⁺Sca1⁺CD48^-CD150⁺. Significance for panels (B, D, and E) was determined with a Student’s t test (*P<0.05; **P<0.01; ***P<0.001).

Figure 5.

Differential TLR4 stimulation and p53 activation in Tifab^-/-;miR-146a^-/- hematopoietic stem and progenitor cells with low-dose lipoploysaccharide. (A) Outline of RNA-sequencing using hematopoietic stem and progenitor cells (HSPC) from wild-type (WT) and Tifab^-/-;miR-146a^-/- mice in the presence of low-dose lipopolysaccharide (LD-LPS). (B) Heatmap of differentially expressed genes in WT or Tifab^-/-;miR-146a^-/- LSK cells treated with LPS or phosphate-buffered saline (PBS) (1.5-fold; P<0.05; N=3 per group). (C) Venn diagram of upregulated/downregulated genes (1.5-fold; P<0.0.5) in WT (relative to WT LSK cells treated with PBS) or Tifab^-/-;miR-146a^-/- LSK cells (relative to Tifab^-/-;miR-146a^-/- LSK cells treated with PBS). (D) Heatmap of differentially expressed genes in LPS-stimulated WT or Tifab^-/-;miR-146a^-/- LSK cells (1.5-fold; P<0.05; N=3 per group). (E) Enrichment of transcription factors was determined with the ENCODE and CHIP Enrichment Analysis (ChEA) libraries using genes that are overexpressed (left panel) or downregulated (right panel) in LPS-stimulated Tifab^-/-;miR-146a^-/- vs. WT LSK. (F) Gene set enrichment analysis plots for TP53 targets in LPS-stimulated WT LSK cells (relative to WT LSK cells treated with PBS) and LPS-stimulated Tifab^-/-;miR-146a^-/- LSK cells (relative to Tifab^-/-;miR-146a^-/- LSK cells treated with PBS). NES: normalized enrichment score.

Figure 6.

Low-dose lipoploysaccharide induces p53 expression in Tifab^-/-;miR-146a^-/- hematopoietic stem and progenitor cells via IRAK1/4-TRAF6. (A) Outline of in vivo administration of lipopolysaccharide (LPS) following by immunoblot analysis. (B) Immunoblot analysis of wild-type (WT) and Tifab^-/-;miR-146a^-/- c-Kit⁺ bone marrow (BM) cells from WT and Tifab^-/-;miR-146a^-/- mice treated with low-dose LPS (LD-LPS) (1 μg /g) or vehicle twice a week for 30 days. (C) Immunoblotting of Tifab^-/-;miR-146a^-/- BM cells treated with LPS (1 mg/mL) and the IRAK1/4 (1 mM) or UBE2N (5 mM) inhibitor for 60 minutes. (D) Immunoblot analysis of a patient-derived del(5q) myelodysplastic syndromes (MDS) cell lines (MDSL) was treated with LPS (100 ng/mL) for the indicated time points. (E) Immunoblot analysis of MDSL cells treated with LPS (100 ng/mL) and the IRAK1/4 inhibitor (1 mM) for 2 hours.

Figure 7.

Deletion of p53 restores exhaustion of Tifab^-/-;miR-146a^-/- hematopoietic stem cells following low-dose lipoploysaccharide. (A) Outline of competitive bone marrow (BM) transplantations using Tifab^-/-;miR-146a^-/- or Tifab^-/-;miR-146a^-/-;p53^+/- mice in the presence of low-dose chronic inflammation. (B) Immunoblot analysis of Tifab^-/-;miR-146a^-/- or Tifab^-/-;miR-146a^-/-;p53^+/- c-Kit⁺ BM cells from wild-type (WT) and Tifab^-/-;miR-146a^-/- mice treated with low-dose lipopolysaccharide (LD-LPS) (1 μg/g) or vehicle twice a week for 30 days. (C, D) Proportion of donor-derived peripheral blood (PB) cells from the recipient mice transplanted with Tifab^-/-;miR-146a^-/- or Tifab^-/-;miR-146a^-/-;p53^+/- mice treated with either phosphate-buffered saline (PBS) or LPS. Error bars represent the standard error of the mean (SEM). (E) Proportion of donor-derived SLAM hematopoietic stem cells (HSC) in total cells from the recipient mice transplanted with Tifab^-/-;miR-146a^-/- or Tifab^-/-;miR-146a^-/-;p53^+/- mice treated with either PBS or LPS. Error bars represent the SEM. (F) Proportion of bromouridine (BrdU)-positive cells within HSC from Tifab^-/-;miR-146a^-/- or Tifab^-/-;miR-146a^-/-;p53^+/- mice treated with LPS (N=3 per group). Error bars represent the SEM. (G) Summary of findings. Del(5q) myelodysplastic syndromes (MDS) hematopoietic stem and progenitor cells (HSPC) (blue) exhibit impaired hematopoiesis and increased innate immune signaling because of derepression of TRAF6 and IRAK1 following deletion of miR-146a and TIFAB. Loss of TIFAB also results in increased p53 activation due to diminished USP15 activation. During inflammation, del(5q) MDS HSPC activated IRAK-TRAF6 signaling and induce p53, which results in clonal depletion. Loss-of-function mutations (LOF) or deletion of p53 permits clonal expansion of del(5q) MDS HSPC during inflammation. Significance for panels (C, D, E, and F) was determined with a Student’s t test (*P<0.05).