Loss of p53 accelerates the complications of myelodysplastic syndrome in a NUP98-HOXD13-driven mouse model - PubMed (original) (raw)

Loss of p53 accelerates the complications of myelodysplastic syndrome in a NUP98-HOXD13-driven mouse model

Haiming Xu et al. Blood. 2012.

Abstract

The nucleoporin gene NUP98 is fused to several genes including HOXD13 in patients with myelodysplastic syndromes (MDS), acute myeloid leukemia, and chronic myeloid leukemia, blast crisis. Genetically engineered mice that express a NUP98-HOXD13 (NHD13) transgene (Tg) display the phenotypic features of MDS, including cytopenias, bone marrow dysplasia, and transformation to acute leukemia. Here we show that short-term treatment with the p53 inhibitor Pifithrin-α partially and transiently rescued the myeloid and lymphoid abnormalities found in NHD13(+) Tg mice, with no improvement in the anemia, while the genetic deletion of 2 alleles of p53 rescued both the myeloid progenitor cell and long-term hematopoietic stem cell compartments. Nonetheless, loss of one or both alleles of p53 did not rescue the MDS phenotype, but instead exacerbated the MDS phenotype and accelerated the development of acute myeloid leukemia. Our studies suggest that while targeting p53 may transiently improve hematopoiesis in MDS, over the long-term, it has detrimental effects, raising caution about abrogating its function to treat the cytopenias that accompany this disease.

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Figures

Figure 1

Figure 1

HSPC frequencies in NHD13+ Tg and control mice. Bone marrow (BM) cells from wild-type and NHD13+ Tg mice were harvested at 4 months of age and prepared for flow cytometric analysis. (A) Representative FACS plots show LSK, CD34, Flt3, and CD150 expression within WT and NHD13+ Tg mice BM cells. (B) The frequencies of LSK cells, ST-HSCs (LSKCD34+Flt3−) and LMPPs (LSKCD34+Flt3+) in WT and NHD13+ Tg mouse BM (mean ± SD, n = 7, *P < .05, **P < .01). (C) The frequency and (D) absolute number of CD150+ LT-HSCs (CD150+LSKCD34−Flt3−) in WT and NHD13+ Tg mouse BM are shown (mean ± SD, n = 7, *P < .05). (E left panel) The FACS plots and gating strategies used to identify CMPs (Lin−Sca-1− cKit+ IL-7Rα−CD34+FcgRlow), GMPs (Lin−Sca-1−cKit+IL-7Rα−CD34+FcgRhigh), and MEPs (Lin−Sca-1−cKit+IL-7Rα−CD34−FcgRlow) within the Lin−IL-7Rα−cKit+ population. Right panel, The frequencies of CMPs, GMPs, and MEPs in WT and NHD13+ Tg mice BM (mean ± SD, n = 8, **P < .01). (F left panel) Representative FACS plots of the gating strategy to identify the CLPs (Flt3+Lin−IL-7Rα+ckitintSca-1int) population. (Right panel) The frequency of CLPs in WT and NHD13+ Tg mice BM (mean ± SD, n = 8, **P < .01).

Figure 2

Figure 2

Increased p53 level in NHD13+ bone marrow cells. The intracellular p53 level was measured by flow cytometry. (A) Representative FACS profiles of p53 protein levels in WT (gray) and NHD13+ Tg (black) mouse bone marrow (BM) LSK (Lin−Sca-1+cKit+) cells. The graph on the right indicates the mean percentage of p53+ cells present (n = 5, ***P < .001). (B) Representative FACS profiles of p53 protein levels in WT (gray) and NHD13+ Tg (black) mice BM CD71+Ter119− cells. The bar graph on the right indicates the mean percentage of p53+ cells present (n = 5, *P < .05). (C) Representative FACS profiles of p53 protein levels in WT (gray) and NHD13+ Tg (black) mice BM CD71+Ter119+ cells. Data shown on the right are the mean percentage of p53+ cells present (n = 5, P = .052).

Figure 3

Figure 3

The effects of PFT-α treatment on NHD13+ Tg mice. NHD13+ Tg mice at the MDS stage (∼ 5 months of age) were intraperitoneally injected daily with DMSO vehicle or PFT-α (2 mg/kg body weight) for 8 weeks. Peripheral blood was collected weekly. (A) The white blood cell (WBC) counts, (B) neutrophil (NE) counts, (C) lymphocyte (LY) counts, (D) hemoglobin (HB) value, (E) mean corpuscular volume (MCV), and (F) platelet counts are shown (mean ± SD, n = 5 mice/group, *P < .05). (G) Kaplan-Meier survival curves of NHD13+ mice treated with DMSO vehicle or PFT-α (2 mg/kg body weight) for 8 weeks (n = 5 mice/group).

Figure 4

Figure 4

The loss of 1 or 2 p53 alleles does not rescue the MDS phenotype induced by NHD13 fusion protein. (A) Representative staining profiles for LSK cells, CD34, Flt3, and CD150 expression within BM cells of NHD13+, NHD13+p53+/−, and NHD13+p53−/− mice with MDS, compared with WT controls. (B) The frequencies of LSK cells, ST-HSCs, LMPPs, CD150+ LT-HSCs, and the total number of CD150+ LT-HSCs in NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with MDS are compared with WT control mice (mean ± SD, *P < .05, **P < .01). (C) Representative FACS profiles for CMPs (Lin−Sca-1− cKit+ IL-7Rα−CD34+FcgRlow), GMPs (Lin−Sca-1−cKit+IL-7Rα−CD34+FcgRhigh), and MEPs (Lin−Sca-1−cKit+IL-7Rα−CD34−FcgRlow) within the Lin−IL-7Rα−cKit+ population for the NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with MDS, compared with the WT control mice. (D) The frequencies of CMPs, GMPs, and MEPs in the NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with MDS, compared with WT control mice (mean ± SD, *P < .05, **P < .01). (E) Representative FACS profiles showing CD71 and Ter119 staining of BM cells isolated from NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with MDS, compared with WT control. (F) The frequencies of Ter119+ and CD71−Ter119+ cells in the NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with MDS, compared with WT controls (mean ± SD, *P < .05, **P < .01). The mice used for this figure were between 4 and 5 months old; n = 8 for each genotype.

Figure 5

Figure 5

The inactivation of p53 decreases the survival of NHD13+ Tg mice with MDS or AML. (A) Kaplan-Meier survival curves for the NHD13+ (n = 23), NHD13+p53+/− (n = 25), NHD13+_p53_−/− (n = 18) mice with MDS or AML, and the p53+/− (n = 24), _p53_−/− (n = 23) control mice are shown. The P value represents the comparison of survival between the NHD13+_p53_−/− and _p53_−/− mice, or a comparison of survival between NHD13+p53+/− and p53+/− mice (***P < .001). (B) Survival curves for the NHD13+ (n = 23), NHD13+p53+/− (n = 25), and NHD13+_p53_−/− (n = 18) mice with MDS or AML. The P value represents a comparison of survival between the NHD13+_p53_−/− and NHD13+ mice, or between the NHD13+p53+/− and the NHD13+ mice (***P < .001). (C) Survival curves for the NHD13+ (n = 7), NHD13+p53+/− (n = 14), and NHD13+_p53_−/− (n = 10) mice with MDS. The P value represents the comparison of survival between NHD13+_p53_−/− and NHD13+ MDS mice (***P < .001). (D) Survival curves for the NHD13+ (n = 16), NHD13+p53+/− (n = 11), and NHD13+_p53_−/− (n = 8) mice with AML. The P value represents a comparison of survival between NHD13+_p53_−/− and NHD13+ mice, or between NHD13+p53+/− and NHD13+ mice (***P < .001).

Figure 6

Figure 6

The loss of one or two p53 alleles accelerates the development of AML. (A) Representative FACS profiles show Mac-1 and Gr-1, c-Kit and Sca-1, c-Kit and Mac-1, c-Kit and Gr-1staining of BM cells isolated from NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with AML, compared with WT control mice. (B) The frequencies of Mac-1+ or Gr-1+, c-Kit+Sca-1−, Mac-1+c-Kit+, and Gr-1+c-Kit+ cells in NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mouse BM with AML, compared with WT control mice (mean ± SD, *P < .05, **P < .01). (C) Representative H&E-stained BM cytospin samples from NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with AML compared with WT control. (D) Representative staining profiles for LSK cells, showing also CD34, Flt3, and CD150 expression within the BM of NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with AML, compared with WT controls. (E) The frequencies of LSK cells, CD150+ LT-HSCs, ST-HSCs, and LMPPs in NHD13+, NHD13+p53+/−, and NHD13+_p53_−/− mice with AML, compared with WT control mice (mean ± SD, *P < .05, **P < .01). The mice used for this figure: NHD13+ mice with AML (n = 10, age between 9 months to 12 months), NHD13+p53+/− mice with AML (n = 10, age between 5 months to 10 months), NHD13+_p53_−/− mice with AML (n = 8, age between 4 and 6 months), and WT control (n = 6, age between 4 and 7 months).

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