Role of p21 in a Mouse Model of NUP98-HOXD13 Fusion Driven Myelodysplastic Syndromes (MDS)

The myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective hematopoiesis leading to cytopenias and a high rate of progression to acute myeloid leukemia (AML). The NUP98-HOXD13 (NHD13) fusion has been found in patients with MDS and AML. A transgenic (Tg) mouse model, generated by Peter Aplan’s group, which utilizes Vav 1 regulatory elements to direct expression of the NHD13 transgene in hematopoietic tissues, displays the phenotypic features of MDS including a chronic phase of cytopenias followed by transformation to AML (Lin et al., 2005). We previously reported that loss of one or both alleles of p53 did not rescue the MDS phenotype in NHD13⁺ Tg mice, but rather exacerbated the MDS phenotype and accelerated the development of AML (Xu et al., 2012). Expression of p21^WAF1/CIP1(p21) was increased in the Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells isolated from NHD13⁺ Tg mice, so we generated and analyzed NHD13⁺p21^+/– and NHD13⁺p21^–/– mice to further investigate whether the accelerated MDS and AML that occurs in the absence of p53 relates to the defective expression of the p53 target gene. Deletion of p21 significantly altered the fate of the NHD13⁺ Tg mice. All of the NHD13⁺p21^–/– mice died of AML, rather than MDS. Only 18% (4 out of total 22 mice) of the NHD13⁺p21^+/– mice developed MDS with a median survival of 289 d; in contrast 31% (9 out of total 29 mice) of NHD13⁺ Tg mice died from MDS, with a median survival of 230 d (p<0.05). We examined the peripheral blood counts of the “clinically healthy” NHD13⁺p21^–/– mice at 3 to 5 months, and found increased white blood cell (WBC) and neutrophil (NE) counts, compared to the age matched NHD13⁺ Tg mice. Clearly the deletion of one or two alleles of p21 increases the median survival of NHD13⁺ Tg mice with MDS, and the complete loss of p21 rescues the fatal MDS induced by NHD13 fusion gene. However the deletion of one or two p21 alleles does not significantly affect the development of AML, which in 82% of NHD13⁺p21^+/– mice resulting in a median survival of 291 d, AML in the NHD13⁺p21^–/– mice was with a median survival of 320 d, and 69% of NHD13⁺ Tg mice showed AML with a median survival of 315 d. p21 is important for maintaining a normal-sized HSPC pool (Cheng et al., 2000), and both p21 and p53 have been shown to be involved in the determination of asymmetric vs symmetric cell divisions of epithelial cells (O’Brien et al., 2012; Cicalese et al., 2009). To determine whether the symmetric division of NHD13⁺ HSCs is affected by the loss of p21 or p53 in vitro, we performed paired daughter cell assays. Single LSKCD34^–Flt3^–CD150⁺ cells isolated from wild type (WT), NHD13⁺, NHD13⁺p21^–/– and NHD13⁺p53^–/– mice bone marrow were sorted into 96-well plates one cell/well. After the first cell division, the two daughter cells were split into two wells for 12 days in culture. We examined the ability of sorted single LSKCD34^–Flt3^–CD150⁺ cells to generate daughter cells that retain multipotent lineage differentiation potential and found that NHD13⁺p53^–/– CD150⁺CD34^–Flt3^–LSK cells underwent symmetric self-renewal divisions 85% of the time (both daughter cells are multipotent), with 15% asymmetric divisions (only one daughter cell is multipotent); the NHD13⁺p21^–/– CD150⁺CD34^–Flt3^–LSK cells produced 13% symmetric self-renewal divisions, 50% asymmetric divisions and 37% symmetric commitment divisions (both daughter cells are not multipotent); the NHD13⁺ CD150⁺CD34^–Flt3^–LSK cells produced 50% symmetric self-renewal divisions, 40% asymmetric divisions and 10% symmetric commitment divisions; and the WT CD150⁺CD34^–Flt3^–LSK cells produced 27% symmetric self-renewal divisions, 42% asymmetric divisions and 31% symmetric commitment divisions. These data indicate that loss of p53 increases symmetric self-renewal divisions of NHD13⁺ HSCs, and loss of p21 increases asymmetric self-renewal divisions of NHD13⁺ HSC in vitro. Collectively, our data indicate that loss of p21 maintains the survival of MDS driven by NUP98-HOXD13 fusion, which is independent of the function of p53; and the increased asymmetric self-renewal divisions of NHD13⁺p21^–/– HSCs may contribute to the increased survival observed in NHD13⁺p21^–/– MDS mice.