Characterization of Hematopoiesis in Tp53 R172H Mutant Mice
Authors: Liu Tuoen , Ndonwi Matthew , Walter Matthew J. 
Published: October 2019

In a cohort of 150 patients with myelodysplastic syndrome (MDS), the most common adult myeloid malignancy, missense mutations in TP53 were more common than frameshift, nonsense, splice site mutations or gene deletions predicted to be inactivating (29 missense compared to 11 inactivating mutations). However, mouse models of hematopoietic cancers often use Tp53 knock-out mice (i.e., modeling inactivating mutations) to study the role of Tp53 mutations on disease pathogenesis. We hypothesize that the type of TP53 mutation (missense vs. deletion) has different effects on hematopoiesis. To test our hypothesis, we characterized the hematopoiesis, including stem and progenitor cells, of wild-type (WT) and Tp53mutant mice. We analyzed two Tp53 mutant mouse models: i) knock-in mice harboring a G-to-A substitution at nucleotide 515 of Tp53 coding for the R172H substitution corresponding to the R175H mutation in humans [Cell. 2004;119(6):861-72], and ii) Tp53 knockout mice [Nature. 1992;356(6366):215-21]. We analyzed Tp53 heterozygous and homozygous knock-in mice (R172H/WT and R172H/R172H, respectively) and knock-out mice (Tp53 +/- and Tp53-/-, respectively). There was no difference in spleen/body weight ratio, complete blood counts, and distribution of mature hematopoietic lineages and stem/progenitor cells in the bone marrow and spleen of R172H/WT, R172H/R172H and WT mice (except for a minor reduction in myeloid-erythroid progenitors in R172H/WT and R172H/R172H mice) (7-15 weeks old, N=8). Compared to all other mice, Tp53-/- mice have significantly higher spleen/body weight ratio (2.24-fold increase compared to WT, p<0.001), and Tp53+/- and Tp53-/- mice have a higher percentage of lineage-/Sca+/Kit+ bone marrow progenitor cells (LSK, 2-fold increase in Tp53+/- mice compared to WT, p<0.001; 2.67-fold increase in Tp53-/- mice compared to WT, p<0.01), and bone marrow hematopoietic stem cells (HSC), including LSK-signaling lymphocyte activating molecule (SLAM) cells (5.6-fold increase in Tp53+/- and Tp53 -/- mice compared to WT, p<0.01), short-term HSC (2-fold increase in Tp53+/- mice compared to WT, p<0.01; 2.3-fold increase compared to WT, p<0.05), long-term HSC (5-fold increase in Tp 53+/- and Tp53-/- mice compared to WT, p<0.05), and dormant HSCs (5.6-fold increase in Tp53+/- mice, p<0.01; 4-fold increase in Tp53-/- mice compared to WT, p<0.01).

Competitive bone marrow repopulation transplant studies showed that stem cells from Tp53-/-, R172H/WT and R172H/R172H mice have a competitive advantage over wild-type competitor stem cells (N=10, Tp53-/- vs WT, p<0.001; R172H/WT vs WT, p<0.001; R172H/R172H vs WT, p<0.001; Tp53-/- vs R172H/WT and Tp53-/- vs R172H/R172H, p<0.01; R172H/WT vs R172H/R172H, p>0.05), with Tp53 -/- stem cells having the largest advantage based on peripheral blood chimerism (4 months after transplant, the contribution of test cells to the peripheral blood was 55±1.3% in WT mice compared to 92±1.5% in Tp53-/- mice; p<0.001). Competitive bone marrow repopulation studies are currently in progress for Tp53+/- mice and will be presented at the meeting. The overall survival of bone marrow chimeric mice was dependent on the Tp53 genotype of donor test cells, with recipients of Tp53 -/- bone marrow cells having the shortest overall survival compared to other genotypes (median overall survival of Tp53-/- recipients = 125.5 days after transplant, R172H/R172H recipients = 166.5 days, R172H/WT recipients = not reached, WT recipients = not reached)(overall survival of Tp53 -/- vs. WT recipient mice, p<0.001; Tp53-/- vs. R172H/WT, p<0.001; Tp53-/- vs. R172H/R172H, p<0.01; R172H/R172H vs. WT, p<0.01; R172H/R172H vs. R172H/WT, p<0.01; R172H/WT vs. WT, p=0.32; N=10 mice per genotype). In conclusion, our studies show that the type of Tp53 mutation affects the hematopoietic phenotype in mice, with Tp53-/- mice having the most severe phenotype compared to heterozygous and homozygous R172H Tp53 mutant mice. We are currently testing whether the penetrance or latency of hematopoietic cancers is influenced by cooperating mutations in these various Tp53 mutant mouse models. Collectively, these results may have implications for modeling hematopoietic cancers in vivo.

Disclosures

No relevant conflicts of interest to declare.