Abstract 841: Mutant Trp53
R172H has gain-of-function or dominant-negative effects in response to hematopoietic stressors in mice
Authors: Liu Tuoen , Ahmed Tanzir , Alberti Michael O. , Wadugu Brian A. , Shao Jin , Grieb Sarah , Przybyl Grace , Ndonwi Matthew , Walter Matthew J. 
Published: June 2022

Background: Missense mutations of the TP53 gene frequently occur in patients with myelodysplastic syndrome and acute myeloid leukemia. The functional consequences of mutant p53 expression on hematopoiesis remain incompletely studied. Whether TP53 mutations confer a loss-of-function, gain-of-function, or dominant-negative effect in response to chemotherapies has not been fully explored. One of these hotspot mutations is a G-to-A substitution at nucleotide 525 of TP53, which codes for an Arg175His (R175H) substitution. Thus, we characterized hematopoiesis in Trp53R172H constitutive knock-in mice, corresponding to R175H TP53 mutation in patients, and compared the results to Trp53 knock-out and control mice.

Methods: Five genotypes of mice were analyzed (Trp53+/+ [WT], Trp53R172H/+, Trp53R172H/R172H, Trp53+/-, and Trp53-/-). Frequencies of peripheral blood (PB) and bone marrow (BM) cells were measured by flow cytometry and fluorouracil (5-FU) and N-ethyl-N-nitrosourea (ENU) were used as hematopoietic stressors.

Results: There was no change in the PB counts or lineage distribution of mature PB and BM cells in Trp53R172H/+ and Trp53R172H/R172H mice compared to WT or Trp53 knock-out mice at 8-15 weeks of age. We found that expression of Trp53R172H mutation in mice does not cause hematopoietic stem cell (HSC) expansion under basal conditions, in contrast to heterozygous and homozygous deletion of Trp53, but does confer a functional advantage to HSCs in a competitive repopulation assay. Trp53R172H HSCs expand further following ENU exposure, similar to Trp53-/- cells and consistent with a dominant-negative effect of Trp53R172H. Mechanistically, mutant p53R172H has a dominant-negative effect on p21-induced expression following ENU treatment. Repeated 5-FU injections drive proliferation of progenitors. To test the hematopoietic cell-intrinsic effects of mutant p53R172H on white blood cell count recovery, we exposed mice reconstituted with Trp53+/+, Trp53+/-, Trp53-/- or Trp53R172H/+ BM cells to a single dose of 5-FU and followed mice weekly for a month. Trp53R172H/+ mice were least affected by the 5-FU challenge and showed the fastest and largest recovery of WBC count compared to Trp53+/+, Trp53+/- and Trp53-/- mice. These results demonstrate that mutant p53R172H confers a gain-of-function property following 5-FU exposure that was not seen with deletion of Trp53.

Conclusion: Our data suggest that in vivo expression of mutant Trp53R172H has minimal effects on basal hematopoiesis. However, mutant Trp53R172H induces either a gain-of-function or a dominant-negative effect depending on the type of hematopoietic stress and hematopoietic phenotype being observed. Overall, our results indicate that the functional consequences induced by various Trp53 mutations (e.g., deletion and point mutation) are not identical, but are influenced by the type of hematopoietic stress.

Citation Format: Tuoen Liu, Tanzir Ahmed, Michael O. Alberti, Brian A. Wadugu, Jin Shao, Sarah Grieb, Grace Przybyl, Matthew Ndonwi, Matthew J. Walter. Mutant Trp53 R172H has gain-of-function or dominant-negative effects in response to hematopoietic stressors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 841.