Gain of Function Mutant p53 Drives Clonal Hematopoiesis and the Pathogenesis of Myelodysplastic Syndromes

Clonal hematopoiesis increases with age, where a single mutant hematopoietic stem or progenitor cell (HSPC) contributes to a significant, measurable clonal proportion of mature blood lineages. Evolution of mutant clonal hematopoiesis with age predisposes the elderly to myelodysplastic syndromes (MDS) and other aging-associated diseases, suggesting that mutations identified in HSPCs contribute to disease development. Previous studies using whole exome sequencing of blood DNAs identified a high frequency of TP53 mutations in aged healthy individuals . TP53 mutations are also present in 10% of MDS cases and correlate with worse clinical outcomes. However, the role of TP53 mutations in clonal hematopoiesis and the pathogenesis of MDS is largely unknown. Ample data indicate that mutant p53 proteins not only lose their tumor suppressive functions, but also gain new abilities that promote tumorigenesis. Here, we report that specific gain-of-function (GOF) mutant p53 proteins drive the development of pre-leukemic hematopoietic stem cells (HSCs), predisposing aged mutant p53 mice to MDS development.

Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. We found that GOF mutant p53 (p53^R248W) enhances the competitive advantage of HSCs without affecting terminal differentiation in steady state. Further, we observed that p53^R248W mutant promotes clonal expansion of HSCs following genotoxic stresses. RNA-seq analysis revealed that HSC and leukemia stem cell signatures were significantly enriched in mutantHSCs compared to wild-type HSCs. Thus, HSCs expressing GOF mutant p53 recapitulate several key features of pre-leukemic HSCs. GOF p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. We discovered that GOF mutant p53 proteins, including p53^R248W, p53^R273H, and p53^R175H, but not wild-type p53, interact with histone methyltransferase EZH2 and enhance H3K27me3 in HSCs. EZH2, a catalytic component of the Polycomb repressive complex 2 (PRC2), trimethylates histone H3 at lysine 27 (H3K27me3) to repress the transcription of target genes. Indeed, we found that the expression of HSC regulators, including Elf4 and Gadd45g, was significantly downregulated in mutant HSCs compared to wild-type HSCs. ChIP-seq analysis show enrichment of H3K27me3 in the promoter regions of Elf4 and Gadd45g in mutant HSCs. Importantly, we discovered that EZH2 inhibitor treatment decreased the replating potential of mutantbone marrow cells to wild-type cell level. Thus, we demonstrated that GOF mutant p53 drives the development of pre-leukemic HSCs through enhancing EZH2 activity.

Given that most homozygous p53^-/- and p53^R248W/R248Wmice develop spontaneous tumors, including lymphoma, thymoma, and sarcoma, and die within 3 to 6 months after birth, it was not clear whether heterozygous mutant p53 mice (p53^R248W/+) would develop myeloid malignancies during aging. To evaluate the role of GOF mutant p53 in the pathogenesis of myeloid malignancies, we maintained p53^+/+ and p53^R248W/+ mice for more than a year and monitored their survival and tumor development. While p53^R248W/+ mice show extended life span compared to p53^-/- and p53^R248W/R248W mice, most of these mice died within 16-18 months after birth. During the observation period, approximately 30 % of heterozygous mutant p53mice developed MDS based upon pathological analysis of BM, spleen, and peripheral blood smear. Histological assessment revealed hypercellular BM in some aged p53^R248W/+ mice. BM of some aged p53^R248W/+ mice show neutrophil, erythroid precursor and megakaryocyte dysplasia. We further demonstrated that mutant p53 driven-MDS is transplantable. Thus, we provided evidence that GOF mutant p53 drives MDS development during aging.

In summary, we have identified a critical role for GOF mutant p53 in clonal hematopoiesis and the pathogenesis of MDS.