Presence of mutant p53 increases stem cell frequency and is associated with reduced binding to classic TP53 binding sites in cell lines and primary AMLs [RNA-seq]

With an overall 5%-10% incidence rate in acute myeloid leukemia (AML), the occurrence of TP53 mutations is low compared with that in solid tumors. However, when focusing on high-risk groups including secondary AML (sAML) and therapy-related AMLs, the frequency of mutations reaches up to 35%. Mutations may include loss of heterozygosity (LOH) or deletion of the 17p allele, but are mostly missense substitutions that are located in the DNA-binding domain. Despite elaborate research on the effects of TP53 mutations in solid tumors, in hematological malignancies, the effects of TP53 mutations versus loss of TP53 remain unclear and under debate. Here, we compared the cellular effects of a TP53 mutant and loss of TP53 in human hematopoietic stem and progenitor cells (HSPCs). We found that when expressing TP53 mutant or loss of TP53 using siRNA, CD34+/CD38- cells have a significantly enhanced replating potential, which could not be demonstrated for the CD34+/CD38+ population. Using RNA-sequencing analysis, we found a loss of expression of p53 target genes in cells with TP53 knockdown. In contrast, an increased expression of a large number of genes was observed when expressing TP53 mutant, resulting in an increase in expression of genes involved in megakaryocytic differentiation, plasma membrane binding, and extracellular structure organization. When binding of p53 wild type and p53 mutant was compared in cell lines, we found that mutant p53 binds to a large number of binding sites genomewide, contrary to wild-type p53, for which binding is restricted to genes with a p53 binding motif. These findings were verified in primary AMLs with and without mutated TP53. In conclusion, in our models, we identified overlapping effects of TP53 mutant and loss of TP53 on in vitro stem cell properties but distinct effects on DNA binding and gene expression. RNA sequencing of Cordblood cells transfected with TP53 shRNA constructs or TP53 mutants

急性髓系白血病（acute myeloid leukemia, AML）中TP53突变的总体发生率为5%~10%，较实体瘤中偏低。但若聚焦于继发性急性髓系白血病（secondary AML, sAML）与治疗相关急性髓系白血病等高危群体，该突变的检出率可高达35%。TP53突变可包含杂合性缺失（loss of heterozygosity, LOH）或17p等位基因缺失，但多数为定位于DNA结合结构域的错义替换。尽管学界已对实体瘤中TP53突变的生物学效应开展了详尽研究，但在血液系统恶性肿瘤领域，TP53突变与TP53功能缺失的效应差异仍未明确，且存在广泛争议。
本研究对比了TP53突变体与TP53功能缺失在人类造血干细胞及祖细胞（human hematopoietic stem and progenitor cells, HSPCs）中的细胞效应。研究发现，通过小干扰RNA（siRNA）介导TP53敲低或表达TP53突变体后，CD34+/CD38-细胞的再铺板潜能显著增强，而CD34+/CD38+细胞未观察到该变化。通过RNA测序（RNA-sequencing）分析，我们在TP53敲低的细胞中检测到p53靶基因表达下调；与之相反，表达TP53突变体的细胞中大量基因表达上调，其中巨核细胞分化、质膜结合及细胞外结构组织相关基因的表达水平显著升高。
在细胞系中对比野生型p53与突变型p53的全基因组结合特征时，我们发现突变型p53可结合大量基因组位点，而野生型p53的结合仅局限于携带p53结合基序的基因。上述发现在携带与未携带TP53突变的原发性AML样本中均得到验证。
综上，在本研究模型中，我们发现TP53突变体与TP53功能缺失对体外干细胞特性存在重叠调控效应，但在DNA结合与基因表达层面则表现出截然不同的特征。本研究同时对转染了TP53短发夹RNA（shRNA）构建体或TP53突变体的脐带血细胞开展了RNA测序。