MUTANT P53 INCREASES STEM-CELL FREQUENCY BUT IS ASSOCIATED WITH REDUCED BINDING TO TP53 BINDING SITES IN PRIMARY AMLS AND CELL LINES
In AML, TP53 alterations have been shown in 10% of the patients. Disruption of p53 function can be caused by loss of the short arm of chromosome 17 or by TP53 mutations. Most mutations are missense substitutions that lead to loss of tumour suppressive functions of p53. TP53 mutations have also been associated with gain-of-function, probably related to an altered protein stability, but it is unknown if GOF plays a role in the pathogenesis of AML.
We overexpressed TP53 mutant (TP53R273H) or performed a knockdown of TP53 in CD34+ umbilical cord blood (CB) cells and performed cell culture assays. We then performed ChIP-sequencing to identify mutant p53 binding sites in cell lines and primary AMLs. We aimed to identify differences in RNA expression by comparing TP53wt and TP53mut AMLs in different AML cohorts.
In CB CD34+/CD38-, TP53 mutant resulted in an enhanced replating potential of myeloid colony formation in association with an significantly enhanced stem-cell frequency in LTC-IC assays without affecting differentiation. Similar to the TP53R273H, shTP53 resulted in an enhanced replating potential of CFU-GMs, suggesting overlapping effects.
In order to identify p53 binding by wt and mutant protein, ChIP-sequencing was performed against endogenous p53. By motif search in primary AMLs with and without TP53 mutation and detected that the p53 motif was present in p53 binding sites in TP53wt AMLs, but not in TP53mut AMLs.
To identify the transcriptional program induced by p53R273H we sorted GFP+/CD34+/CD38- cells transduced shTP53 or TP53R273H expressing vectors and performed RNA-sequencing. Loss of TP53 resulted in decreased expression of pro-apoptotic genes. In contrast, expression of TP53mut was associated with decreased expression of genes involved in ribosome biogenesis and metabolism but increased expression of genes involved in differentiation, plasma membrane binding and extracellular structure organization. To identify whether comparable pathways are affected in AMLs with TP53mut we used 3 large datasets and identified the differentially expressed genes between TP53mut and TP53wt AMLs per dataset. We identified genes that are linked to megakaryocyte and erythroid differentiation and bone marrow homing of AML cells.
Our results indicate that loss of TP53 expression or the presence TP53mut lead to increased stem-cell frequency and replating potential in the stem cell enriched fraction. In addition, the TP53mut triggered a number of additional programs in conjunction with an altered p53 binding. The binding of p53 to the non-classical bindings sites may be linked to the increased protein stability and altered protein complex formation. Also, by using large AML data sets a gene set was identified that was consistently differentially expressed in TP53mut AML.