Dynamic Telomere Shortening and Chromosomal Instability in Irradiated CD34+ Cells Transduced with TP53 Hotspot Mutations R175H, R248W and R249S

Patients with MDS and a complex karyotype have a very short median survival and a high risk of transformation into AML. We showed earlier that TP53 mutations are associated with complex karyotype and disease progression. However, it is poorly understood how TP53 mutations contribute to the induction of chromosomal instability in hematopoietic stem and progenitor cells.

We therefore established a long-term cell culture (LTC) model and investigated the role of TP53 in human CD34+ hematopoietic stem and progenitor cells (HSCs) isolated from cord blood and in HT1080 cells over 6 weeks. We chose 3 different modifications: 1) TP53-deficient HSCs via shRNA knockdown, 2) HSCs with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S) and 3) HT1080 cells with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S). We performed each LTC at least three times. In order to stress the cells and induce chromosomal instability, we irradiated half of the cells. Besides functional assays in the first week, we performed detailed cytogenetic analysis including telomere length measurement at weeks 1, 3 and 6.

TP53 mutations and the downregulation of TP53 led to impaired hematopoiesis with decreased erythroid differentiation, increased apoptosis and decreased proliferation. None of the modifications induced chromosomal instability in cells without irradiation. In the irradiated cells, all cells carrying a TP53 mutation or a TP53 downregulation developed a chromosomal instability in comparison to the cells transduced with control vectors. However, no stable complex clones developed. Telomeres shortened during follow-up in HSCs carrying the mutations R175H, R248W and R249S. No other cells showed a dynamic response in telomere length. In order to analyze the DNA repair capacity, we performed yH2AX foci assay. Surprisingly, the same cells which showed a telomere shortening showed a lower amount of foci. This could be due to a lower amount of double-strand breaks or to a lower ability to form foci.

In summary, TP53 mutations and the downregulation of TP53 led to an increased chromosomal instability in irradiated cells only. Modifications alone did not lead to the development of complex karyotypes. Furthermore, only the mutations R175H, R248W and R249S led to a telomere shortening. In conclusion, a TP53 mutation seems to require additional passenger mutations in order to lead to MDS or AML with complex karyotype.