TP53 mutations in myeloid neoplasms (MDS/AML) are associated with high-risk disease, poor outcome, and complex karyotype. The molecular mechanisms which lead to global chromosomal instability remain poorly understood. Loss of 5q [del(5q)] is the most frequent cytogenetic abnormality associated with TP53 mutations suggesting that haploinsufficiency of genes on 5q contributes to chromosomal instability.
We reprogrammed MDS/AML patient samples to establish genetically accurate iPSC lines from preleukemic subclones. We generated iPSCs with TP53 mutations and del(5q), differentiated them to hematopoietic progenitors (HPCs), and determined the contribution of del(5q) to genome instability.
By reprogramming MDS/AML complex karyotype patient samples, we identified iPSCs with heterozygous TP53 mutations (TP53-only), as well as iPSCs with TP53 mutations and del5(q22-q31) (TP53;del5q), and an otherwise normal karyotype. HPCs derived from TP53;del5q iPSCs had decreased multilineage differentiation potential compared to the TP53-only HPCs. Gene expression analysis of TP53;del5q HPCs revealed downregulation of genes involved in chromosome segregation and DNA damage repair. Following irradiation TP53;del5q cells had significantly delayed DNA damage repair kinetics. In order to evaluate the effects of TP53 and del(5q) on chromosomal segregation during stress, we arrested the cells in mitosis by disrupting the mitotic spindle and quantified the induction of micronuclei, a marker of chromosomal instability that occurs due to lagging chromosomes. TP53;del5q cells had an increased frequency of micronuclei formation compared to TP53-only cells. We also detected micronuclei in primary AML patient samples. Micronuclei in iPSC-HPCs and primary patient cells had disrupted nuclear envelope and DNA damage marked by y-H2AX.
Our reprogramming approach revealed that TP53 mutations are disease-initiating and frequently followed by 5q loss. We propose that del(5q) cooperates with mutant TP53 to promote genome instability via two distinct mechanisms: classical double-stranded break repair and micronuclei formation. The latter is associated with global chromosomal instability, aneuploidy, and chromothripsis. We propose that loss of 5q accelerates genome instability in TP53-mutant cells which over time impedes normal hematopoietic differentiation and leads to complex karyotype.
Becker:The France Foundation: Honoraria; Accordant Health Services/Caremark: Consultancy; AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding.
Asterisk with author names denotes non-ASH members.