Impact of Mono- and Bi-Allelic TP53 Aberrations on Transformation of Human HSPCs

Introduction

Clonal hematopoiesis with mono-allelic TP53 mutations confers a high-risk for progression to myeloid neoplasms, particularly in the context of exposure to cytotoxic stress. During leukemic transformation, bi-allelic TP53 aberrations frequently evolve - however, the exact molecular mechanisms driving transformation are not well understood. Here, we use genetically engineered human HSPCs to investigate the impact of mono- and bi-allelic TP53 aberrations on HSPC function and genomic integrity.

Methods

Cord blood derived HSPCs were engineered using a CRISPR-Cas9 and adeno-associated viral vector-mediated knock-in strategy to generate TP53^R175H/WT, TP53^R175H/KO, TP53^R273H/WT, TP53^R273H/KO, TP53^KO/WT and TP53^KO/KO aberrations, respectively. To control for gene editing, the AAVS1 safe harbor locus was targeted. To understand the functional impact of different TP53 allelic states, cell cycle analysis, colony formation and serial replating as well as bulk mRNA sequencing were performed. The emergence of structural chromosomal variants was investigated by single-cell template strand sequencing upon doxorubicin exposure.

Results

Successful editing of HSPCs at the TP53 locus was confirmed at the genomic DNA, RNA and protein levels, respectively. Bi-allelic TP53 aberrant HSPCs showed an increase of cells in S-phase and formed significantly more colonies with a high proportion of BFU-E as compared to control cells. Mono-allelic TP53 aberrant cells lost their replating capacity after three passages whereas bi-allelic ones were not exhausted after five replatings. Transcriptome analysis revealed the induction of an inflammatory signature in cells with both, mono-and bi-allelic TP53 aberrations, with a skewed expression rate of erythroid and myeloid genes. Single-cell template strand sequencing indicated that mono-allelic TP53 aberrations, especially in conjunction with doxorubicin treatment, already induced a substantial increase of structural genomic variants including chromothripsis.

Conclusions

Our data indicate that already mono-allelic TP53 aberrations induce an inflammatory signature and structural chromosomal alterations in human HSPCs. Alterations of the second TP53 allele increase the proliferative and self-renewing capacity of HSPCs further promoting their leukemic transformation and progression. These data contributes towards a better understanding of human, TP53 aberrant leukemogenesis.

Woelfler:BMS/Celgene, AOP, Novartis, CTI, Janssen, Abbvie, GSK, Merck, CTI, Kartos, Telios.: Consultancy; Novartis, BMS/Celgene, CTI: Research Funding. Zebisch:Apollo Therapeutics: Research Funding; BMS, Otsuka: Honoraria; Astellas, BMS, Abbvie, Novartis, Laboratoires Delbert, JAZZ: Other: Consultancy; Pfizer: Other: Travel support.