Novel p53 Mutant Mouse Model Recapitulates the Biology of Human Therapy-Related Myeloid Neoplasms

Therapy-related myeloid neoplasms (t-MNs) represent a serious and devastating complication of cancer chemotherapy. The pathogenesis of t-MNs, driven by the selective expansion of mutant hematopoietic stem cells (HSCs, also known as clonal hematopoiesis [CH]) under chemotherapy, remains inadequately understood. TP53 mutations, present in 25-40% of t-MNs, are the most common genetic alterations, with chemotherapies such as alkylating agents and platinum compounds promoting the expansion of p53 mutant HSCs. Despite this knowledge, the mechanisms that drive the transition from clonal expansion to malignant transformation are unclear.

To investigate how chemotherapy affects the clonal expansion and malignant transformation of p53-mutant cells, we have established a mouse model of p53-mutated clonal hematopoiesis. This model uses a chimeric bone marrow transplantation approach, where a defined number of bone marrow (BM) cells carrying the Trp53^R172H mutation were harvested from Vav-Cre;Trp53^wmR172H/fl;mTmG mice, identifiable by GFP. Additionally, congenic wildtype BM cells from mTmG mice, marked by RFP, were co-transplanted into irradiated recipients. This dual-marker system allows for tracking and comparison of the mutant and wildtype cell populations. Six weeks after transplant, the levels of p53-mutant cells in the peripheral blood of recipient mice were measured using flow cytometry. The mice were then split into two groups: one received weekly intraperitoneal injections of carboplatin, and the other was given a placebo, over 4 weeks. The proportion of Trp53^R172H mutant cells in the blood was assessed at baseline and again after the treatment period on weekly basis.

As anticipated, the mice treated with carboplatin showed a significant expansion of p53-mutant cells compared with both baseline and the control group. Notably, by the fourth month after treatment, all mice in the carboplatin group developed therapy-related acute myeloid leukemia (t-AML) and died, whereas none of the placebo treated mice developed myeloid neoplasms. BM analysis revealed AML cells were all GFP positive (i.e., Trp53 mutant) with nuclear accumulation of the mutant p53 protein.

CyTOF analysis on BM samples from three mice in each group 3 months after treatment revealed a distinct population of p53-mutant cells in the carboplatin group, expressing c-Kit, Ki67, p53mut, and CD71, representing proliferative erythroid progenitors. Whole exome sequencing on GFP-sorted p53 mutant cells identified recurrent deletions in specific chromosomal regions, such as 1q, 4q, and chromosome 10, indicating genomic instability and the potential involvement of these regions in leukemogenesis. Furthermore, gene mutations in RAS pathway genes were frequently identified in the AML cells, Ptpn11 gene in 6 of 8 mice, with Kras and Nf1 mutations found in the remaining two.

To better understand the cellular heterogeneity and the specific pathways activated during this transformation, we sorted the GFP+c-Kit+ population from three t-MN mice and controls and performed scRNA-seq. Our scRNA-seq analysis identified distinct clusters of specific progenitor cells that were highly proliferative and characterized by robust upregulation of genes in DNA damage response and repair pathways, particularly through Brca1 and homologous recombination. These cells also showed advanced regulation of the cell cycle via Chk proteins, with active DNA synthesis and replication controls. These findings identified critical survival pathways and potential targets for therapeutic intervention in t-AML.

In conclusion, we developed a novel p53-mutant mouse model that faithfully recapitulates therapy-related AML following platinum chemotherapy. This model not only allows the clonal tracing of p53 mutant cells during chemotherapy treatment but also clone-specific molecular analysis during clonal expansion and malignant transformation. In contrast to previous models that demonstrated the expansion of p53 mutant HSCs under chemotherapy treatment, our model uniquely captures the subsequent malignant transformation into t-AML. This advancement is crucial as it bridges the gap between clonal expansion and the onset of full malignancy, providing a more complete and clinically relevant model of the disease process in t-MNs. This model will be useful to study the pathogenesis of t-MN development and strategies to mitigate the process.