The Pathogenesis of Therapy-Related Myeloid Neoplasms from TP53-Mutant Clonal Hematopoiesis

Introduction

Therapy-related myeloid neoplasms (t-MN) arise from pre-malignant hematopoietic stem and progenitor cells (HSPCs) carrying mutations in leukemia driver genes. Patient outcomes are dismal, especially when harboring mutations in TP53. The molecular mechanisms underlying the progression from TP53-mutant clonal hematopoiesis (CH) to t-MN are poorly understood. Particularly, the impact of the TP53 allelic state (i.e., mono- vs. biallelic) remains controversial, reflected by inconsistencies in the clinical classification systems of myeloid neoplasms.

Methods and Results

To investigate the pathogenesis of t-MN derived from TP53-mutant CH, we developed novel in vitro and in vivo model systems. By enforced Hoxb8 expression in bone marrow cells derived from Trp53-floxed-R245W-GFP mice, we generated reversibly immortalized murine HSPC lines carrying inducible and traceable Trp53 missense mutations in an either mono- or biallelic configuration. This allowed us to compare the functional impact of the Trp53 allelic states in the absence of confounding genomic alterations as well as to test for their potential to promote malignant transformation by monitoring for cytotoxic therapy-induced, Hoxb8-independent immortalization.

Upon treatment with chemotherapeutic agents, both mono- or biallelic loss of wild-type Trp53 promoted clonal expansion due to resistance to p53-dependent G1 arrest and apoptosis, but only cells with biallelic mutations showed signs of persistent genomic instability as measured by γH2AX staining. Consequently, only cells with biallelic Trp53 mutations showed continued proliferation in the absence of Hoxb8 expression after drug-induced DNA damage indicative of immortalization. These transformed cell lines uniformly exhibited multiple numerical and structural chromosomal aberrations, reminiscent of the clinical association of biallelic TP53 mutations with complex karyotypes.

To test the importance of the Trp53 allelic state in vivo, we generated mice with 5-10% of mono- or biallelic Trp53-mutant HSPCs without prior transplantation using SCL-CreERT2;Trp53-floxed-R245W-GFP mice. After γ-irradiation to mimic anti-cancer therapy, mono- and biallelic Trp53 mutations promoted clonal expansion of HSPCs on all levels of the hematopoietic hierarchy. Subsequently, nearly 100% of mice with biallelic Trp53-mutant HSPCs progressed to t-MN. These t-MNs were characterized by cytopenias, splenomegaly, leukemic blasts with an erythroid progenitor-like immunophenotype, and an enrichment of erythroid as well as Myc target gene transcriptional signatures – compatible with an acute erythroid leukemia (AEL). Notably, mice with monoallelic Trp53 mutations similarly developed AELs, although less frequently and with a significantly longer latency. Whole genome sequencing revealed extensive structural and numerical chromosomal aberrations as well as copy number alterations, and – importantly – all cases derived from mice with monoallelic Trp53 mutations showed spontaneous loss of heterozygosity (LOH) of the Trp53 allele including copy number-neutral LOH as well as hemizygous deletions.

To confirm that complete loss of wild-type p53 functionality is required for malignant transformation, we examined the impact of non-mutational p53 inactivation by overexpressing its negative regulator MDM2, which is often amplified in cancer. While this had no additional effect on cells carrying biallelic Trp53 mutations, combining monoallelic Trp53 mutations with MDM2 overexpression phenocopied the biallelic state regarding abrogated p53 target gene expression, resistance to apoptosis, increased genomic instability, and potential for in vitro transformation.

Discussion and Outlook

We established novel and well-controlled in vitro and in vivo model systems to investigate the role of the Trp53 allelic states (mono- or biallelic) during the progression from CH to t-MN. While monoallelic Trp53 mutations are sufficient for clonal expansion in the context of DNA-damaging insults, full inactivation of wild-type p53 activity, either by a second mutation or through non-mutational inactivation via MDM2 overexpression, is necessary to induce genomic instability and transformation into highly aggressive myeloid neoplasms, characterized by complex karyotypes. Together, these findings provide novel insight into TP53-driven leukemogenesis.