Modelling the Evolution of Clonal Hematopoiesis to Myeloid Malignancies Under the Selective Pressure of Anti-Cancer Therapy

Introduction

Myeloid neoplasms post cytotoxic therapy (MN-pCT) are a catastrophic complication of cancer therapy with an aggressive disease course and dismal patient outcomes. They frequently arise from preexisting clonal hematopoiesis (CH). Large sequencing studies have reported differences in the mutational spectra of age-related CH (ARCH), therapy-related CH (t-CH), de novo MN, and MN-pCT, respectively, suggesting functional differences in the capacity of the various driver gene mutations to initiate MN-pCT. Here, we utilized clinical sequencing data, meta-analytic statistical methods, newly established CRISPR/Cas9-edited mouse hematopoietic stem and progenitor cell (HSPC) lines, and murine mixed bone marrow chimeras to model the evolution of MN-pCT from CH under the selective pressure of anti-cancer therapy.

Results

We compared targeted next-generation sequencing data from MN-pCT and de novo MN patients collected at our institution. We found a significantly lower frequency of DNMT3A mutations in the MN-pCT group, while mutations in PPM1D and TP53 were more frequent. To determine the frequency of typical ARCH mutations (i.e., in the genes DNMT3A, TET2, ASXL1) as opposed to mutations in genes associated with t-CH (i.e., ATM, CHEK2, PPM1D, and TP53) in cohorts comprising the general population, therapy-exposed non-myeloid cancer patients, or MN-pCT patients, we performed a meta-analysis from 21 publicly available sequencing studies encompassing over 760,000 individuals. Estimation of cohort-specific mutational frequencies using a random effects logistic regression model revealed a significantly higher frequency of above mutations in therapy-exposed non-myeloid cancer patients (i.e., t-CH) as compared to the general population (i.e., ARCH) with highest odds ratios for PPM1D and TP53. Importantly, only TP53 mutations were strongly enriched in MN-pCT samples compared to patients with t-CH.

To study the development of MN-pCT from CH in vitro, we generated CRISPR/Cas9-edited conditionally immortalized HSPC lines by reversible Hoxb8 overexpression with mutations in Dnmt3a, Tet2, Asxl1, Atm, Chek2, Ppm1d, or Trp53. First, we tested the capacity of HSPCs with these ARCH or t-CH mutations to clonally expand upon treatment with common anti-cancer drugs. Chek2- and Ppm1d-mutant HSPCs moderately outcompeted their wild-type counterparts mostly after treatment with double-strand break-inducing agents, whereas Trp53^-/- HSPCs demonstrated rapid and almost complete outgrowth in the presence of a wide range of conventional cytostatic drugs as well as various targeted agents - e.g., PARP, BRAF, and BCL2 inhibitors. By contrast, cells with ARCH mutations did not expand upon treatment with anti-cancer drugs. Accordingly, Trp53^-/- HSPCs displayed strongest defects in apoptosis and G1 cell cycle arrest as well as signs of persistent DNA damage as shown by γ-H2AX staining when compared to HSPCs with mutations in the other tested genes. Next, we assessed the capacity for malignant transformation after treatment with anti-cancer drugs in in vitro immortalization assays. Only Trp53^-/- HSPCs were able to generate immortalized cell lines that harbored complex karyotypes reminiscent of those found in patients with TP53-mutant MN.To investigate clonal expansion and malignant transformation in vivo, we generated murine mixed bone marrow chimeras with 5-10% CRISPR/Cas9-edited HSPCs with mutations in Atm, Chek2, Ppm1d, or Trp53. After sublethal irradiation to mimic anti-cancer therapy, clonal expansion was observed in peripheral blood of Chek2-, Ppm1d- and again strongest in Trp53-mutant mice. Strikingly, only mice with Trp53^-/- HSPCs displayed significantly shortened survival driven by the development of MN-pCT.

Discussion and Outlook

Using clinical sequencing data together with pre-clinical modelling in vitro and in vivo, we demonstrate that mutations in CHEK2, PPM1D, and TP53 but not classic ARCH mutations confer a clonal advantage to HSPCs under the selective pressure of anti-cancer drugs, thereby explaining their enrichment in t-CH. However, only TP53 mutations are sufficient to drive MN-pCT development in vitro and in vivo. Our findings further support the development of mitigation strategies to prevent MN-pCT in cancer patients at risk - in particular those with TP53 mutations.

Boettcher:Astellas: Consultancy; Pfizer: Consultancy; Servier: Consultancy.