Abstract PR007: High-throughput CRISPR screens uncover selective EZH2 inhibitor EPZ-6438 driving expansion of preleukemic hematopoietic stem cell with mutation in Asxl1
Authors: Ieva Budriunaite, Phoebe E. Crawley, Elena V. Knatko, Kasper D. Rasmussen
Journal: Cancer Research (2025)

Abstract Hematopoetic stem and progenitor cells (HSPCs) are a small subset of cells responsible for the generation of all mature blood cell lineages. Somatic mutations acquired with age can cause expansion of mutated HSPCs in bone marrow and subsequently in blood stream. This phenomenon known as clonal haematopoiesis (CH) is driven by ∼60 genes observed to be mutated in about 10% of the population aged 65 and over. CH has been linked to increased risk of cardiovascular disease, type II diabetes and haematological malignancies, such as acute myeloid leukemia (AML). Several studies have shown that therapeutic agents, such as topoisomerase II inhibitors and alkylating agents used in treatment of solid tumours can increase the risk of therapy-related AML (t-AML) in adult cancer survivors. Classed as a distinct type of AML, t-AML has proven harder to treat and has higher risk of mortality. Improved chemotherapy options and subsequent prolonged overall survival following primary cancer treatment has led to the prediction that the incidence of t-AML will increase. To understand mechanisms driving expansion of preleukemic HSPCs, we have developed a series of isogenic CRISPR-editing capable murine HSPC-like cell lines. These cells can be stably maintained in vitro and, upon transplantation into irradiated mice, demonstrate robust engraftment and multipotency, much like normal HSPCs. We used these HSPC-like cell lines in unbiased CRISPR-based screens to assess the expansion of preleukemic HSPCs and investigate the impact of various cancer therapies on these cells. Results of CRISPR-based screens conducted without drug treatment showed expansion of HSPCs with mutations in Dnmt3a, Tet2, Asxl1, Tp53 and others. This profile aligns with the mutational patterns seen in population studies investigating CH-related mutations. Additional experiments conducted by adding common therapeutic agents revealed three-fold greater expansion of HSPCs with mutation in Asxl1 upon treatment with EPZ-6438, a selective EZH2 inhibitor that is currently used in treatments of follicular lymphoma and epitheliod sarcoma as well as in on-going clinical trials for other cancer types. Asxl1 mutations have previously been linked to decreased polycomb repressive complex 2 (PRC2)-mediated H3K27me3 levels, which provides a potential explanation on how inhibiting EZH2, a catalytic subunit of the PRC2 complex might further promote expansion of Asxl1-mutated HSPCs. Based on these findings, a careful consideration should be given to the use of EPZ-6438 in patients carrying mutation in Asxl1 for the treatment of their primary tumours. Patients receiving EPZ-6438 should be closely monitored for clonal expansion of mutated-HSPCs and treatment adjustments should be considered to mitigate the risk of t-AML. In conclusion, we developed and used HSPC-like cells combined with CRISPR-based screening approach to study drivers of CH. We successfully replicated mutational patterns observed in individuals with CH and identified the selective EZH2 inhibitor EPZ-6438 as a driver of Asxl1-mutated HSPC expansion. Citation Format: Ieva Budriunaite, Phoebe E. Crawley, Elena V. Knatko, Kasper D. Rasmussen High-throughput CRISPR screens uncover selective EZH2 inhibitor EPZ-6438 driving expansion of preleukemic hematopoietic stem cell with mutation in Asxl1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr PR007.