Chemotherapy Modulates the Immune Microenvironment and Epigenetic Landscape in MLL-AF9-Driven Acute Myeloid Leukemia

Introduction Acute Myeloid Leukemia (AML) is an aggressive blood cancer characterized by the expansion of immature myeloid cells in the bone marrow (BM), leading to hematopoietic failure. AML remains associated with poor prognosis, due to early treatment failure and high relapse rates following intensive chemotherapy. Here, we investigated how chemotherapy reshapes the immune microenvironment in a murine model of AML to identify tumour vulnerabilities and enhance immune responses and treatment outcomes. Materials and Methods MLL-AF9-transformed murine AML cells were transplanted into immunocompetent mice without myeloablation, followed by a standard chemotherapy regimen (5+3 days of Daunorubicin and Cytarabine). BM and spleen (SPL) were analysed two days post-treatment for immune cell composition using flow cytometry. Single-cell RNA sequencing (scRNA-seq) examined transcriptional changes, and histone modifications were assessed via western blot. Results scRNA-seq identified five leukemic clusters: one monocytic non-cycling and four granulocyte-monocyte progenitor (GMP)-like clusters with distinct cell cycle and inflammatory states. While chemotherapy reduced overall leukemic burden, all clusters were maintained but cycling and inflammatory GMP-like clusters were slightly increased. However, none of the tumour clusters upregulated senescence signatures, and instead downregulated inflammatory and interferon (IFN) pathways, indicating weak immune activation. In the non-leukemic compartment, T and NK cells showed no significant IFN activation post-chemotherapy, while inflammatory pathways were upregulated in monocyte/macrophage subpopulations. Quantitative Flow cytometry revealed a decrease in macrophages and an increase in monocytes in the BM. To investigate potential immune suppression mechanisms, we analysed the epigenetic remodelling in leukemic cells. Expression of EZH2, the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), was heterogeneous but upregulated in most GMP-like tumour clusters post-chemotherapy. Additionally, genes associated with H3K27me3 histone modification were downregulated, suggesting increased H3K27me3 levels, further confirmed via western blot analyses, highlighting EZH2 as a potential immunotherapeutic target. Conclusions Our findings indicate that chemotherapy reshapes the immune microenvironment in MLL-AF9 AML by modulating myeloid populations but fails to induce a strong IFN response or robust immune activation. The weak senescence signature in BM-derived leukemic cells suggests that chemotherapy alone is insufficient to trigger immunogenic senescence. Additionally, the upregulation of EZH2 and increased H3K27me3 levels suggest an epigenetic mechanism contributing to immune evasion and leukemia persistence. Targeting EZH2 may enhance immune activation, offering a potential therapeutic strategy to improve AML clearance.