Abstract Background Acute megakaryoblastic leukemia (AMKL), a high-risk pediatric AML subtype (FAB-M7), carries a particularly poor prognosis in non-Down syndrome-associated cases (non-DS-AMKL). This aggressive malignancy is characterized by frequent relapse, intrinsic chemoresistance, and limited efficacy of hematopoietic stem cell transplantation (HSCT), creating an urgent unmet therapeutic need. While epigenetic dysregulation is increasingly associated with tumorigenesis, the therapeutic potential of histone deacetylase (HDAC) inhibitors in non-DS-AMKL remains elusive. Methods Through single-cell transcriptomics of BBMCs from pediatric AMKL patients, we identified aberrant pathways of cell death and epigenetic regulation, prompting us to screen chemical libraries of around 400 compounds specifically targeting cell death and histone modification in MEG-01 cells. This screen identifies HDAC inhibitors as top candidates. Their efficacy was validated across AMKL cell lines and 3 primary patient samples, alone or combined with chemotherapy and B7H3-CAR T cells(Flow cytometry confirmed high expression of B7H3 across AMKL cell lines). RNA-seq and Western blot were used to explore mechanisms. CRISPR-generated knockout cell lines were used to dissect pathways. In vivo efficacy was tested using MEG-01-Luc-GFP CDX models in NSG mice treated with vehicle (Group A), Chidamide (Group B), DT2216 (Group C), or combination (Group D). Tumor burden was monitored via IVIS, and survival was recorded. Results HDAC inhibitors showed potent cytotoxicity in AMKL models, by inducing apoptosis and pyroptosis. Mechanistically, western blot confirmed caspase-3 activation and GSDME cleavage. This cell death was significantly blocked by pan-caspase inhibitor Z-VAD-FMK. RNA-seq revealed downregulation of BCL-XL, XIAP, and upregulation of pro-apoptotic genes (BIK, BMF, NOXA). Notably, knockout of caspase-9, but not caspase-8, reduced caspase-3-mediated apoptosis and GSDME-mediated pyroptosis, demonstrating mitochondrial activation as an initiating signal to drive HDAC inhibitors induced cell death. Neither necroptosis nor GSDMD-mediated pyroptosis was induced in AMKL treated with HDAC inhibitors. Recent studies have found that BCL-2 family inhibitors and CAR-T therapy benefit AML patients. Our research demonstrates that in AMKL cell lines and patient-derived ex vivo drug sensitivity assays, the combination of HDAC inhibitors with BCL-2 family inhibitors or CAR-T therapy significantly enhances treatment efficacy, particularly with BCL-XL inhibitors. In vitro experiments confirmed that HDAC inhibitor and BCL-XL inhibitor co-treatment induces caspase9-dependent cell death and the release of HMGB1 which suggested the occurrence of immunogenic cell death (ICD). In vivo studies showed that the combination of HDAC inhibitors and BCL-XL inhibitors markedly reduces tumor burden in AMKL mouse models and improves survival rates. Thus, this combination therapy may serve as a potential treatment strategy for AMKL. Conclusion HDAC inhibitors activate apoptosis and pyroptosis, inducing ICD in non-DS-AMKL. Combining HDAC inhibitors with BCL-XL-targeting agents or CAR T cells shows strong preclinical efficacy. This multimodal strategy offers a promising avenue for treating high-risk AMKL.
Hu Liu (Mon,) studied this question.