Targeting Mitochondrial Calcium Homeostasis via novel S100A9 Inhibitor B2 as a Promising Agent Against AML

Acute myeloid leukemia (AML) remains difficult to treat in the relapsed or refractory (R/R) setting due to limited therapeutic targets and the emergence of drug resistance. Through integrative analysis of BeatAML, TCGA, and GEO datasets, we identified S100A9 as a gene highly associated with disease progression and poor treatment response in R/R AML. Functional assays demonstrated that modulating S100A9 expression significantly suppressed AML cell proliferation and led to marked changes in mitochondrial calcium (Ca²⁺) levels. To therapeutically target this axis, we performed structure-based virtual screening and identified compound 2(C2), a novel small-molecule compound that binds to S100A9 and downregulates its expression. C2 treatment restored mitochondrial Ca²⁺ accumulation and induced mitochondrial permeability transition pore (MPTP) opening. Transcriptomic analysis via RNA-Seq confirmed that C2 alters mitochondrial signaling and suppresses STAT5 and AKT activation, two key pathways in AML survival and resistance. Meanwhile, this compound also exhibited potent and selective cytotoxicity in S100A9-high AML cell lines and primary patient samples, with minimal toxicity to normal hematopoietic cells. In vivo, it significantly reduced leukemia burden in xenograft models. Our findings reveal a critical role of S100A9 in mitochondrial calcium regulation in AML and propose C2 as a promising therapeutic candidate that targets mitochondrial vulnerabilities and downstream oncogenic signaling in R/R AML.