Aberrant activation of clpp exerts multifaceted anti-AML activity beyond tumor cells

Abstract Background and Objectives The progression of acute myeloid leukemia (AML) relies not only on tumor cells but also involves leukemia stem cells (LSCs), therapy-resistant cells, and the leukemic microenvironment, which collectively drive disease progression through oxidative phosphorylation (OXPHOS) mechanisms. Current OXPHOS inhibitors face clinical limitations due to insufficient selectivity, limited efficacy, and neglect of their immunomodulatory effects beyond tumor cells. Homo sapiens caseinolytic protease P (ClpP), which is specifically overexpressed in AML, plays a critical role in maintaining OXPHOS functional integrity. Thus, targeted activation of ClpP may represent a novel therapeutic strategy. This study aimed to develop IMP125, a highly potent ClpP agonist, and comprehensively evaluate its anti-AML activity and immunomodulatory effects on the tumor microenvironment. Methods The novel ClpP agonist IMP125 was designed and synthesized, and its anti-leukemic activity was validated through in vitro cell experiments, in vivo AML mouse models, and primary patient samples. Single-cell RNA sequencing was employed to analyze the regulatory effects of IMP125 on T cell states, combined with dynamic pseudotime trajectory analysis and sorted T cell functional assays to investigate its impact on OXPHOS and mitochondrial function. Additionally, the combined therapeutic effect of IMP125 and anti-PD-L1 antibodies was assessed in AML mouse models. Results IMP125 demonstrated significant anti-leukemic activity in vitro, in vivo, and in primary AML samples. Beyond directly killing tumor cells, IMP125 markedly enhanced T cell-mediated cytotoxicity against AML. Single-cell transcriptomic analysis revealed that IMP125 induced a distinct T cell activation state compared to conventional chemotherapy. Mechanistic studies showed that IMP125 promoted T cell activation by upregulating OXPHOS and improving mitochondrial function. Furthermore, the combination of IMP125 with anti-PD-L1 antibodies synergistically inhibited AML progression and significantly prolonged survival in mice. Conclusion As a novel ClpP agonist, IMP125 exhibits multidimensional anti-AML effects: it directly targets tumor cells while also enhancing T cell function by modulating the immune microenvironment. Its synergistic effect with immune checkpoint inhibitors provides a new strategy for AML treatment, holding significant potential for clinical translation. Keywords Acute myeloid leukemia; ClpP agonist; Mitochondrial metabolism; Oxidative phosphorylation; Tumor immunity; T-cell cytotoxicity