Ophiopogonin D′ Targets PPM1B to Suppress B-Cell Acute Lymphoblastic Leukemia via Disruption of Wnt/β-Catenin Signaling

Abstract B-cell acute lymphoblastic leukemia (B-ALL) carries a dismal prognosis in adults due to the scarcity of effective targeted therapies, underscoring an urgent need for novel agents. Ophiopogonin D′ (OPD′), a steroidal saponin structurally distinct from Ophiopogonin D (differing in aglycone and glycosylation), is isolated from Ophiopogon japonicus. While OPD has been studied in solid tumors, the potential of OPD′ in hematological malignancies remains completely unexplored. Here, we identify OPD′ as a potent and selective agent against B-ALL. OPD′ suppresses B-ALL cell proliferation, induces G0/G1 arrest and apoptosis, and markedly inhibits xenograft tumor growth in vivo without overt toxicity. Using Cellular Thermal Shift Assay coupled with Mass Spectrometry (CETSA-MS), we discover that protein phosphatase magnesium/manganese-dependent 1B (PPM1B) is the direct cellular target of OPD′. Mechanistically, OPD′ acts as a competitive inhibitor of PPM1B phosphatase activity, with mutational mapping identifying Arg-33 and Glu-35 as critical binding residues. Global phosphoproteomics reveals that OPD′-mediated PPM1B inhibition disrupts Wnt/β-catenin signaling by elevating β-catenin phosphorylation at Ser37, leading to the downregulation of c-Myc and Cyclin D1, upregulation of p27, and a pro-apoptotic Bax/Bcl-2 imbalance. Genetic knockdown of PPM1B phenocopies OPD′-induced cytotoxicity, while PPM1B depletion attenuates OPD′’s antileukemic effects, confirming on-target specificity. Collectively, this study establishes OPD′ as a natural PPM1B inhibitor and unveils PPM1B as a novel druggable vulnerability in B-ALL, providing a promising therapeutic lead for clinical translation.