AP-3 targets the mrgbp/Tip60/MYC axis to exert potent anti T-ALL effects in a Notch1 independent manner

Abstract Background: T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive and lethal hematologic malignancy, accounting for 15%–30% of acute lymphoblastic leukemia cases. Adult T-ALL has a poor prognosis, with a 5-year overall survival (OS) rate below 40%. The prognosis for relapsed or refractory T-ALL is even worse, with a 3-year OS rate under 15%, and outcomes remain dismal even after transplantation. There is an urgent need for new therapeutic targets and drugs. This study utilized a self-established T-ALL cell line to screen high-efficacy anti-T-ALL natural compounds through high-throughput drug screening, aiming to identify novel targets and mechanisms for T-ALL treatment. Methods Using the ZYXY-T1 T-ALL cell line developed by our team, we screened 2,541 natural compounds via high-throughput drug screening. Compounds with IC50 values below 10 nM were selected for further dose-response experiments to identify the most potent anti-T-ALL candidate. Cell proliferation, apoptosis, and cycle assays were performed to confirm its anti-T-ALL effects, followed by validation in CDX and PDX models. Drug affinity responsive target stability (DARTS) technology was employed to identify binding targets, which were further verified by surface plasmon resonance (SPR), auto-docking, and Western blot (WB). Molecular cloning techniques were used to knockdown or overexpress the target to assess its regulatory role in T-ALL. RNA-seq and WB were conducted to elucidate the underlying mechanisms. Results Three rounds of high-throughput screening identified 13 natural compounds with IC50 values below 10 nM. Among these, AP-3 exhibited the highest sensitivity against T-ALL, with an IC50 of 170 pM. Across multiple T-ALL cell lines and primary T-ALL cells, AP-3 demonstrated potent anti-T-ALL activity (IC50: 50 pM~1 nM). Further experiments revealed that AP-3 induced apoptosis and M phase cell cycle arrest in T-ALL cells. In vivo studies showed that AP-3 reduced tumor burden and prolonged survival in CDX and PDX T-ALL models. DARTS identified MRGBP as the binding target of AP-3, which was confirmed by auto-docking and SPR. WB demonstrated that AP-3 downregulated MRGBP expression in T-ALL cells. Knockdown of MRGBP inhibited T-ALL cell proliferation and promoted apoptosis. RNA-seq analysis of AP-3-treated and MRGBP knockdown T-ALL cells revealed that AP-3 targets MRGBP to suppress the Tip60/MYC axis independently of Notch1. Conclusion This study identifies AP-3 as a novel, highly potent anti-T-ALL natural compound that directly binds MRGBP and inhibits the Tip60/MYC axis via a Notch1-independent pathway, providing a promising candidate drug and therapeutic target for T-ALL. Key Words：T-ALL AP-3 MRGBP/Tip60/MYC DRATS Targets