Background <p>Acute myeloid leukemia (AML) has a poor prognosis due to high chemoresistance and recurrence. TRIM24, an E3 ubiquitin ligase, is oncogenic in solid tumors, but its role in AML and regulation of the oncoprotein p110 CUX1 remain unclear.</p> Methods <p>We performed bioinformatic analysis to assess TRIM24 expression, prognosis, and protein-protein interactions. Functional studies used genetic manipulation and pharmacological inhibition in AML cells. Protein interactions were examined by Co-IP, IP-MS, and immunofluorescence. The stability and ubiquitination of p110 CUX1 were examined via CHX chase assays and ubiquitination profiling. The biological and therapeutic significance of the identified axis was validated using cell proliferation, apoptosis assays, drug sensitivity tests, and a CDX mouse model.</p> Results <p>TRIM24 is overexpressed in AML and predicts poor prognosis. TRIM24 promotes proliferation and inhibits apoptosis. Mechanistically, TRIM24 functions as a scaffold-like adaptor to recruit USP10 to deubiquitinate and stabilize p110 CUX1 via K48-linked ubiquitin chain cleavage. The TRIM24/USP10/p110 CUX1 axis activates the MAPK signaling pathway, with CUX1 regulating ERK transcription. TRIM24 overexpression enriches DNA repair pathways, conferring cytarabine resistance. Targeting TRIM24 genetically or with dTRIM24 sensitizes AML cells to cytarabine in vitro and in vivo.</p> Conclusion <p>Our study reveals a novel non-canonical role of TRIM24 as a stabilizer of the p110 CUX1 oncoprotein by recruiting USP10. This TRIM24/USP10/p110 CUX1 axis is critical for AML progression, clinical prognosis, and chemoresistance, presenting a promising therapeutic target for AML treatment.</p> Graphical Abstract <p></p>