Spatial-single cell multiomics reveals TRIM44-driven Treg differentiation and drug resistance in AML: Therapeutic reversal by Sinomenine

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by genetic diversity. These features of AML contribute to immune evasion and therapeutic resistance. Relapse after chemotherapy remains a major clinical challenge in the management of AML. The bone marrow microenvironment is believed to play a critical role in resistance; however, the underlying mechanisms are not fully understood.

We combined spatial single-cell analyzes, ligand-receptor network inference, and Treg pseudotime modeling with functional genetics and pharmacology. A full experimental cascade (reverse transcription [RT]-quantitative polymerase chain reaction [qPCR], western blotting, immunofluorescence, and flow cytometry/function) and in vivo xenografts were used to validate these mechanisms. Structure-guided modeling supported the direct recognition of TRIM44 by the natural alkaloid sinomenine, complemented by cellular evidence of its on-target activity.

Single-cell profiling revealed increased immune heterogeneity and enrichment of regulatory T cells (Tregs) in relapsed AML. TRIM44 was overexpressed during relapse and is associated with poor outcomes. Spatial mapping and trajectory analysis revealed a TRIM44-driven transition from Treg_naïve to Treg_effector, and tumor-Treg co-culture showed that TRIM44 knockdown in AML cells dampens Treg activation/effectorization. Mechanistically, TRIM44 coordinated a dual resistance program-apoptosis escape and epithelial-mesenchymal transition (EMT)-like features in leukemic blasts-converging with enhanced tumor→Treg communication. Sinomenine disrupted this TRIM44-dependent axis, suppressed Treg activity, and re-sensitized resistant AML cells to cytarabine, yielding synergistic anti-leukemic effects in vitro and efficacy in xenograft models.

This study coupled spatial single-cell multiomics with functional genetics and pharmacology to decode an immune-metabolic driver of AML relapse, providing experimental evidence that pharmacological TRIM44 blockade overturns Treg-mediated immune evasion and chemoresistance. We nominate sinomenine as a tractable TRIM44 inhibitor and propose the TRIM44-Treg axis as an actionable therapeutic entry point for precision combinations with cytarabine in relapsed AML.