This study examined expression of the homeobox gene SIX1 in human bone marrow stromal cells (HS-5) and a panel of acute myeloid leukemia (AML) cell lines (HL-60, SKM-1, THP-1, KG-1, NB4, and MOLM13) using RT-qPCR. The authors report that SIX1 was significantly up-regulated in AML cells relative to stromal cells, establishing a rationale for functional studies in leukemia models.
To investigate causal effects, the investigators engineered MOLM13 AML cells with reduced or increased SIX1 expression. SIX1-knockdown lines were produced by RNA interference, and SIX1-overexpressing cells were generated by plasmid transfection. These genetically modified MOLM13 models were used throughout the study to assess how SIX1 alters leukemia cell behavior, metabolic parameters, and interactions with immune cells.
Functional assays compared proliferation and cell cycle distribution between control and SIX1-manipulated MOLM13 cells. The study used flow cytometry to analyze cell cycle phases, EdU incorporation to measure DNA synthesis, and MTT assays to estimate cell proliferation. The authors report that SIX1 promoted MOLM13 cell proliferation and affected cell cycle distribution, indicating a pro-growth role for SIX1 in AML cells.
Metabolic phenotyping focused on glycolysis-related readouts. Intracellular glucose, lactate, and ATP levels were quantified using flow cytometry and ELISA to evaluate glycolytic flux and energetic status. Transmission electron microscopy was used to examine mitochondrial content. The authors found that SIX1 enhanced glycolytic metabolism in leukemia cells, consistent with a metabolic shift favoring glycolysis over oxidative metabolism.
To model tumor–immune metabolic interactions, peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with genetically modified MOLM13 cells under low-glucose conditions. The study assessed T-cell subset proportions and cytokine production (IL-6, TNF-α, and IFN-γ). Findings indicated that SIX1 expression in leukemia cells suppressed CD8⁺ T-cell effector function in vitro and altered cytokine profiles, supporting a mechanism in which metabolic reprogramming by tumor cells can impair antitumor T-cell responses.
An AML mouse model was established to evaluate SIX1 effects in vivo. Flow cytometry was used to examine T-cell composition and function within the experimental animals. The authors report that SIX1 suppressed CD8⁺ T-cell effector function in vivo as well, mirroring the in vitro co-culture results and suggesting that SIX1-driven changes in leukemia cell metabolism can translate into impaired antitumor immunity in a whole-animal setting.
To characterize systemic and tumor-associated metabolic changes, global metabolomics was applied to samples from the AML mouse model. The metabolomics analysis associated SIX1 expression with extensive alterations across amino acid, glucose, and lipid metabolism. These in vivo metabolic signatures support the conclusion that SIX1 drives broad metabolic reprogramming in the AML context.
Taken together, the data in this report support a model in which SIX1 facilitates AML progression by promoting a glycolytic metabolic phenotype in leukemia cells and by impairing CD8⁺ T-cell effector function—likely through metabolic competition and alteration of the tumor microenvironment. The study highlights SIX1 as a potential therapeutic target for disrupting leukemia metabolic reprogramming and restoring antitumor immunity.
The authors note institutional ethics approval for human and animal components: approvals were obtained from the Ethics Committee of Inner Mongolia University and the Maternal and Child Health Care Hospital of the Inner Mongolia autonomous region; experiments complied with national animal care guidance, the ARRIVE guidelines, and the Declaration of Helsinki. Written informed consent was obtained from participants. The authors declared no competing interests.
Limitations and details not reported in the abstract: the abstract does not provide quantitative values for expression changes, proliferation or metabolic assay results, specific metabolites altered by metabolomics, or survival and clinical outcome data in the mouse model. Those details would need to be consulted in the full text for effect sizes, statistical analyses, and experimental replicates.
Overall, the study links SIX1 to leukemia cell glycolysis, systemic metabolic perturbations, and suppression of CD8⁺ T-cell function, and proposes SIX1 as a candidate target for therapeutic intervention in AML.