Experimental study on treatment of relapsed and refractory acute myeloid leukemia with DNA methyltransferase 1 inhibitor combined with extracellular signal-regulated kinase 1, homeodomain-interacting protein kinase 2, and glycogen synthase kinase 3β inhibitors

Objective To investigate the mechanism of DNA methyltransferase 1 (DNMT1) inhibitor combined with extracellular signal-regulated kinase 1 (ERK1), homeodomain-interacting protein kinase 2 (HIPK2), and glycogen synthase kinase 3β (GSK3β) inhibitors in synergistically inducing apoptosis and protein arrest in relapsed and refractory acute myeloid leukemia (AML) cells.

Methods Three therapeutic targets, including ERK1, HIPK2, and GSK3β, were screened based on the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA) database, and Expression 2 Kinases database. Human AML cell line U937 cells were treated with DNMT1 inhibitor alone or combined with ERK1, HIPK2, or GSK3β inhibitors. Cell viability was detected using the CCK-8 method. Apoptosis rate was analyzed by flow cytometry, and cell cycle distribution was determined by propidium iodide (PI)staining. The mRNA expression levels of DNMT1, ERK1, HIPK2, and GSK3β were detected by real-time fluorescent quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR). Protein expressionlevels of DNMT1, ERK1, HIPK2, and GSK3β were detected by immunoblotting.

Results DNMT1 inhibitor significantly inhibited the cell viability of U937 cells, and significantly induced apoptosis and cell cycle arrest in U937 cells (P < 0.05). When DNMT1 inhibitor was combined with ERK1, HIPK2, or GSK3β inhibitors, cell viability and apoptosis rate were significantly reduced (P < 0.05). DNMT1 inhibitor alone or its combination with ERK1, HIPK2, and GSK3β inhibitors induced U937 cell arrest in the G₀/G₁ phase, with a significant increase in the proportion of cells in the G₀/G₁ phase in the combination group (P < 0.05). The combination of DNMT1 inhibitor with ERK1, HIPK2, and GSK3β inhibitors significantly reduced the mRNA expression levels in DNMT1, ERK1, HIPK2, and GSK3β in U937 cells (P < 0.05). Similarly, the combination therapy significantly reduced the protein expression levels of DNMT1, ERK1, HIPK2, and GSK3β in U937 cells (P < 0.05).

Conclusion DNMT1 inhibitor combined with ERK1, HIPK2, and GSK3β inhibitors can synergistically induce apoptosis and protein arrest in relapsed and refractory AML cells, providing a novel strategy for combined targeted therapy of AML.