F-36P

Levels of DNMTs and Methyl-Binding Proteins Are Higher in F-36P/AZA Cells than in F-36P Cells

MDS is an age-related clonal disease with poor prognosis. Azacitidine (AZA) is a standard of care for MDS but only 30–40 % of patients respond to the treatment and mechanisms of drug resistance are still unknown. Kim et al. attempted to identify DNA-methylation-based biomarkers to predict AZA resistance, with a view to implementing an earlier treatment modification.

In order to identify the mechanisms of AZA resistance, they established an AZA-resistant cell model using F-36P cell line. As shown in Fig. 1, F-36P/AZA cells were highly resistant to AZA when compared to F-36P cells. The IC₅₀ of AZA for F-36P/AZA was 125 μmol/L, which was 125 times higher than that of F-36P (1 μmol/L) (Fig. 1D). In a previous report, up-regulated mRNA levels of DNMT1, DNMT3a, and DNMT3b were found to be associated with AZA resistance of leukemia cells. They also found that the expression of DNMTs was higher in F-36P/AZA cells than in F-36P cells (Fig. 2A and B). The expression of DNMT3B was significantly higher than that of DNMT1 and DNMT3A. In addition, DNMTs, MBD2, and MeCP2 proteins were also more highly expressed in F-36P/AZA cells (Fig. 2C–H). Therefore, they confirmed that the expression of DNMTs was significantly upregulated in F-36P/AZA cells.

Fig. 1. Establishment of azacitidine resistant cell line (Kim DY, Shin D-Y, et al., 2024).

Fig. 2. Gene expression and protein levels of DNMTs in F-36P and F-36P/AZA cells (Kim DY, Shin D-Y, et al., 2024).

Gene Expression Profiles Identify Biomarkers of Resistance to Decitabine in Myelodysplastic Syndromes

MDS is an incurable clonal stem-cell disease. DEC is only useful for a part of patients and the development of resistance is related to the short survival. Kim et al. attempted to identify gene-expression signatures that will predict resistance to DEC and lead to a more personalized therapy.

To analyze the mechanism of DEC resistance, they established a DEC-resistant cell line (F-36P/DEC) from the F-36P cell line (Fig. 3A). F-36P cells were round and were seen as single cells under the light microscope, but F-36P/DEC cells aggregated with each other (Fig. 3B). F-36P/DEC cells were able to maintain stable proliferation for 72 h (Fig. 3C). The IC50 for DEC was 17.27 μmol/L in F-36P cells but exceeded 30 μmol/L in F-36P/DEC cells; accurate IC50 calculation wasn't possible even at 1000 μmol/L (Fig. 3D). F-36P/DEC cells had a higher proportion of cells in the G2/M phase than F-36P cells (Fig. 3E). Taken together, the results show that the F-36P/DEC model was greatly resistant to DEC and had different properties from F-36P cells. To find genes associated with DEC resistance, NanoString analysis was performed to identify DEGs between F-36P and F-36P/DEC, and 189 DEGs were found (Fig. 4A and B). The heatmap (Fig. 4A) and volcano plot (Fig. 4B) show this difference, and a scatter plot revealed more deviation from linearity with larger expression differences (Fig. 4C). GO terms found for DEGs were mainly related to transcription regulation, cell cycle, proliferation, nucleus, extracellular region, transcription-factor binding and kinase activity.

Fig. 3. Validation of DEC resistance in the F-36P/DEC cell line (Kim S, Shin D-Y, et al., 2021).

Fig. 4. Analysis of DEGs between F-36P/DEC and F-36P cells (Kim S, Shin D-Y, et al., 2021).