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. 2022 Jan 11;11(2):223.
doi: 10.3390/cells11020223.

Analysis of 5-Azacytidine Resistance Models Reveals a Set of Targetable Pathways

Lubomír Minařík  1   2 , Kristýna Pimková  1 , Juraj Kokavec  1 , Adéla Schaffartziková  1 , Fréderic Vellieux  1 , Vojtěch Kulvait  1 , Lenka Daumová  1 , Nina Dusilková  1   2   3 , Anna Jonášová  1 , Karina Savvulidi Vargová  3 , Petra Králová Viziová  4 , Radislav Sedláček  4 , Zuzana Zemanová  5 , Tomáš Stopka  1   2
Affiliations

Analysis of 5-Azacytidine Resistance Models Reveals a Set of Targetable Pathways

Lubomír Minařík et al. Cells. .

Abstract

The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy.

Keywords: Azacytidine; CDX mice; PI3K/AKT signaling; myelodysplastic syndrome; resistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Analysis of the AZA resistance model. (A) WST1 assay of AZA-sensitive (S) vs. AZA-resistant (R) cells (clone #1); IC50AZA is indicated. (B–D) AZA-S & AZA-R cells were transplanted into NSGS mice and treated with AZA or vehicle (Control). Therapy of 150 μg AZA/mouse was applied i.p. three times weekly. Survival was monitored (days indicated, 4 or 5 mice per each group). AZA-S vs. AZA-R p = 0.004, AZA-S v Ctrl p = 0.0014, AZA-R vs. Ctrl p = 0.0953. (C,D) Luciferase detection (y-axis Mean Rad) in control vs. AZA-treated mice bearing AZA-S (left) or AZA-R (right) xenograft cells. Analysis utilized unpaired Mann–Whitney t-test. * p-value < 0.05, ns not significant.
Figure 2
Figure 2
Transcriptomic analysis of cells either resistant or sensitive to AZA. (A) Volcano plot of differential gene expression; significance indicated by adjusted p < 0.05; log2 fold change expression > 1 is marked by red. Selected mRNAs are displayed with HGNC symbol. (B) Heatmaps show mRNA expression log2(AZA-R/AZA-S) in two replicates as revealed by KEGG pathway analysis.
Figure 3
Figure 3
Validation of AZA resistance pathways. (A left) structural model of AKT1 variant c.430C>T (p.Arg144Cys), unmutated Arg (top, blue) vs. mutated Cys (bottom, yellow), phosphorylated Ser473 in orange; (A middle) AKT1-Ser473 phosphorylation (Western blotting, densitometry on top); (A right) WST1 assay using AKT1 inhibitor (MK2206, AZA-S vs. AZA-R). (B) WST1 assay; Idelalisib (IDE); (C) BET inhibitor JQ1; (D) HDAC inhibitor Panabinostat (PAN); AZA-R clones indicated by #.
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