Mechanism of acquired resistance to histone deacetylase inhibitor, romidepsin, in myeloid leukemia associated with down syndrome Free

Acquired resistance is one of the major limitations to improving patient outcomes. Enhancing the clinical efficacy of treatments requires a deeper understanding of the mechanisms underlying acquired resistance. In our previous work, we showed that EZH2 inhibitor GSK126, and class I histone deacetylase (HDAC) inhibitor romidepsin, synergistically induced ML-DS cell death (Cicek et al., 2022). To further improve their therapeutic potential, we aim to investigate potential resistance mechanisms by generating drug resistant cell lines. While we were unable to establish cell lines resistant to GSK126 or the GSK126/romidepsin combination, we generated romidepsin resistant cells. Romidepsin is a FDA approved HDAC inhibitor for peripheral T cell lymphomas. However, the mechanisms underlying resistance to romidepsin are not fully understood, especially in myeloid malignancies. Here, we investigated the mechanisms of romidepsin resistance to identify potential therapeutic targets that can be combined with romidepsin to overcome drug resistance.

CMY, a myeloid leukemia associated with Down syndrome (ML-DS) line, cells were exposed to increasing concentrations of romidepsin. As a result, we established romidepsin-resistant CMY sublines (CMY-R) exhibiting over 300-fold resistance to romidepsin (EC50 =933.10 nM) compared to parental cells (EC50 =3.03 nM). To identify the molecular alterations associated with romidepsin resistance, we performed bulk transcriptome analysis on both parental cells and CMY-R cells. Differential expression analysis by DESeq2 revealed 2,217 upregulated and 884 downregulated genes in CMY-R (genes with log₂FC > 1 or < -1 and padj < 0.05 stated as differently regulated genes). Gene set enrichment analysis (GSEA) identified significant upregulation of pathways related to inflammatory and cytokine signaling, including Hallmark interferon alpha response, Hallmark inflammatory response and KEGG cytokine receptor interaction (FDR q-value = 0.0). Pathways related to extracellular matrix (ECM) and cell adhesion, such as Reactome non-integrin ECM interactions, Hallmark epithelial mesenchymal transition and KEGG cell adhesion molecules, were also upregulated (FDR q-value= 0.0) in CMY-R.

To validate increased inflammatory signaling in resistant cells, we performed western blotting analysis. CMY-R cells showed increased expression of total and phosphorylated STAT1, one of the key mediators of inflammatory response, compared to parental cells. To further explore functional relevance, we treated cells with BMS-34554, an IκB/IKK inhibitor. CMY-R cells showed reduced sensitivity to BMS-34554, with an EC50 of 3.15 µM compared to 1.43 µM in parental cells, providing further verification of enhanced inflammatory signaling in resistant cells.

Given the upregulation of ECM-related pathways, we examined the expression of ITGB3, a potential therapeutic target in AML. RNA-Seq revealed a 4.5-fold increase in ITGB3 TPM, which was confirmed by 8-fold elevated integrin b3 (CD61) protein expression in CMY-R cells. RNA-Seq indicated an upregulation of CD34 expression in resistant cells, and we confirmed increased CD34, CD44 and CD117 expression in CMY-R cells by flow cytometry, which indicates a shift toward stem-like, adhesion-dependent phenotype.Our findings suggest that resistance to romidepsin in ML-DS involves upregulation of inflammatory signaling, increased ECM and cell adhesion signaling, and elevated expression of stem cell markers. Targeting inflammatory signaling and/or integrin-mediated adhesion signaling may enhance the efficacy of HDACi and overcome drug resistance in ML-DS and other leukemias.