Abstract
Two hypomethylating agents (HMAs), azacitidine and decitabine, have demonstrated clinical activities in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML); however, potential problems include development of acquired resistance. HMA-resistant patients have very poor prognosis and this cohort of patients constitutes an important area of research. To understand the mechanisms underlying HMA-resistance and to overcome it, we established an azacitidine-resistant cell line, MOLM/AZA-1 and a decitabine-resistant cell line, MOLM/DEC-5 using MOLM-13. For cytogenetic characterization, we performed microarray-based comparative genomic hybridization (array-CGH), which identified a total of 15 copy number alterations (CNAs). Among these CNAs, eight regions in HMA-resistant cell lines showed CNA patterns distinct from the parental MOLM-13 genome. Single nucleotide polymorphism (SNP) microarray was also performed to obtain a more reliable interpretation of the identified CNAs, and all HMA-resistance-specific CNAs except one detected by array-CGH were successfully validated. In addition to CNAs, copy neutral loss of heterozygosity and mosaic loss events were identified in HMA-resistant cell lines. In our resistant cell lines, MDR-1 was not overexpressed, while DNMT3b was upregulated. Azacitidine and decitabine did not inhibit DNMT1, DNMT3a, or DNMT3b in both HMA-resistant cell lines, while they inhibited the enzymes in parental MOLM-13. We also developed mouse xenograft models using MOLM/AZA-1 and MOLM/DEC-5. Our in vitro and in vivo models of HMA-resistant cell lines will provide clues for the elucidation of molecular mechanisms related to the development of resistance to HMA and tools for the application of novel therapeutics for AML and MDS.
Highlights
Post-mitotic modification of DNA methylation constitutes a major epigenetic regulatory mechanism for inactivating gene expression
We compared the cytogenetic and phenotypic features of parental and hypomethylating agents (HMAs)-resistant MOLM-13 cell lines to provide the platforms for the clarification of HMA resistance mechanisms and we developed HMA-resistant xenograft models using HMA-resistant MOLM-13 cell lines for the application of novel therapeutics for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)
The IC50 values for azacitidine and decitabine in MOLM-13 were 0.03804 μM and 0.06294 μM, respectively, while the IC50 value for azacitidine in MOLM/AZA-1 was 1.376 μM (36-fold increase compared to the parental cell line) and that for decitabine in MOLM/ DEC-5 was 9.242 μM (147-fold increase compared to the parental cell line) (Figure 1, Table 1)
Summary
Post-mitotic modification of DNA methylation constitutes a major epigenetic regulatory mechanism for inactivating gene expression. Drug-resistant cell line models can be useful in vitro tools for assessing anticancer drug resistance in the clinical scenario. Cell line models with acquired resistance to anticancer drugs provide us with valuable information in elucidating the mechanisms underlying clinical anticancer drug resistance. We developed two HMA-resistant cell lines that show in vitro resistance to clinical doses of azacitidine and decitabine, respectively, from a human monocytic leukemia cell line, MOLM-13. We compared the cytogenetic and phenotypic features of parental and HMA-resistant MOLM-13 cell lines to provide the platforms for the clarification of HMA resistance mechanisms and we developed HMA-resistant xenograft models using HMA-resistant MOLM-13 cell lines for the application of novel therapeutics for AML and MDS
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