biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsShilpee DuttIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13769Anthracyclines, topoisomerase 2 enzyme poison that results in DNA damage, are currently used in acute myeloid leukemia (AML) treatment. Identifying the mechanisms underlying drug resistance remains an important question. Here, using a mitoxantrone-resistant cell line (HL-60/MX2), we found upregulation of DNA-PKcs, independent of the DNA damage response. We demonstrated that anthracyclines failed to induce DNA damage in resistant cells owing to the loss of expression of their target enzyme-TOP2B, rendered by DNA-PKcs directly binding to its promoter upstream region as a transcription repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone and abrogated their tumorigenic potential in a xenograft mouse model. However, to explore other putative dysregulated pathways and genes, we performed RNA-seq experiments on HL-60/MX2 cells after siRNA-mediated knockdown of PRKDC.biostudies-arrayexpressNucleic Acid Extraction - RNA was isolated from the cell pellet resuspended in TRIzol Reagent (Thermo Fisher Scientific) using the chloroform-isopropanol method. The RNA samples were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high-sensitivity RNA screen tape to determine the RNA integrity number (RIN). Samples with 28S/18S peak integrated area ratios >2 were used for library preparation.Library Construction - 1.5 μg of QC verified RNA samples was used for poly (A) mRNA isolation from total RNA using NEB Next Poly (A) mRNA Kit (Cat. no. NEB# 7490) according to the manufacturer’s protocol. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat. no. E7760L) was used to construct double-stranded cDNA libraries from the polyA RNA fraction, using RNA fragmentation, first strand cDNA synthesis with random primers, second strand cDNA synthesis, end repair of double-stranded cDNA, adapter ligation, removal of excess adapter using sample purification beads, PCR enrichment of adapter ligated DNA, and clean-up of PCR products as instructed by the manufacturer. The cleaned libraries were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high sensitivity D1000 screen tape to determine the size range of the fragments and the average library size.Sequencing - Prepared libraries were sequenced on an Illumina NovaSeq 6000 sequencer to generate 2 × 150 bp reads per sample.Sample Collection - PRKDC knockdown: 0.2X106 cells/mL were seeded per well in a 6-well plate and transfected the next day with siRNA at 80pmols PRKDC (siRNA select-ThermoFisher, 4390824) per well using the standard 6-well protocol for Lipofectamine3000 reagent (Invitrogen, L3000015). Cells were collected 48 h post-transfection and tested for the knockdown of the respective proteins by qPCR and immunoblotting.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesShilpee DuttfalseRNA-seq for siPRKDC and siControl in human HL-60/MX2 mitoxantrone resistant derivative of the HL-60 cell lineAnthracyclines, topoisomerase 2 enzyme poison that results in DNA damage, are currently used in acute myeloid leukemia (AML) treatment. Identifying the mechanisms underlying drug resistance remains an important question. Here, using a mitoxantrone-resistant cell line (HL-60/MX2), we found upregulation of DNA-PKcs, independent of the DNA damage response. We demonstrated that anthracyclines failed to induce DNA damage in resistant cells owing to the loss of expression of their target enzyme-TOP2B, rendered by DNA-PKcs directly binding to its promoter upstream region as a transcription repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone and abrogated their tumorigenic potential in a xenograft mouse model. However, to explore other putative dysregulated pathways and genes, we performed RNA-seq experiments on HL-60/MX2 cells after siRNA-mediated knockdown of PRKDC.2025-12-31T00:00:00Z2025-12-31T02:02:11.668Z2024-02-06T11:17:26.254ZE-MTAB-13769ERP157172EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184