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Chronic myeloid leukemia (CML), caused by BCR::ABL1 fusion gene, is known to regulate disease progression by altering the expression of genes. However, the molecular mechanisms underlying these changes are largely unknown. In this study, we identified RNA Exonuclease 5 (REXO5) as a novel gene with elevated mRNA expression levels in chronic myeloid leukemia (CML) patients. Additionally, using the REXO5 knockout (KO) K562 cell lines, we revealed a novel role for REXO5 in the DNA damage response (DDR). Compared to wild-type (WT) cells, REXO5 KO cells showed an accumulation of R-loops and increased DNA damage. We demonstrated that REXO5 translocates to sites of DNA damage through its RNA recognition motif (RRM) and selectively binds to R loops. Interestingly, we identified that REXO5 regulates R-loop levels by degrading mRNA within R-loop using its exonuclease domain. REXO5 KO showed ATR-CHK1 activation. Collectively, we demonstrated that REXO5 plays a key role in the physiological control of R-loops using its exonuclease domain. These findings may provide novel insights into how REXO5 expression contribute to CML pathogenesis.

case control design

Transcription profiling by array

Biotin cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays

Standard Illumina hybridization protocol

BeadStation Plarform/iSCAN system, standard Illumina scanning protocol

PBMCs were obtained from 6 CML-CP patients and 6 healthy donors. Mononuclear cells were isolated using Ficoll-Paque density gradient centrifugation. Samples were frozen in 10% DMSO in FBS, stored in liquid nitrogen, and thawed for analysis. Samples were provided by the Korea Leukemia Bank under IRB NO: UEMC 2022-01-019, with written informed consent.

For microarray analysis, mononuclear cells (MNC) were prepared from PBMCs using Lymphoprep™ (AXIS-SHIELD, Oslo, Norway) and total RNA was extracted from the prepared MNC using the Trizol reagent (Invitrogen, Carlsbad, USA) according to manufacturer's instruction. Two micrograms of total RNA was reverse transcribed into first-strand cDNA using Transcriptor First Strand cDNA Synthesis kit (Roche, Mannheim, Germany). QC with gel electrophoresis, spectrophotometer, Agilent Bioanalyzeror RiboGreen. Ratio of 28S to 18S = 2:1, OD 260/280 range = 1.7-2.1, OD 260/230 > 2.0

The data were normalised using quantile normalisation with IlluminaGUI in R