Role of R-loops in chemotherapy-induced cell death in acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cells, characterized by excessive proliferation and aberrant self-renewal. Young AML patients are treated with the same induction therapy consisting of cytarabine (ARA-C) and daunorubicin (DNR), irrespective of the genetic background or expected long-term outcome. Identifying better biomarkers is crucial for stratifying patients into standard or experimental treatment approaches. To investigate the biological behavior of AML cells, we analyzed three AML cell lines (HL60, MOLM13 and OCI-AML3) upon treatment with ARA-C and DNR. We assessed proliferation, apoptosis, DNA damage, and R-loop formation. Moreover, RNA-seq was performed to examine the expression of genes involved in R-loop regulation. Finally, we blocked the induction of R-loop formation by overexpression of RNaseH1 to explore the role of R loops in treatment sensitivity. Our findings revealed differential drug sensitivity among the cell lines. HL60 cells exhibited the highest sensitivity to ARA-C, whereas MOLM13 cells responded best to DNR. DNA damage analysis, measured by ƴH2AX foci, revealed that DNR-induced DNA damage was lower in HL60 cells compared to MOLM13. Conversely, ARA-C treatment resulted in higher levels of ƴH2AX foci in HL60 than in MOLM13 cells. Co-localization studies with RAD51 and 53BP1 foci indicated preferential activation of specific DNA repair pathways in response to damage. Furthermore, we quantified R-loop formation before and after DNR exposure. R-loop levels increased in MOLM13 but not HL60 cells, correlating with their sensitivity to DNR. Similarly, ARA-C treatment led to elevated R-loop formation in HL60 cells. Interestingly, RNA-seq analysis demonstrated downregulation of genes involved in R-loop prevention and removal upon treatment. The persistence of R-loops following genotoxic stress may induce replication stress and DNA damage, suggesting a potential link between R-loop accumulation and drug sensitivity. To validate this hypothesis, we utilized inducible RNAseH1 overexpression models, including wild type (WT), catalytically inactive (D210N), and catalytically inactive/mutant hybrid-binding domain (WKKD). Overexpression of RNAseH1 WT conferred increased resistance to chemotherapy, supporting the hypothesis that R-loop accumulation contributes to therapy-induced cell death. These findings provide new insights into the role of R-loops in AML chemotherapy response and suggest a potential therapeutic strategies targeting R-loop metabolism to improve AML therapy.