Investigating the chromatin-targeted activity of DNA intercalators in acute and chronic myeloid leukemia

Abstract

DNA intercalating agents such as aclarubicin (ACLA) disrupt transcription and chromatin organization. However, the early mechanistic events underlying these effects at clinically relevant exposures remain poorly defined. A key unresolved question is whether transcriptional disruption precedes RNA polymerase II (RNAP II) degradation and how rapidly dynamic histone modifications respond to intercalator-induced stress. In addition, it remains unclear whether previously reported chromatin alterations, such as histone eviction, represent primary mechanistic effects or secondary consequences of cellular stress. This study examines whether H2B monoubiquitinated at lysine 120 (H2BK120ub), a transcription-coupled and highly dynamic chromatin mark, serves as an early indicator of transcriptional perturbation in myeloid leukemia cell models. Human leukemia cell lines (K562, MOLM-13, and THP-1) were treated with ACLA and the structurally distinct intercalator BMH-21. ACLA was evaluated under a pharmacokinetically relevant condition (0.34 µM, 1 h) and at a higher dose (1 µM, 2 & 4 h). Cell viability (IC₅₀) was measured using the MTS assay. Chromatin responses were analyzed through acid-based histone extraction and immunoblotting for H2BK120ub, H3 di- and tri-methylated at lysine 79 and lysine 36 (H3K79me2, H3K36me3), respectively. Total cell lysates were analyzed for RNAP II and histone H4. ACLA at 0.34 µM rapidly and reproducibly reduced H2BK120ub levels across all cell lines within 1 hour. In contrast, H3K79me2 and H3K36me3 levels did not change, indicating selective disruption of H2BK120ub. RNAP II and H4 levels also did not change, showing that H2BK120ub reduction occurs before detectable RNAP II degradation or histone eviction. At the higher dose, we observed modest, cell line–dependent reductions in RNAP II, whereas histone H4 levels remained unchanged, suggesting that histone eviction is not an immediate response to ACLA. BMH-21 similarly reduced H2BK120ub but required a higher concentration and longer exposure period. These findings identify H2BK120ub as a rapid and sensitive marker of transcriptional disruption following ACLA intercalation. The data support a model in which ACLA perturbs transcription coupled chromatin dynamics at nucleosome-free regions, leading to immediate loss of H2BK120ub prior to RNAP II degradation and histone eviction. This study underscores the importance of examining drug mechanisms under clinically relevant conditions.