Busulfan resistance in AML is associated with changes in mitochondrial copy number and lipid metabolism

Busulfan (BU) is DNA-damaging and also a reactive oxygen species (ROS) generating agent. Both mitochondrial DNA copy number (mtDNA-CN) and ROS levels have been associated with the sensitivity of solid tumor cells to ROS-generating cisplatin. However, the changes of mtDNA-CN during the development of resistance to other ROS-generating agents or chemotherapeutics remain unclear. In this study, we investigated how the resistance of acute myeloid leukemia (AML) cell lines to the BU or the DNA synthesis inhibitor cytarabine (Cyt) is associated with alterations in mtDNA-CN and global gene expression. To follow BU resistance acquisition, MOLM13 myeloid leukemia cells, which are sensitive to BU, were subjected to five consecutive BU treatment rounds, generating BU-resistant cells (5TBU). Both parental control cells and the 5TBU cells underwent five consecutive Cyt treatments to generate cells resistant to both Bu and Cyt. We assessed mtDNA-CN using RT-qPCR targeting the mitochondrial gene MTND1 and investigated global gene expression profiles using bulk RNA-sequencing before and after resistance acquisition. We estimated the BU 50% inhibitory concentration (BU-IC₅₀) relative to mtDNA-CN both in MOLM13-derived cells and 28 lymphoblastoid cells (LCLs). MOLM13 cells showed a progressive increase in resistance from 1.86-fold (p < 0.01) to 2.87-fold (p < 0.05) after 3 and 5 cycles of BU treatment, respectively. An additional 5 cycles of Cyt increased cellular resistance to Cyt by 1.90-fold (p < 0.01) and resistance to BU by 1.60-fold (p < 0.01). BU-resistant cells showed a 1.42-fold (p < 0.01) increase in mtDNA-CN after 3 and 5 cycles of BU treatment. In contrast, no significant changes in mtDNA-CN were observed following Cyt treatment. LCLs exhibiting higher BU-IC₅₀ also showed increased mtDNA-CNs. Transcriptomic analysis revealed that BU resistance was associated with dysregulation in cholesterol and fatty acid transport and synthesis pathways. Expression profiling of the BU-resistant AML cell line revealed significant changes in cholesterol transport and fatty acid biosynthesis, along with alterations in mtDNA-CNs. These changes could represent key adaptations resulting from long-term exposure to the electrophilic agent BU. The current findings thus provide new insights into the molecular mechanisms underlying BU resistance and suggest potential targets for preventing or overcoming this resistance in future therapeutic strategies.