Abstract

Ethnopharmacological relevance Realgar is an arsenic-containing mineral medicine and a key component of Realgar-Indigo naturalis formula, an oral traditional Chinese medicine preparation clinically used in China for acute promyelocytic leukemia (APL), a biologically distinct subtype of acute myeloid leukemia (AML). Realgar Transforming Solution (RTS) is a microbially processed realgar preparation. Its activity in AML stem cell-like contexts outside APL remains insufficiently defined, particularly in cell-line-derived CD34 + CD38 − LSC-like models. Aim of the study: To evaluate whether RTS suppresses stemness-associated phenotypic features in KG-1a-derived CD34 + CD38 − LSC-like cells and to explore its association with endoplasmic reticulum stress (ERS), mitochondrial injury, and mitophagy-related alterations. Materials and methods CD34 + CD38 − LSC-like cells were enriched from KG-1a cells and used as a cell-line-derived in vitro model. Cell viability, apoptosis, cell cycle, CD34 + CD38 − fraction, colony formation, and TIM3/ HAVCR2 expression were assessed after RTS treatment. RNA sequencing, Ca 2+ measurement, ROS detection, mitochondrial membrane potential assay, MDC staining, western blotting, RT-qPCR, immunofluorescence, and transmission electron microscopy were performed. 4-phenylbutyric acid and Mdivi-1 were used as pharmacological rescue agents. Results RTS reduced the CD34 + CD38 − fraction, colony formation, and TIM3/ HAVCR2 expression, while inducing apoptosis in KG-1a-derived LSC-like cells. Transcriptomics implicated ER protein processing, unfolded protein response, apoptosis, autophagy, and mitophagy-related pathways. RTS increased Ca 2+ levels, ROS accumulation, mitochondrial depolarization, PERK-eIF2α-ATF4-CHOP signaling, and PINK1/Parkin-associated mitophagy-like marker changes. 4-phenylbutyric acid and Mdivi-1 partially rescued RTS-impaired LSC-like phenotypic features, supporting pathway involvement. Conclusions RTS suppressed stemnes