Abstract

Arsenic trioxide (ATO) has revolutionized the treatment of acute promyelocytic leukemia (APL), establishing a paradigm for targeted leukemia therapy. ATO, an inorganic arsenic compound derived from realgar (arsenic sulfide), was later purified and developed as a modern drug. Realgar is also the principal active component of the traditional Realgar-Indigo naturalis formula (RIF, Compound Huangdai Tablets), which has demonstrated clinical efficacy comparable to ATO in APL. Inspired by this success, we investigated other constituents of RIF for potential anti-leukemic activity and identified 5-Hydroxyindirubin (5-OH-indirubin), a natural indirubin derivative isolated from Polygonum tinctorium , one of the major sources of Indigo naturalis . 5-OH-indirubin selectively inhibited FLT3 phosphorylation and downstream STAT5/ERK/AKT signaling, inducing apoptosis and G0/G1 cell-cycle arrest in FLT3 -ITD-mutated acute myeloid leukemia (AML) cells. Notably, 5-OH-indirubin retained potent activity against secondary FLT3 -TKD mutations (D835Y, D835V) and gilteritinib-resistant AML cells, thereby overcoming key resistance mechanisms. Furthermore, 5-OH-indirubin exhibited strong synergy with the BCL-2 inhibitor venetoclax, promoting mitochondrial apoptosis and durable growth suppression. In xenograft models, 5-OH-indirubin significantly inhibited leukemia progression, prolonged survival, and was well-tolerated. Collectively, these findings highlight 5-OH-indirubin, a natural indirubin derivative derived from the traditional RIF, as a promising candidate to overcome resistance to FLT3 inhibitors and enhance venetoclax-based therapy for FLT3 -mutated AML.