Rhoifolin, an inhibitor targeting the ALDH2-PKC delta axis, eradicates MRD in chronic myeloid leukemia by eliminating leukemia stem cells

Abstract Background: Chronic myeloid leukemia (CML) patients frequently develop minimal residual disease (MRD) following tyrosine kinase inhibitor (TKI) therapy, primarily due to the persistence and intrinsic drug resistance of leukemia stem cells (LSCs). Moreover, this study pioneers the discovery that Rhoifolin, a naturally occurring flavonoid, selectively eliminates therapy-resistant LSCs through precise targeting of the ALDH2-PKC delta signaling axis, thereby effectively eradicating MRD in CML models. While TKI remains the therapeutic backbone, A small proportion of patients experience relapse due to chemoresistance. This clinical impasse primarily stems from LSCs, a self-renewing subpopulation that orchestrates disease recurrence through their unique capacity to evade conventional therapies and seed MRD. Emerging evidence positions LSCs-targeted strategies as the next frontier in CML therapeutics. However, the development of precision interventions faces dual challenges: molecular heterogeneity of LSCs survival pathways across genetic subtypes and microenvironment-mediated protection of LSCs niches. Method: Our mechanistic studies reveal that ALDH2 orchestrates mitochondrial metabolic plasticity in LSCs through coordinated upregulation of protein kinase C delta (PKC delta), establishing an adaptive metabolic reprogramming axis that sustains chemoresistance. Through high-throughput compound screening, we identified rhoifolin, a flavone glycoside, as an exquisitely selective disruptor of the ALDH2-PKC delta signaling hub. This agent demonstrates: LSCs-specific cytotoxicity, MRD-eradicating capacity in patient-derived xenograft models, suppression of oxidative phosphorylation via ALDH2-PKC delta axis blockade. Notably, rhoifolin's therapeutic window arises from LSCs' distinct metabolic dependencies on ALDH2-PKC delta signaling compared to their normal counterparts. These findings position rhoifolin as a therapeutic candidate targeting LSCs mitochondrial adaptability, with translational potential for preventing CML relapse and eliminating MRD. To determine the impact of ALDH2 high expression on the prognosis of CML through bioinformatics and clinical sample analysis. Quantitative proteomics, metabolome analysis, qRT-PCR, Western blotting, coimmunoprecipitationexperiments, and other biological methods were employed to explore and validate the related pathways and metabolic pathways in LSCs. Various cell line models and animal models were used for biological function validation. Results: In this study, we investigated the role of aldehyde dehydrogenase 2 (ALDH2) in patients with relapsed acute transformation of CML and demonstrated that elevated ALDH2 expression in LSCs is closely associated with disease relapse and treatment resistance. Mechanistically, mitochondrial ALDH2 regulates its downstream effectors, PKC delta to increase oxidative phosphorylation (OXPHOS), promote mitophagy, and sustain LSC self-renewal. The ALDH2‒PKC delta axis plays a pivotal role in conferring resistance to chemotherapy in CML. Notably, rhoifolin, a compound designed to inhibit the specific binding site of ALDH2–PKC delta, significantly increased chemosensitivity. It could target LSCs, work synergistically with TKI. Conclusions:Rhoifolin, as the therapeutic candidate drug targeting the mitochondrial adaptive target of LSCs, exerts its effect by acting on the ALDH2-PKC delta axis. It has potential translational application value in preventing the recurrence of CML and eliminating MRD.