Inhibition of CPT1A reverses AML1-ETO-induced differentiation blockage by activating BCL-2/MYC signaling and metabolic reprogramming

Abstract Objectives: Recent studies have increasingly highlighted the pivotal role of fatty acid metabolism in regulating hematopoietic stem cells (HSCs) fate and its influence on tumorigenesis, metastasis, and the development of drug resistance. Carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme of fatty acid β-oxidation, is essential for the maintenance of normal HSCs function. However, its role in aberrant hematopoiesis, particularly in aberrant stem cells, remains largely unexplored. In this study, we tried to address this issue and provided novel insights into the therapeutic strategy for the eradication of aberrant stem cells in AML. Methods The expression and regulation of CPT1A by the AML1-ETO fusion protein were assessed using public ChIP-seq and Cut&Run data, and verified in a doxycycline-inducible U937 cell model. Metabolomic profiling, pharmacological inhibition were performed in Runx1Runx1t1/+; Mx1-Cre mouse (AML1/ETO mouse), and CRISPR-Cas9-mediated knockout of Cpt1a was performed in AML1/ETO; R26-CAG-Cas9 mice. Flow cytometry was used to evaluate metabolic activity, HSPCs populations, and mature hematopoietic lineages. Colony formation was performed to evaluate the function of HSPCs. The downstream functional patterns were studied following RNA-seq in c-kit+ cells and RT-qPCR was used to verify molecular changes. Results In this study, we found that CPT1A was highly expressed in leukemic stem and progenitor cells, and elevated CPT1A levels were significantly associated with poor clinical prognosis. Further analysis demonstrated that the common leukemia fusion protein, AML1-ETO oncoprotein directly regulated the expression of CPT1A. To further investigate the functional role of CPT1A, we employed AML/ETO mouse, which is characterized by pathological expansion of aberrant HSCs and HPCs populations and blockade in myeloid differentiation. RNA sequencing revealed a marked enrichment of Cpt1a expression in the aberrant stem/progenitor compartments of AML/ETO mice. Given the central role of CPT1A in cellular metabolism, we next examined metabolic alterations in AML1/ETO aberrant HSCs compared to wild-type counterparts. We observed that carnitine metabolism was selectively upregulated in the context of globally suppressed metabolic activity, which corresponded with elevated Cpt1a expression. To determine whether CPT1A represents a functional vulnerability, we assessed the effects of pharmacologic and genetic Cpt1a inhibition in the AML/ETO model. Cpt1a suppression induced extensive metabolic reprogramming, notably enhancing glycolysis–OXPHOS coupling, which led to partial resolution of the differentiation blockade and restoration of the myeloid developmental hierarchy. Furthermore, Cpt1a knockout activated the Myc/Bcl-2 signaling axis, promoting cell cycle progression and facilitating myeloid maturation. These findings indicate that CPT1A maintains aberrant stem cell quiescence and contributes to differentiation arrest in AML1-ETO related leukemogenesis. Conclusions In summary, our study demonstrates that targeting CPT1A induces a critical state transition in aberrant stem cells, converting them from a quiescent, primitive phenotype to a proliferative and differentiation-competent state. This metabolic intervention effectively alleviates AML1-ETO mediated differentiation block and restores myeloid lineage commitment. Our findings position CPT1A as a metabolic checkpoint sustaining LSC stemness and resistance, and suggest that CPT1A inhibition may provide a promising therapeutic strategy for the eradication of chemoresistant leukemic stem cells in t(8;21) AML.