Abstract
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a poor prognosis. Despite recent advances in therapeutic interventions, relapse and treatment resistance remain prevalent, highlighting the need for improved therapeutic approaches. Aminoacyl-tRNA synthetase (aaRS) is an essential enzyme for protein synthesis, which ligates each amino acid to its corresponding tRNA. Recent studies have reported that aaRS could serve as an attractive therapeutic target in cancer, in part through the activation of the integrated stress response triggered by amino acid deprivation. Notably, the inhibition of prolyl-tRNA synthetase (ProRS) activity has demonstrated potent antitumor activity in preclinical models of multiple myeloma and T-cell acute lymphoblastic leukemia. However, the potential therapeutic effect of ProRS inhibition in AML remains unexplored. First, we analyzed the prognostic impact of EPRS, which encodes the bifunctional glutamyl-prolyl-tRNA synthetase, on AML using the GEPIA2 platform. The results demonstrated that patients with high EPRS expression had significantly worse overall survival than those with low expression (p = 0.031). To experimentally validate the functional relevance of EPRS, we silenced EPRS using shRNA in MOLM-14 cells. This resulted in marked suppression of cell growth, indicating that EPRS is critical for the survival of AML cells. We next evaluated the effect of pharmacological inhibition of ProRS in AML cell lines. We employed NCP26, a novel, pyrazinamide-based inhibitor of ProRS that functions in an ATP-competitive manner. NCP26 decreased cell viability and induced G0/G1 cell cycle arrest as determined by propidium iodide-based flow cytometry. Furthermore, NCP26 triggered apoptosis in both AML cell lines and patient-derived primary cells, as confirmed by cleavage of caspases and PARP, and increased Annexin V staining. To investigate the mechanism underlying the pharmacological activity of NCP26, we performed RNA sequencing using AML cell lines (MOLM-14 and Kasumi-1). Gene set enrichment analysis revealed that NCP26 treatment altered the expression of genes involved in cell cycle and RNA splicing, and enriched gene sets associated with negative regulation of the KRAS signaling pathway. Furthermore, we observed that NCP26 treatment resulted in increased expression of Bcl-2-modifying factor (BMF), a member of the pro-apoptotic Bcl-2 homology 3 domain-only (BH3-only) proteins, which are known to be regulated by the RAS signaling pathway. Following the results of RNA-seq analysis, we performed qRT-PCR to confirm changes in BMF transcription. We observed an increase in BMF expression in MOLM-14 and Kasumi-1 cells treated with NCP26, whereas no such increase was observed in THP-1 cells. Given that MOLM-14 harbors an FLT3-ITD mutation and Kasumi-1 carries a KIT mutation, whereas THP-1 lacks receptor tyrosine kinase (RTK) mutations, these findings suggest that NCP26 upregulates BMF transcription by suppressing the RAS pathway, particularly in RTK-mutant AML cells. Since BMF is known to neutralize the activity of anti-apoptotic proteins, we hypothesized that NCP26-induced upregulation of BMF could potentially act synergistically with the BCL-2 inhibitor venetoclax. To evaluate this hypothesis, we treated MOLM-14 and Kasumi-1 cells with a combination of NCP26 and venetoclax, which exhibited marked synergistic effects in these cell lines. Furthermore, NCP26 and venetoclax also exhibited synergistic effects in primary cells obtained from the bone marrow samples of RTK-mutant AML patients. Similarly, halofuginone, a quinazoline alkaloid derived from febrifugine and recognized for its ability to inhibit ProRS, demonstrated synergistic antileukemic effects when combined with venetoclax in AML cell lines. These results indicate that ProRS inhibition has standalone antitumor activity and can potentiate BCL-2 blockade in AML. In conclusion, our findings demonstrated that ProRS plays a critical role in AML cell survival and that its inhibition induces apoptosis in both AML cell lines and primary cells. Furthermore, ProRS inhibition exhibited synergistic effects with venetoclax, particularly in RTK-mutant AML, through the upregulation of BMF via suppression of the RAS signaling pathway. These findings highlight a promising therapeutic strategy that warrants further investigation for the treatment of AML.
