G Protein-Coupled Receptor 183 Mediates Homoharringtonine Resistance Via NF-Κb Pathway in Acute Myeloid Leukemia
Authors: Zhuang Haihui , Li Fenglin , Xu Rongzhen , Pei Renzhi , Jin Jie , Lu Ying 
Published: December 2024

Background: Homoharringtonine (HHT) is commonly used as a first-line chemotherapy treatment for acute myeloid leukemia (AML) in China. However, drug resistance presents a significant challenge to treatment efficacy. Identifying key genes and elucidating the mechanisms underlying drug resistance is essential for overcoming HHT resistance. In our previous research, we established HHT-resistant cell lines with gradually increasing resistance indices (RIs) to investigate the underlying resistance mechanisms. Utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), we identified G protein-coupled receptors (GPR) and related molecules as potential mediators of resistance. Among these, GPR183 was a highly expressed hub gene in the HHT-resistant cell lines. This study aims to clarify the specific roles and mechanisms by which GPR183 mediates HHT resistance in AML by using cell lines, animal models, and clinical samples.

Methods: The expression of GPR183 in HHT-resistant cell lines was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting. Spearman correlation analyses were conducted to explore the relationship between GPR183 expression and the half-maximal inhibitory concentration (IC50) of HHT in clinical AML samples. The binding affinity between HHT and GPR183 was investigated by Autodock Vina and surface plasmon resonance (SPR) techniques. Additionally, gene set enrichment analysis (GSEA) together with Western blotting were applied to decipher the underlying mechanisms by which GPR183 regulates HHT resistance. An AML mouse model was utilized to validate the effects of GPR183 on tumor burden and animal survival.

Results: Expression levels of GPR183 were significantly elevated in HHT-resistant AML cell lines compared to sensitive cell lines, both at the mRNA and protein levels. To further elucidate the relationship between GPR183 and HHT resistance, we assessed HHT IC50 and GPR183 expression levels in various AML cell lines and primary patient samples. Our data revealed a positive correlation between HHT IC50 values and GPR183 expression levels in AML cell lines. Furthermore, Spearman correlation analysis confirmed a strong positive association between GPR183 expression and HHT IC50 in primary AML patient samples, with a correlation coefficient of 0.806. In addition, overexpression of GPR183 led to an increase in IC50 from 4.475 nM to 9.520 nM, resulting in a resistance index of 2.13. This evidence strongly indicates that GPR183 mediates HHT resistance in AML cells. Treatment of MOLM-13 cells with varying concentrations of HHT resulted in a dose-dependent upregulation of GPR183 protein expression. We then explored the direct interaction between HHT and GPR183 using AutoDock Vina and SPR experiments. Our findings identified a direct binding site between HHT and GPR183, with a binding energy of -9.599 kcal/mol. SPR experiments confirmed this interaction, showing an affinity of 74.3 μM. These results indicated that GPR183 mediates HHT resistance in AML cells by directly binding to HHT. To gain insights into the mechanisms underlying GPR183-mediated HHT resistance, we performed GSEA on transcriptomic data from drug-resistant and sensitive AML cell lines. Results indicated activation of the NF-κB signaling pathway was the top 1 pathway in the HHT resistance cell line. Western blot analysis revealed that NF-κB p65 protein levels were significantly elevated in the HHT-resistant cell line compared to HHT-sensitive cells. Notably, overexpression of GPR183 correlated with elevated NF-κB p65 levels, while knockdown of GPR183 resulted in reduced NF-κB p65 levels. These findings suggested that GPR183 may facilitate HHT resistance through activation of the NF-κB signaling pathway. Furthermore, knocking down the expression of GPR183 inhibits the growth of AML cells both in vitro and in vivo.

Conclusion: GPR183 mediates HHT resistance in AML cells by directly binding to HHT and plays a critical role in promoting HHT resistance through the activation of the NF-κB signaling pathway.