Cancer Res Treat.  2023 Jul;55(3):851-864. 10.4143/crt.2022.1527.

Combination of the LARS1 Inhibitor, BC-LI-0186 with a MEK1/2 Inhibitor Enhances the Anti-Tumor Effect in Non–Small Cell Lung Cancer

• Lee SH
• Kim EY
• Han JM
• Han G
• Chang YS

• KMID: 2544166
• DOI: http://doi.org/10.4143/crt.2022.1527

Abstract

Purpose
The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth and proliferation by growth factor coordination and amino acid availability. Leucyl-tRNA synthetase 1 (LARS1) senses the intracellular leucine concentration and mediates amino acid-induced activation of mTORC1. Thus, LARS1 inhibition could be useful in cancer treatment. However, the fact that mTORC1 can be stimulated by various growth factors and amino acids suggests that LARS1 inhibition alone has limitations in inhibiting cell growth and proliferation. We investigated the combined effects of BC-LI-0186, a LARS1 inhibitor, and trametinib, an MEK inhibitor, on non–small cell lung cancer (NSCLC).
Materials and Methods
Protein expression and phosphorylation were observed by immunoblotting, and genes differentially expressed between BC-LI-0186–sensitive and –resistant cells were identified by RNA sequencing. The combined effect of the two drugs was inferred from the combination index values and a xenograft model.
Results
LARS1 expression was positively correlated with mTORC1 in NSCLC cell lines. BC-LI-0186 treatment of A549 and H460 cells maintained in media supplemented with fetal bovine serum revealed paradoxical phosphorylation of S6 and activation of mitogen- activated protein kinase (MAPK) signaling. Compared with BC-LI-0186–sensitive cells, –resistant cells showed enrichment of the MAPK gene set. The combination of trametinib and BC-LI-0186 inhibited the phosphorylation of S6, MEK, and extracellular signal-regulated kinase and their synergistic effects were confirmed in a mouse xenograft model.
Conclusion
The combination of BC-LI-0186 and trametinib inhibited the non-canonical mTORC1-activating function of LARS1. Our study demonstrated a new therapeutic approach for NSCLC without targetable driver mutations.

Keyword

BC-LI-0186; LARS1; Non-small cell lung cancer; mTORC1; Trametinib

Figure

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  Fig. 1 Expression of leucyl-tRNA synthetase 1 (LARS1) correlates positively with the mammalian target of rapamycin complex 1 (mTORC1) signal in non–small cell lung cancer (NSCLC). (A) Immunoblotting of LARS1 and molecules involved in mTOR signaling in various NSCLC cells. (B) The correlation plot between LARS1 and pS6 (r=0.8156, p=0.0040), pAkt (r=0.5879, p=0.0739), pERK (r=0.4092, p=0.2403), and pMEK (r=0.7478, p=0.0129). There was a significant positive correlation between LARS1 and pS6. p-values were obtained from Pearson’s correlation analysis and r denotes Pearson’s correlation coefficient. (C) Immunoblotting and heatmap of LARS1 and molecules associated with mTOR signaling according to leucine concentration in the A549 cell line and H460 cell line. (D) Effects of BC-LI-0186 (10 μM) on the A549 and H460 cell lines over time in fetal bovine serum (FBS) (−) media. (E) Effects of the BC-LI-0186 concentration on the A549 and H460 cell lines in FBS (+) media. It was observed that BC-LI-0186 concentrations less than 10 μM had a paradoxical effect on both A549 and H460 cell lines. R*, each cell line was treated with rapamycin (10 μM) for 10 minutes and subjected immunoblotting; S**, serum shock (used for positive control).

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  Fig. 2 Paradoxical response of BC-LI-0186 to lung cancer cell line in the presence of growth factors. (A) Immunoblotting and line graph of pMEK, pERK, and pS6 expression according to the concentration of BC-LI-0186 in the A549 and H460 cell lines. (B) H460 and A549 cell lines were treated with BC-LI-0186 (10 μM) or cisplatin (50 μM) for 48 hours, and cell death was measured by flow cytometry unsing annexin V and propidium iodide (PI) staining. (C) Volcano plot. The log2 fold-change indicates the mean expression level for each gene and each dot represents one gene. Genes with significantly increased expression in the A549 cell line relative to the H460 cell line was indicated. (D) Results of Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis.

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  Fig. 3 The combination of BC-LI-0186 and trametinib shows a synergistic effect in human lung cancer cell lines. (A) Immunoblotting of pS6, pMEK, and pERK according to trametinib concentration in the A549 and H460 cell lines. (B) Colocalization images of LC3A/B and LAMP2, and histogram for the ratio of colocalization obtained from slides with 10 or more cells according to the use of BC-LI-0186 and trametinib in A549 and H460 cells. (C) Expression of p62, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase 3 according to the use of BC-LI-0186 and trametinib in A549 and H460 cells. (D) Immunoblotting results pAKT, pS6, pMEK, and pERK according to the use of BC-LI-0186 and trametinib. (E) Fa-dose plot and combination index plot using for treatment of A549, H460, H356, H1703, and H1650 cells.

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  Fig. 4 Synergistic anti-tumor effect of BC-LI-0186 and trametinib combination treatment. (A) Diagram of the treatment schedule in BALB/c nude mice lung cancer model. (B) Photographs of typical BALB/c nude mice bearing tumors. (C) Change rate in tumor volume of each group according to differences before and after treatment. There was a significant tumor volume change in control group comparing with combination group (p=0.004). (D) Representative photograph of haematoxylin and eosin and immunohistochemistry staining for activated caspase-3, pMEK, and pS6 on lung sections obtained from mice after 2 weeks’ treatment, and quantification of caspase-3, pMEK, and pS6 in the tumor of each treatment group. *p < 0.05. p-values were obtained by one-way analysis of variance followed by Tukey’s post-hoc multiple comparison tests. (E) Body weight change rate among mouse groups during the course of treatment.

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  Fig. 5 Schematic diagram of effective cancer suppression process through combination therapy of BC-LI-0186 and trametinib (created in BioRendor.com). mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; Rag, Ras-related GTP-binding protein.

Reference

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