Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

Abstract

The emergence of secondary resistance is the main failure cause of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) as a targeted therapy for non-small cell lung cancer (NSCLC). EGFR mutations of NSCLC cells can markedly increase glutamine transporter (SLC1A5) expression, thereby increasing glutamine metabolism. Glutamine metabolites can activate EGFR downstream signals, including mTOR, ERK1/2, STAT3, etc., which is an important cause for the decreased sensitivity of NSCLC to EGFR-TKIs. CCK8 and Annexin V/PI assays were conducted to detect the effects of Almonertinib and/or V9302 on the proliferation and apoptosis of NSCLC cells. Proteomics was used to determine the effect of Almonertinib on energy metabolism-related proteins in NSCLC. siRNA transfection was performed to study the effect of SLC1A5 down-regulation on cell proliferation. In addition, the effects of drugs on colony formation capacity were determined by colony formation assay. Immunofluorescence and Western blot were utilized to detect the apoptosis- and autophagy-related proteins expression. DAPI staining was utilized to detect the effect of drugs on the nucleus. Transmission electron microscope was used to observe the changes of submicroscopic structure such as autophagosomes and nucleus of cells. mCherry-GFP-LC3B tandem fluorescent protein was to used to detect the level of autophagy flux. Tumor-bearing nude mouse model was utilized to detect the effect of V9302 on the anti-tumor effect of Almonertinib in vivo. As a result, Almonertinib suppressed H1975 and A549 cell proliferation depended on its dosage and treatment duration, and it also induced apoptosis. A549 cells with wild-type EGFR had lower sensitivity to Almonertinib. The expression of SLC1A5 was up-regulated by stimulating with low concentration of Almonertinib in NSCLC cells. SLC1A5 was highly expressed in A549 cells with wild-type EGFR. Glutamine deletion or SLC1A5 inhibition/silencing inhibited the proliferation of NSCLC cells, and decreased cellular glutamine uptake. The combination of SLC1A5 inhibitor V9302 and Almonertinib had a synergistic inhibitory effect on the proliferation of NSCLC. V9302 enhanced the effect of Almonertinib in apoptosis-inducing in NSCLC cells. The combination of V9302 and Almonertinib might induce apoptosis by inhibiting autophagy.

Therapeutic Potential of Glutamine Pathway in Lung Cancer

“…Induction of Gln deficiency or SLC1A5 inhibition/silencing inhibited NSCLC cell proliferation and reduced cellular uptake of Gln. Thus, the combination of SLC1A5 inhibitor V9302 and Almonertinib (an EGFR-TKI) had a synergistic inhibitory effect on NSCLC proliferation (81). In a study by Meng Xia et al, we found that Gln utilization was higher in KRAS mutant NSCLC than in KRAS wild type.…”

Targeting Pulmonary Fibrosis by SLC1A5 dependent Glutamine Transport Blockade

“…We performed MTT and XTT assay (cell viability and proliferation assay) which confirmed no significant effect of V-9302 on cellular proliferation (i.e., toxicity) at 10μM concentrations that significantly impacted profibrotic TGF-β signaling (Supplementary Figure 2A and 2B). This concentration was subsequently used throughout this study as reported in different tumor models (28, 31, 32). There were also no significant changes in gene expression predominantly associated with DNA repair ( ATM, ATR and BRCA1 ), recombination ( RAD51 and RAD52 ) and cell cycle ( CCNB1 and CCND1 ), under V-9302 treatment conditions (Supplementary Figure 2C).…”

Role and therapeutic targeting of glutamine metabolism in non‑small cell lung cancer (Review)

“…EGFR TKIs are targeted therapeutic drugs for patients with NSCLC with EGFR mutations; however, secondary drug resistance is the main reason for the limited antitumor effect (88). The expression of SLC1A5 increased after treatment with the EGFR TKI almonertinib (89), which may be related to secondary resistance since glutamine can activate the downstream signal of EGFR, such as mTOR. SLC1A5 inhibitors, such as GPNA, can enhance the antitumor effect of almonertinib independently of the presence of EGFR mutations, and the combination of these two drugs may be the reason for inducing apoptosis by inhibiting autophagy (89).…”

“…The expression of SLC1A5 increased after treatment with the EGFR TKI almonertinib (89), which may be related to secondary resistance since glutamine can activate the downstream signal of EGFR, such as mTOR. SLC1A5 inhibitors, such as GPNA, can enhance the antitumor effect of almonertinib independently of the presence of EGFR mutations, and the combination of these two drugs may be the reason for inducing apoptosis by inhibiting autophagy (89). The GAC inhibitor 968 can reverse the secondary resistance of erlotinib by preventing glutamine metabolism; their combination not only restricts glutamine and glycolytic metabolism, but also maximizes the antitumor effect and safety, which provides a direction for the treatment of patients with NSCLC resistant to EGFR TKIs (81).…”