NNMT promotes acquired EGFR-TKI resistance by forming EGR1 and lactate-mediated double positive feedback loops in non-small cell lung cancer

Abstract

Background: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are remarkably effective for treating EGFR-mutant non-small cell lung cancer (NSCLC). However, patients inevitably develop acquired drug resistance, resulting in recurrence or metastasis. It is important to identify novel effective therapeutic targets to reverse acquired TKI resistance.

Results: Bioinformatics analysis revealed that nicotinamide N-methyltransferase (NNMT) was upregulated in EGFR-TKI resistant cells and tissues via EGR1-mediated transcriptional activation. High NNMT levels were correlated with poor prognosis in EGFR-mutated NSCLC patients, which could promote resistance to EGFR-TKIs in vitro and in vivo. Mechanistically, NNMT catalyzed the conversion of nicotinamide to 1-methyl nicotinamide by depleting S-adenosyl methionine (the methyl group donor), leading to a reduction in H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3) and subsequent epigenetic activation of EGR1 and ALDH3A1. In addition, ALDH3A1 activation increased lactic acid levels, which further promoted NNMT expression via p300-mediated histone H3K18 lactylation on its promoter. Thus, NNMT mediates the formation of a double positive feedback loop via EGR1 and lactate, EGR1/NNMT/EGR1 and NNMT/ALDH3A1/lactate/NNMT. Moreover, the combination of a small-molecule inhibitor for NNMT (NNMTi) and osimertinib exhibited promising potential for the treatment of TKI resistance in an NSCLC osimertinib-resistant xenograft model.

Conclusions: The combined contribution of these two positive feedback loops promotes EGFR-TKI resistance in NSCLC. Our findings provide new insight into the role of histone methylation and histone lactylation in TKI resistance. The pivotal NNMT-mediated positive feedback loop may serve as a powerful therapeutic target for overcoming EGFR-TKI resistance in NSCLC.

Keywords: EGFR-TKI resistance; Histone lactylation; Histone methylation; Lung cancer.

Fig. 1

Bioinformatics analyses of RNA expression profiling data reveal the role of NNMT in EGFR-TKI resistance in lung cancer. (A) Activated genes in TKI-resistant cells in these GEO datasets (GSE193258, GSE114647, and GSE123066). (B) Higher NNMT expression is associated with a poorer prognosis in patients.(C) The ROC curve suggests that NNMT expression serves as a predictive marker for EGFR-TKI resistance. (D) NNMT was expressed at higher levels in tissues from TKI-resistant patients than in tissues from TKI-sensitive patients. (E, F) IC50 values of gefitinib in gefitinib-resistant cells (PC9/GR, HCC827/GR) and their respective parental cells (PC9, HCC827) were examined using the MTT assay. (G, H) The IC50 values of osimertinib in osimertinib-resistant cells (PC9/OR, HCC827/OR) and their respective parental cells (PC9, HCC827) were examined using the MTT assay. (I) Treatment with EGFR-TKIs (gefitinib or osimertinib) increased NNMT expression in TKI-resistant cell lines. Three independent experiments were performed. **P < 0.01

Fig. 2

NNMT regulates EGFR-TKI resistance in NSCLC cells. (A) The IC50 values of NNMT in cells after its knockdown or overexpression. (B) MTT assays were performed to determine cell viability and drug resistance after NNMT knockdown or overexpression. (C) Colony formation assays were used to evaluate the colony formation capacity after NNMT knockdown or overexpression. (D) Cell proliferation was analyzed using EdU after NNMT knockdown or overexpression. (E) Cell apoptosis was determined in TKI-resistant cells after NNMT knockdown. Three independent experiments were performed. **P < 0.01

Fig. 3

EGR1 directly transcriptionally activates NNMT in TKI resistance. (A) Schematic diagram of the EGR1 binding site in the NNMT promoter. (B) NNMT expression was determined after EGR1 was knocked down or overexpressed. (C-E) NNMT promoter activity was tested using a dual-luciferase reporter assay after the corresponding treatments. (F) ChIP assays were performed to detect the enrichment of EGR1 in the NNMT promoter. (G-I) IC50, MTT and colony formation assays were used to determine cell proliferation and resistance to TKIs after the corresponding treatments. Three independent experiments were performed. **P < 0.01, n.s., not significant

Fig. 4

NNMT regulates EGR1 and ALDH3A1, leading to the resistance of NSCLC to EGFR-TKIs. (A) RNA sequencing was performed to identify downstream genes after the inhibition of NNMT. (B) Differential gene expression was analyzed using a volcano plot. (C) GO analysis after NNMT knockdown. (D) qRT‒PCR was used to verify representative genes. (E) Western blotting was used to detect the expression of EGR1 and ALDH3A1 after NNMT knockdown or overexpression. (F) EGR1 and ALDH3A1 expression was analyzed in paired tissues from TKI-sensitive and TKI-resistant patients with lung cancer. (G) A positive correlation between NNMT and EGR1 or ALDH3A1 in tissues from TKI-resistant patients. (H) EGR1 and ALDH3A1 protein expression in TKI-resistant cells and sensitive cells. (I-K) IC50, MTT and colony formation assays were used to determine cell proliferation and resistance to TKIs after the corresponding treatments. (L-N) Tumor volumes and weights were measured after the corresponding treatments. (O) Immunohistochemistry analysis was performed to analyze the tumor tissue. Three independent experiments were performed. *P < 0.05, **P < 0.01

Fig. 5

NNMT reduces H3K9me3 and H3K27me3, leading to epigenetic activation of EGR1 and ALDH3A1 and resistance of NSCLC to EGFR-TKIs. (A) SAM and SAM/SAH were increased after NNMT knockdown in PC9/OR cells. (B) Western blot assays were used to detect the expression of H3K9me3 and H3K27me3 after NNMT knockdown or overexpression. (C-E) Western blot assays for the corresponding treatments. (F) ChIP assays showing the levels of H3K9me3 and H3K27me3 modifications in the EGR1 promoter after NNMT knockdown or overexpression. (G) ChIP assays showing the levels of H3K9me3 and H3K27me3 modifications in the promoter of ALDH3A1 after NNMT knockdown or overexpression. Three independent experiments were performed. *P < 0.05, **P < 0.01

Fig. 6

ALDH3A1 mediates histone lactylation by targeting NNMT to promote EGFR-TKI resistance. (A) Glycolysis capacity was measured in resistant cells after the corresponding treatments. (B, C) The levels of lactate and ATP were detected in resistant cells after the corresponding treatments. (D) Western blot analysis of lactic acid-treated cells. (E) Expression of pan-Kla and H3K18la following lactic acid treatment. (F, G) Increased level of global lactylation in tissues from TKI-resistant patients and lactated proteins (red) located in the nucleus. (H) The expression of pan-Kla was positively correlated with NNMT. (I, J) H3K18la levels were greater in tissues from TKI-resistant patients than in those from TKI-sensitive patients. (K) H3K18la expression was positively correlated with NNMT. (L) Pan-Kla and H3K18la levels were increased in TKI-resistant cells. (M, N) Glycolysis inhibitors (2-DG or oxamate) decreased lactate levels in a dose‒dependent manner. (O, P) The expression of pan-Kla, H3K18la and NNMT was determined after the corresponding treatments. (Q) Western blot assays were used to detect H3K18la, NNMT and p300 expression after p300 was knocked down. (R) Western blot assays detected H3K18la and NNMT expression after treatment with the HDAC inhibitor trichostatin A (TSA). (S) Luciferase reporter assays after p300 knockdown. (T) ChIP assay after p300 knockdown. (U) Luciferase reporter assay after treatment with lactic acid. (V) ChIP assay after treatment with lactic acid. Three independent experiments were performed. *P < 0.05, **P < 0.01

Fig. 7

NNMT promotes TKI resistance by mediating histone lactylation to generate a lactate-mediated positive feedback loop. (A) Western blot assays after knockdown or overexpression of ALDH3A1. (B) Glycolysis capacity was measured in resistant cells after the corresponding treatments. (C, D) Lactate and ATP levels were detected in resistant cells after the corresponding treatments. (E-J) NNMT promoter activity was tested using a dual-luciferase reporter gene assay after the corresponding treatments. (K) ChIP assay after the corresponding treatments. (L‒N) IC50, MTT and colony formation assays were used to determine cell proliferation and resistance to TKIs after the corresponding treatments. (O‒Q) IC50, MTT and colony formation assays were used to determine cell proliferation and resistance to TKIs after the corresponding treatments. (R) Dual-luciferase reporter assays after the corresponding treatments. (S) ChIP assays after the corresponding treatments. Three independent experiments were performed. **P < 0.01

Fig. 8

NNMT regulates EGFR-TKI resistance in lung cancer in vivo. (A) Tumor tissues from mice. (B) Tumor volumes were calculated at regular intervals 4 days after injection. (C) Mouse body weights were measured at regular intervals of 4 days. (D) Tumor weights are presented as the means ± SD (standard deviation). (E) The tumor sections were subjected to immunohistochemistry (IHC) staining for H&E, Ki67, NNMT, EGR1 and ALDH3A1. (F) Tumor tissue from the mice. (G) Tumor volumes were calculated at regular intervals 4 days after injection. (H) Mouse body weights were measured. (I) Tumor weights are presented as the means ± SD. (J) The tumor sections were subjected to immunohistochemical staining. (K) Tumor tissues from the mice. (L) Tumor volumes were calculated at regular intervals of 4 days after injection. (M) Mouse body weights were measured. (N) Tumor weights are presented as the means ± SD. (O) Tumor sections were subjected to immunohistochemical staining. (P) Proposed mechanisms for NNMT-mediated EGFR-TKI resistance in NSCLC. *P < 0.05, **P < 0.01