ALKBH5 promotes non-small cell lung cancer progression and susceptibility to anti-PD-L1 therapy by modulating interactions between tumor and macrophages

Abstract

Background: Understanding the mechanisms that mediate the interaction between tumor and immune cells may provide therapeutic benefit to patients with cancer. The N6-methyladenosine (m6A) demethylase, ALKBH5 (alkB homolog 5), is overexpressed in non-small cell lung cancer. However, its role in the tumor microenvironment is unknown.

Methods: Datasets and tissue samples were used to determine the relationship between ALKBH5 expression and immunotherapy efficacy. Bioinformatic analysis, colorimetric assay to determine m6A RNA methylation, dual luciferase reporter assay, RNA/m6A-modified RNA immunoprecipitation, RNA stability assay, and RNA sequencing were used to investigate the regulatory mechanism of ALKBH5 in non-small cell lung cancer. In vitro and in vivo assays were performed to determine the contribution of ALKBH5 to the development of non-small cell lung cancer.

Results: ALKBH5 was upregulated in primary non-small cell lung cancer tissues. ALKBH5 was positively correlated with programmed death-ligand 1 expression and macrophage infiltration and was associated with immunotherapy response. JAK2 was identified as a target of ALKBH5-mediated m6A modification, which activates the JAK2/p-STAT3 pathway to promote non-small cell lung cancer progression. ALKBH5 was found to recruit programmed death-ligand 1-positive tumor-associated macrophages and promote M2 macrophage polarization by inducing the secretion of CCL2 and CXCL10. ALKBH5 and tumor-associated macrophage-secreted IL-6 showed a synergistic effect to activate the JAK2/p-STAT3 pathway in cancer cells.

Conclusions: ALKBH5 promotes non-small cell lung cancer progression by regulating cancer and tumor-associated macrophage behavior through the JAK2/p-STAT3 pathway and the expression of CCL2 and CXCL10, respectively. These findings suggest that targeting ALKBH5 is a promising strategy of enhancing the anti-tumor immune response in patients with NSCLC and that identifying ALKBH5 status could facilitate prediction of clinical response to anti-PD-L1 immunotherapy.

Keywords: ALKBH5; JAK2/p-STAT3 pathway; N6-methyladenosine demethylase; Non-small cell lung cancer; Tumor microenvironment; Tumor-associated macrophage.

Fig. 1

ALKBH5 is upregulated in NSCLC and is associated with the immune response. A qRT-PCR analysis of m6A regulators (ALKBH5, FTO, METTL3/14/16, and WTAP) in 40 pairs of NSCLC tissues and adjacent normal tissues. B qRT-PCR analysis of ALKBH5 expression in A549, BEAS-2B, H460, H1299, H1975, HCC-827, PC-9, and SPC-A1 cells. C Western blot analysis of ALKBH5 expression in A549, BEAS-2B, H460, H1299, H1975, HCC-827, PC-9, and SPC-A1 cells. GAPDH was used as the loading control. D Kaplan–Meier analysis of the relationship between ALKBH5 expression and overall survival in patients with lung cancer. E Kaplan–Meier analysis of the relationship between ALKBH5 expression and overall survival in patients with cancer treated with immunotherapy. F Representative immunohistochemical images of ALKBH5 in DCB (n = 30) and NDB (n = 25) groups (left panel; scale bar = 20 µm) and immunohistochemistry correlation analysis (right panel). G Representative immunohistochemical images of CD8, CD68, CD206, and PD-L1 in the low (n = 28) and high ALKBH5 (n = 27) expression groups (upper panel; scale bar = 20 µm) and immunohistochemistry correlation analysis (lower panel). H Pearson correlation coefficient analysis of ALKBH5 and PD-L1 expression in 40 cases of NSCLC. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant

Fig. 2

ALKBH5 upregulates JAK2 expression to activate the JAK2/p-STAT3 pathway. A qRT-PCR analysis of the efficiency of siRNA-mediated knockdown of ALKBH5 in A549 and H1299 cells. B qRT-PCR analysis of JAK2 expression in A549 and H1299 cells transfected with ALKBH5 siRNA. C Western blot analysis of ALKBH5 and JAK2 expression in A549 and H1299 cells transfected with ALKBH5 siRNA (loading control = GAPDH). D The mRNA stability of JAK2 in ALKBH5-knockdown A549 and H1299 cells treated with actinomycin D (5 µg/mL). E RIP–qRT-PCR analysis of the binding affinity of ALKBH5 to JAK2 in A549 and H1299 cells (control = IgG). F Pearson correlation coefficient analysis of ALKBH5 and JAK2 mRNA expression in 40 cases of NSCLC. G Western blot analysis of ALKBH5, JAK2, and p-STAT3 expression in A549 and H1299 cells with or without ALKBH5 knockdown (loading control = GAPDH). H Immunofluorescence analysis of p-STAT3 expression in A549 and H1299 cells with or without ALKBH5 knockdown (scale bar = 20 μm). **P < 0.01; ***P < 0.001; NS, not significant

Fig. 3

ALKBH5 regulates JAK2 expression in an m6A-YTHDF2-dependent manner. A qRT-PCR analysis of the efficiency of ALKBH5 overexpression in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A. B Overall m6A deposited RNAs in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A detected using the colorimetric m6A assay. C Western blot analysis of ALKBH5, JAK2, and p-STAT3 expression in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, or OE-H204A (loading control = GAPDH). D MeRIP–qRT-PCR analysis of m6A modification sequences of JAK2 transcript in ALKBH5 overexpression and knockdown H1299 cells. E Schematic showing the sequence of the wild type and m6A motif mutation (A to T) plasmids containing luciferase reporter gene vectors. F Dual luciferase reporter analysis of wild type and m6A motif mutation reporter vector plasmids in A549 and H1299 cells with or without ALKBH5 knockdown. G qRT-PCR analysis of the efficiency of YTHDF2 knockdown in siRNA-transfected A549 and H1299 cells. H qRT-PCR analysis of JAK2 mRNA expression in A549 and H1299 cells transfected with YTHDF2 siRNA. I Western blot analysis of JAK2 and YTHDF2 expression in A549 and H1299 cells with or without YTHDF2 knockdown (loading control = GAPDH). J RIP–qRT-PCR analysis of the binding affinity of YTHDF2 to JAK2 in A549 and H1299 cells (control = IgG). K The mRNA stability of JAK2 in YTHDF2-knockdown A549 and H1299 cells treated with actinomycin D (5 µg/mL). L Western blot analysis of JAK2 expression in ALKBH5-knockdown A549 and H1299 cells with or without YTHDF2 deficiency (loading control = GAPDH). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant

Fig. 4

ALKBH5 induces PD-L1 expression and the malignant phenotype of NSCLC by regulating the JAK2/p-STAT3 pathway. A qRT-PCR analysis of PD-L1 expression in ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM). B Western blot analysis of PD-L1 and p-STAT3 expression in ALKBH5-overexpressing H1299 and H1975 cells treated with AG490 (50 μM; loading control = GAPDH). C CCK-8 assay of the proliferation of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM). D Colony formation assay of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM). E Transwell assay of the migration of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM). F Quantification of tube formation in HUVECs treated with the supernatant of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM; magnification, × 100). G qRT-PCR analysis of VEGFA expression in ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM). H ELISA of VEGFA secreted into the supernatant of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM). *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

RNA sequencing identifies CCL2 and CXCL10 as targets of ALKBH5. A Volcano plot of up- (red) and down-regulated (green) genes (log2[Fold Change] > 1 and P < 0.05) in H1299 cells transfected in triplicate with si-ALKBH5 or si-NC. B Gene Ontology analysis of differentially expressed genes after ALKBH5 knockdown. C qRT-PCR analysis of the expression of six selected chemokines in A549 and H1299 cells transfected with ALKBH5 siRNA. D ELISA of CCL2 and CXCL10 secreted into the supernatant of ALKBH5-knockdown A549 and H1299 cells. E qRT-PCR analysis of CCL2 and CXCL10 expression in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A. F ELISA of CCL2 and CXCL10 secreted into the supernatant of H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant

Fig. 6

ALKBH5 recruits PD-L1⁺ TAMs and promotes M2 macrophage polarization. A Transwell assay of the migration of PMA-stimulated THP-1 cells co-cultured with conditioned medium from ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins. B qRT-PCR analysis of PD-L1 expression in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins. C Western blot analysis of PD-L1 expression in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins (loading control = GAPDH). D Flow cytometry analysis of the mean fluorescence intensity (MFI) of PD-L1 in CD206⁺ TAMs co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins. E Immunofluorescence analysis of CD206⁺PD-L1⁺ TAMs in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins (blue = DAPI; green = CD206; red = PD-L1; scale bar = 20 µm). F Flow cytometry analysis of the percentage of CD11b⁺CD206⁺ TAMs in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

ALKBH5 has a synergistic effect with IL-6 on the activation of the JAK2/p-STAT3 pathway. A Schematic of the co-culture model (created with BioRender.com). B qRT-PCR analysis of IL-6 expression in A549 or H1299 cells, THP-1 macrophages, and PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown or control A549 and H1299 cells. C ELISA of IL-6 secreted from A549 or H1299 cells, THP-1 macrophages, and PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown or control A549 and H1299 cells. D qRT-PCR analysis of ALKBH5 expression in H1299 and H1975 cells treated with or without IL-6 (20 ng/mL). E Western blot analysis of ALKBH5, p-JAK2, and p-STAT3 expression in H1299 and H1975 cells treated with or without IL-6 (20 ng/mL; loading control = GAPDH). F Western blot analysis of p-STAT3 expression in ALKBH5-overexpressing H1299 and H1975 cells treated with or without IL-6 (20 ng/mL; loading control = GAPDH). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant

Fig. 8

ALKBH5 promotes tumor growth in vivo and is sensitive to anti PD-L1 therapy. A qRT-PCR analysis of the efficiency of ALKBH5 overexpression in LLC cells. B Western blot analysis of the efficiency of ALKBH5 overexpression in LLC cells (loading control = GAPDH). C Left panel: ALKBH5 overexpression promotes tumor growth in C57BL/6 J mice (n = 4). Middle panel: Tumor volumes measured by growth curve every three days from day 6 to day 21 after cell transplantation. Right panel: Tumor weight determined immediately after removal from C57BL/6 J mice. D qRT-PCR analysis of tumor mRNA expression of CCL2, CXCL10, JAK2, and PD-L1. E ELISA of CCL2 and CXCL10 in plasma. F Representative immunohistochemical images of ALKBH5, CD8, CD31, CD206, F4/80, JAK2, Ki-67, and PD-L1 expression in ALKBH5-overexpressing and control mice (upper panel; scale bar = 20 µm) and immunohistochemistry correlation analysis (lower panel). G qRT-PCR analysis of the efficiency of ALKBH5 knockdown in LLC cells. H Western blot analysis of the efficiency of ALKBH5 knockdown in LLC cells (loading control = GAPDH). I Schematic of the cell transplantation and drug administration experiment (created with BioRender.com). J Left panel: ALKBH5 knockdown and anti-PD-L1 therapy attenuated tumor growth in C57BL/6 J mice (n = 4). Middle panel: Tumor volumes measured by growth curve every three days from days 6 to 21 after cell transplantation. Right panel: Tumor weight determined immediately after removal from C57BL/6 J mice. K Tumor inhibition rate of ALKBH5-knockdown and control mice treated with anti-PD-L1 therapy. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant

Fig. 9

Illustrative model of the proposed mechanism.ALKBH5 promotes NSCLC progression and susceptibility to anti-PD-L1 therapy by modulating the interactions between tumor and macrophages (created with BioRender.com)