Fig 1: NEK2 phosphorylates SPTLC1 at S401 to promote kinase metabolic activity.(A) Docking model of NEK2 (2XNM) and SPTLC1/SPTLC2 complex (7K0M). (B) Cell lysates from BMDMs analyzed by immunoprecipitation (IP) and Western blot using the antibodies indicated. (C) Western blot analysis of whole-cell lysates and anti-Flag IP derived from 293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 12 hours before harvesting. (D) IP and Western blot using the antibodies indicated in cell lysates from BMDMs. Representative images are shown. (E) GST pull-down assay of NEK2-His and GST-SPTLC1 protein. Representative images are shown. n = 3 independent experiments. (F) Microscale thermophoresis (MST) analysis of NEK2-SPTLC1 interaction measurements. (G) Representative images of individual immunofluorescence staining of NEK2 and SPTLC1 interaction in BMDM cells by Duolink assay combined with immunofluorescence staining using markers for ER (HSP90B1) and nuclei (DAPI). The red dots (NEK2/PD-L1 interaction) indicate their interaction. (H) Trypsin digestion of ER fractions with or without permeabilization in BMDM cells. Representative image is shown, n = 3 independent experiments. (I) SPTLC1 phosphorylation at the S401/S381/S404/S50 residues were identified by MS analysis. (J) Immunoblotting (IB) analysis of Input and anti-Flag IP derived from 293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 12 hours before harvesting. (K) Representative images and statistical results of individual immunofluorescence staining of the NEK2 and SPTLC1 interaction in BMDM cells by a Duolink assay. The red dots (NEK2/SPTLC1 interaction) indicate their interaction. (L) IB analysis of S401-phosphorylated SPTLC1 in BMDM cells with NEK2 inhibitor (10 μM, 48 hours) and NEK2 KO BMDM. (M) In vitro kinase assay and IB analysis of pS401-SPTLC1 expression of recombinant SPTLC1 WT and NEK2 (active) protein. Representative images are shown. n = 3 independent experiments. IgG, immunoglobulin G; GFP, green fluorescent protein; HA, hemagglutinin; m/z, mass/charge ratio.
Fig 2: NEK2 in macrophages drives hepatocarcinogenesis and progress.(A) Tumor incidence in WT and Nek2cKO mice (n = 9). (B) Representative images and statistical results of tumor in WT/Nek2cKO mice by orthotopic and HTVi model. (C) mIHC analysis of CD8+ T cells and Foxp3+ Tregs in WT and Nek2cKO mice tumors. (D) Survival of WT and Nek2cKO mice in HTVi and orthotopic model. (E) Immunophenotype analysis of NEK2hi TAMs and NEK2low TAMs in patients’ HCC and HTVi mice. (F) Correlation of NEK2hi TAMs with CD8+ T cells or Tregs in a tissue microarray. (G) mIHC spatial staining of NEK2hi TAMs, CD8+ T cells, and FOXP3+ Tregs in patients’ HCC tumors. (H) CFSE-based proliferation of CD8+ T cells cocultured with WT/Nek2cKO TAMs. (I and J) RT-PCR analysis of CD8+ Teff/Tex and GZMB and TNFα expression in CD8+ T cells cocultured with WT/Nek2cKO TAMs. (K) OT-1 T cell–meditated killing of Hep-53.4 OVA with WT/Nek2cKO TAMs. (L and M) RT-PCR analysis of Tregs and TH17 cells and flow cytometric analysis of Foxp3/IL-17A in Tregs cocultured with WT/Nek2cKO TAMs. (N) Ex vivo suppression of CFSE-labeled WT naïve CD8+ T cell proliferation by Tregs cocultured with WT/Nek2cKO TAMs. (O) KEGG analysis by LC-MS energy reanalysis of WT/Nek2cKO TAMs. (P) MitoTracker Green of WT/Nek2cKO TAMs. (Q) TEM analysis of mitochondrial (red arrowheads) from WT and Nek2cKO TAMs. (R) Representative OCR of WT and Nek2cKO TAMs. (S and T) MitoTracker Green and Seahorse analysis of CD8+ T cells and Tregs cocultured with WT/Nek2cKO TAMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Using a two-tailed, unpaired Student’s t test [(B), (C), (H), (J), (K), (M), (N), (Q), (R), (S), and (T)], Pearson correlation test (F), or log-rank test [(A) and (D)]. Data are from ≥3 independent experiments (means ± SD).
Fig 3: NEK2hi TAMs, which was the main source of S1P-related metabolites, were identified as a driver for immunotherapy resistance.(A) Biochemical pathways for de novo S1P and SM synthesis. ADP, adenosine diphosphate; Pi, inorganic phosphate. (B) SPTLC activity of tumor cell and immune cell sorting from HCC tissues (n = 5). (C) Concentration analysis of S1P by ELISA in cell culture lysates and medium of indicated cells. (D) Schematic diagram showing the process of model establishment, sample collection, and analysis. (E) RNA-seq analysis of HCC-associated TAMs in responder and nonresponder groups. (F) Representative histograms further quantification of NEK2 in TAMs isolated from para-tumor, tumor, responder, and nonresponder samples (n = 5). (G) Representative images and statistical results showing immunofluorescence staining of indicated antibodies in para-tumor, tumor, responder, and nonresponder sample (n = 9). (H) Western blot of NEK2 in TAMs isolated from para-tumor (normal), tumor, responder, and nonresponder samples. (I) Mean fluorescence intensity (MFI) analysis of NEK2hi TAMs in tumor tissue from stage I to II (n = 7) and stage III to IV patients (n = 7). (J) Representative images showing immunofluorescence staining of NEK2hi TAMs in well differentiated and poorly differentiated samples. H&E, hematoxylin and eosin. (K) Correlation of NEK2 in TAMs with prognostic factors in patients with HCC. (L) Multiple IHC labeling using indicated antibodies in tumor tissues. (M and N) KEGG and heatmap analysis of TAMs by LC-MS metabonomics from WT and Nek2cKO mice. CGMP-PKG, cyclic guanosine monophosphate-protein kinase G signaling pathway. (O) Concentration analysis of S1P and sphinganine by ELISA in cell culture lysates and medium of TAMs from WT and Nek2cKO mice. (P) SPTLC activity of TAMs from WT and Nek2cKO mice or with a NEK2 inhibitor (10 μM) (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Using a two-tailed, unpaired Student’s t test [(F), (G), (I), (O), and (P)] or log-rank test (K). Data are from ≥3 independent experiments (means ± SD). DAPI, 4′,6-diamidino-2-phenylindole.
Fig 4: Predicted model of the NEK2-mediated sphingolipid synthesis in macrophages.A schematic model is proposed to illustrate how tumor immune surveillance and therapeutic resistance are regulated by NEK2-mediated sphingolipid synthesis in macrophages. Combining the NEK2 inhibitor or S1P antagonist with ICB provides notable antitumor efficacy by reprogramming the TME.
Fig 5: NEK2 deficiency in TAMs sensitizes PD-1–targeted HCC immunotherapy.(A and B) Representative photograph and statistical results of the IVIS imaging system in mice orthotopically implanted with luciferase-expressing Hep-53.4 in Nek2cKO genetic mice in combination with αPD-1 treatment (200 μg per mouse, every 2 days, ip) (n = 7). (C and D) Representative images and statistical results of tumor in Nek2cKO genetic mice in combination with αPD-1 treatment (n = 7). (E) Survival curve of orthotopic tumor implantation in Nek2cKO genetic mice (n = 7). (F) The statistical results of spleen weight of Nek2cKO genetic mice in combination with αPD-1 treatment (n = 7). (G and H) Representative images and statistical results of immune cells in Nek2cKO genetic mice in combination with αPD-1 treatment (n = 7). (I and J) Representative images displaying tumors, tumor weight, and mouse weight of Nek2cKO genetic mice treated with αPD-1(200 μg per mouse, every 2 days, ip) in the HTVi model (n = 5). (K) Survival curve of HTVi model in Nek2cKO genetic mice in combination with αPD-1 (n = 9). (L) Plots of t-distributed stochastic neighbor embedding (tSNE) showing the distinct immune landscape of tumors in the different treatment groups. (M) Proportions of immune cell types in the four treatment groups (n = 5). MHC-II, major histocompatibility complex class II. (N) Flow cytometric analysis and statistical results of functional indication of lymphocytes that have infiltrated the tumors (n = 5). (O) Concentration analysis of S1P by ELISA in tissue lysates and serum from Nek2cKO mice in combination with αPD-1. *P < 0.05, **P < 0.01, and ***P < 0.001. Using a two-tailed, unpaired Student’s t test (O), using one way ANOVA, with post hoc comparisons conducted using Tukey’s test [(B), (D), (F), (H), (J) and (M)] or log-rank test [(E) and (K)]. Data are from ≥3 independent experiments (mean ± SD).
Supplier Page from Abcam for Recombinant human NEK2 protein (His tag N-Terminus)