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. 2023 Dec 26;120(52):e2313693120.
doi: 10.1073/pnas.2313693120. Epub 2023 Dec 20.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer

Affiliations

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer

Songnan Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

Keywords: ENPP1; STING; breast cancer metastasis; extracellular cGAMP; immune checkpoint.

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Conflict of interest statement

Competing interests statement:V.B. and L.L. have filed two patents on ENPP1 inhibitors (PCT/US2020/015968 and PCT/US2018/050018) that are licensed to Angarus Therapeutics. L.L. is a co-founder of Angarus Therapeutics. L.A.G. has filed patents on CRISPR technologies and is a co-founder of Chroma Medicine. All other authors have no additional financial interests.

Figures

Fig. 1.
Fig. 1.
ENPP1’s catalytic activity drives breast tumor growth and metastasis by restricting immune infiltration. (A) Disease-free survival of breast cancer patients in the METABRIC database in the ENPP1-high group (n = 59) and ENPP1-low group (n = 1,926) and the number of patients stratified by stages. Threshold for high vs. low expression was set at which P value was the smallest. (B) ENPP1 expression in patients with stage 1 to 4 breast cancer. Shown as box plots of median and interquartile levels. The P value was determined by the nonparametric Mann-Whitney U test. (C) Primary tumor volumes and quantification of lung metastases of WT BALB/cJ mice bearing ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors (n = 5 and 9 mice). Tumor growth curves were plotted as mean ± SEM. Metastasis data were plotted as mean. P values were determined by the unpaired t test with Welch correction. (D) UMAP plots of the annotated clusters of ENPP1T238A-OE and ENPP1WT-OE 4T1 primary tumors and metastasis colonized lungs. (E) Barplots comparing immune cell compositions (containing C08-C18) between ENPP1T238A-OE and ENPP1WT-OE 4T1 primary tumors and metastasis colonized lungs. *P 0.05; P value is shown if it is between 0.05 and 0.15. See also SI Appendix, Figs. S1 and S2.
Fig. 2.
Fig. 2.
ENPP1’s catalytic activity promotes immune suppression in primary tumors and lung metastases. (AC) Violin plots of indicated transcripts in indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. Arg1 in macrophages in primary tumors and lung metastases (A); Itgae and H2-Ab1 in cDC1s in primary tumors (B); Cd69, Ilr2a, Pdcd1, and Tox in T cells in primary tumors and lung metastases (C). P values were determined by the nonparametric Mann-Whitney U test. cDC1 stands for conventional dendritic cell type 1. See also SI Appendix, Figs. S2 and S3.
Fig. 3.
Fig. 3.
ENPP1 expressed on cancer and responder cells blocks paracrine cGAMP–STING activation. (A) Bar graphs of Cgas and Sting1 expression across the annotated clusters. P values were determined by the nonparametric Mann-Whitney U test. (B) Violin plots of Lrrc8c, Lrrc8a, Lrrc8e, and Ifitm2 across the annotated clusters. Schematic of cGAMP responder cells and their putative transporters: LRRC8A:C in endothelial cells, TAM and cDC; LRRC8A:E in fibroblasts. (CF) Bar graphs of indicated transcripts in indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. Ifitms and Vegfc in endothelial cells in primary tumors (C); Ifitms in macrophages in primary tumors and metastases (D); Ifitms in myCAFs in primary tumors and metastases (E); Enpp1 in myCAFs in primary tumors and metastases (F). P values in C and D were determined by the nonparametric Mann-Whitney U test. (G) Bar graphs of Enpp1 expression across the annotated clusters. (H) Bar graphs of Enpp1 and Ifitms across the annotated macrophage subclusters. P values were determined by the ordinary one-way ANOVA test. (I) Differentially expressed genes in Enpp1-high vs. Enpp1-low groups. Bars represent mean ± SEM. TAM stands for TAMs. cDC stands for conventional dendritic cell, combining both cDC1 and cDC2. myCAF stands for myofibroblastic cancer-associated fibroblast. See also SI Appendix, Figs. S4 and S5.
Fig. 4.
Fig. 4.
The antitumoral and immunostimulatory effect of ENPP1 deficiency is connected to extracellular cGAMP levels. (A) Primary tumor volumes of WT or Enpp1−/− 4T1 BALB/cJ mice orthotopically injected with WT or Enpp1−/− 4T1 (n = 7, 17, 9, 16 mice for Enpp1 KO × KO, KO × WT, WT × KO, and WT × WT cancer × tissue genotype combinations). Data were plotted as mean ± SEM. P values of the last tumor measurement were determined by the multiple unpaired t test with Welch correction. (B) Quantifications of lung metastatic colonies of WT or Enpp1−/− 4T1 BALB/cJ mice intravenously injected with WT or Enpp1−/− 4T1 (n = 4, 5, 3, 3 mice for Enpp1 KO × KO, KO × WT, WT × KO, and WT × WT cancer × tissue genotype combinations). Data were plotted as mean ± SD. P values were determined by the unpaired t test. (C) Schematic of experimental strategies of comparing between wildtype, Enpp1 knockout, and extracellular cGAMP depletion through genetic manipulation and cGAMP neutralization. Structures of dimer ENPP1 (PWD: 4B56), monomer ENPP1 (PWD: 6XKD), mSTING (PWD: 4KCO), and mSTING bound with DMXAA (PWD: 4LOL). (D) Primary tumor volumes of Enpp1 WT mice receiving Enpp1 WT 4T1 and R237A non-binding STING injection (WT + NB) (n = 3 biological replicates), Enpp1 KO mice receiving Enpp1 KO 4T1 and NB STING injection (KO + NB) (n = 4 biological replicates), and Enpp1 KO mice receiving Enpp1 KO 4T1 and WT neutralizing STING injection (KO + Neu) (n = 3 biological replicates). (E) The percentage of Ly6GLy6Clow cells out of MHC-II+CD11b+CD11c macrophages. (F) The geometric mean of pIRF3 of F40/80+CD206 M1-like macrophages. (G) The percentage of MHC-IIhiCD103+ cells out of CD11b-CD11c+ cells. (H and I) The geometric mean of CD69 (H) and CD25 (I) of CD3+CD4CD8+ cytotoxic T lymphocytes (CTLs). (J) The geometric mean of FOXP3 of CD45+CD3hiCD4+CD8 regulatory T cells (Tregs). (D and E) Data were plotted as mean ± SD. P values were determined by the unpaired t test with Welch correction. *P 0.05., **P 0.01; P value is shown if between 0.05 and 0.2; not significant (ns). See also SI Appendix, Fig. S6.
Fig. 5.
Fig. 5.
Selective inhibition of ENPP1’s cGAMP hydrolysis activity abolishes breast cancer metastasis in a STING-dependent manner. (A) Survival of WT, Enpp1H362A, Sting1−/−, and Enpp1H362A × Sting1−/− C57BL/6J mice (n = 27, 32, 10, 15 mice) bearing orthotopic E0771 breast tumors. Survival was measured by time taken for orthotopic E0771 breast tumors to reach 1,000 m3. (B) Tumor-free survival of MMTV and Enpp1H362A × MMTV mice (n = 15 and 18 mice) that developed spontaneous breast tumors. Tumor-free survival was measured by onset of the first spontaneous breast tumors. (C) Representative images and quantification of lung metastatic colonies of WT, Enpp1H362A, Enpp1−/−. Sting1−/− and Enpp1H362A × Sting1−/−C57BL/6J mice (n = 10, 8, 9, 9, 9 mice) intravenously injected with E0771.lmb.PuroR cells. Data are shown as mean. P values comparing the percentage of mice with lung metastasis were determined by the chi-squared test. P values comparing number of colonies were determined by the nonparametric Mann-Whitney U test. (D) Proposed model of mechanism of action in ENPP1 depletion/inhibition. P value for Kaplan–Meier curves were determined by the log-rank Mantel-Cox test. *P 0.05, **P 0.01, ***P 0.001. not significant (ns).
Fig. 6.
Fig. 6.
ENPP1 expression predicts response and outcomes of breast cancer patients receiving anti-PD-1 neoadjuvant therapy. (A and B) ENPP1 expression in Immune+ vs. immune- patients (A) and pCR vs. no pCR patients (B) across 10 treatment arms in the ISPY 2 Trial. P values were determined by the nonparametric Mann-Whitney U test. (C) DMFS of patients in the pembrolizumab arm in the ISPY 2 Trial with high ENPP1 expression (n = 33) vs. low ENPP1 expression (n = 32). P value was determined by the log-rank Mantel-Cox test. Immune+ stands for immune-positive; immune- stands for immune-negative; Carbo stands for Carboplatin; pCR stands for pathological complete response; DMFS stands for distant metastasis-free survival; HR stands for hazard ratio.

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