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EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

Nature Cancer volume 4, pages 872–892 (2023)Cite this article

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Abstract

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.

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Fig. 1: NK cell immunity induced in the lung but not the pancreas TME after therapy-induced senescence.
Fig. 2: The proinflammatory SASP is transcriptionally and epigenetically repressed in the pancreas TME.
Fig. 3: SMA+ fibroblasts constrain SASP-mediated NK cell and T cell immunity in the pancreas TME.
Fig. 4: Targeting EZH2 expression or its methyltransferase activity reactivates the proinflammatory SASP in PDAC.
Fig. 5: EZH2 blockade activates NK cell and T cell-mediated, long-term tumor control after therapy-induced senescence in PDAC models.
Fig. 6: EZH2 suppression reinstates SASP-associated chemokines to drive NK cell and T cell accumulation in PDAC.
Fig. 7: Pharmacological EZH2 methyltransferase inhibition in combination with T/P reactivates cytotoxic NK cell and T cell immunity and enhances tumor control in preclinical PDAC models.
Fig. 8: EZH2 is associated with suppression of inflammatory chemokine signaling, reduced NK cell and T cell immune surveillance and poor survival in PDAC patients.

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Data availability

RNA-seq and CUT&Tag data that support the findings of the present study have been deposited in the GEO under accession nos. GSE141684, GSE201495 and GSE203623. Datasets derived from this resource that support the findings of the present study are available in Supplementary Tables 1–46. Gene expression data for human LUAD and PDAC cell lines treated with T/P were obtained under accession no. GSE110397. Gene expression data from 145 primary human PDAC specimens were obtained under accession no. GSE71729. Source data are provided with this paper. All other data supporting the findings of the present study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank K. Hatzi for providing shRNA constructs, R. Mezzadra for generating Ccl2 O/E cell lines, G. Cottle for technical assistance, C. Baer and C. Hung in the Sanderson Center for Optical Experimentation (SCOPE) facility (RRID:SCR_022721) and Y. Liu in the Morphology Core at UMass Chan Medical School for assistance with IHC analysis and quantification, J. Pitarresi for assistance with coimmunofluorescence staining and analysis, and W. Xue, A. Mercurio, M. Kelliher, M. Green, J. Chuprin, L. Zhou and other members of the Ruscetti laboratory for helpful suggestions and comments on the manuscript. Graphics in Figs. 1a–c, 2a, 3a and 5a were created with BioRender.com. This work was supported by a K99/R00 CA241110 grant from the National Cancer Institute to M.R. and a Memorial Sloan Kettering Cancer Center Support grant (no. P30 CA008748) to S.W.L. We acknowledge support from Our Danny Cancer Fund (no. U6035343000000W to L.C.), the National Institutes of Health (grant nos. R01 HD072122 to T.G.F. and P30 CA008748 S5 to E.d.S.) and the National Center for Advancing Translational Sciences (grant no. UL1-TR001453 to K.S.). S.W.L. is the Geoffrey Beene Chair for Cancer Biology and a Howard Hughes Medical Institute investigator.

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Authors and Affiliations

  1. Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Loretah Chibaya, Katherine C. Murphy, Kelly D. DeMarco, Sneha Gopalan, Haibo Liu, Chaitanya N. Parikh, Yvette Lopez-Diaz, Junhui Li, Sachliv K. Chana, Karl Simin, Lihua Julie Zhu, Thomas G. Fazzio & Marcus Ruscetti

  2. Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Melissa Faulkner & Lihua Julie Zhu

  3. Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan & Scott W. Lowe

  4. Department of Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Amanda Kulick & Elisa de Stanchina

  5. Program in Bioinformatics and Integrative Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Lihua Julie Zhu

  6. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Scott W. Lowe

  7. Immunology and Microbiology Program, University of Massachusetts Medical Chan School, Worcester, MA, USA

    Marcus Ruscetti

  8. Cancer Center, University of Massachusetts Medical Chan School, Worcester, MA, USA

    Marcus Ruscetti

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  1. Loretah Chibaya
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Contributions

L.C. and M.R. conceived the study, designed and performed experiments, interpreted results and wrote the paper with assistance from all authors. K.C.M., K.D.D., Y.L.-D., J.P.M. and S.C. designed, performed and analyzed in vitro and in vivo experiments. K.C.M., K.D.D., C.N.P., Y.L.-D., J.S., W.L., A.K. and E.d.S. produced and treated animal models. H.L., J.L., Y.-j.H. and L.J.Z. analyzed transcriptomic datasets. S.G., T.G.F., H.L. and L.J.Z. designed and performed CUT&Tag analysis. M.F. and K.S. provided human PDAC patient specimens and data. M.R. and S.W.L. supervised the study.

Corresponding authors

Correspondence to Scott W. Lowe or Marcus Ruscetti.

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Competing interests

S.W.L. is a founder and member of the scientific advisory board of Blueprint Medicines, Mirimus Inc., ORIC Pharmaceuticals, Senescea and Faeth Therapeutics, is on the scientific advisory board of PMV Pharmaceuticals, and is a consultant for Fate Therapeutics. M.R. is a consultant for Boehringer Ingelheim. L.C. and M.R. have filed a US patent application (US22/45163) related to this work. The remaining authors declare no competing interests.

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Nature Cancer thanks Manuel Collado and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 T/P treatment induces cellular senescence across tumor conditions in vivo.

a, Representative Hematoxylin and eosin (H&E) (top) and Masson’s trichrome (bottom) staining of indicated KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD (LIL, LIP, LILiver) derived-tumors grown in different organs from 2-3 independent experiments. Scale bars, 100μm. b, Immunohistochemical (IHC) staining of indicated KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD (LIL, LIP, LILiver) derived-tumors treated with vehicle (V) or combined trametinib (1 mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Quantification of the percentage of SA-β-gal+ area and the number of Ki67+ and pRb+ cells per field are shown inset (n = 2-4 independent tumors per group). Scale bar, 50μm. c, Immunofluorescence staining of indicated KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD (LIL, LIP, LILiver) derived-tumors treated as in (b). Quantification of the percentage of GFP+ (green) tumor cells expressing p21 (cyan) is shown inset (n = 2-4 independent tumors per group). Scale bar, 50μm. d, GFP+ tumor cells were FACS sorted from indicated tumors and extracted RNA subjected to RNA-seq analysis (n = 2-4 independent samples per group). Gene Set Enrichment Analysis (GSEA) of RNA-seq data using an established senescence gene set is shown. NES, normalized enrichment score. P values in d were calculated using two-sided, Kolmogorov-Smirnov test. Error bars, mean ± SEM. IHC experiments were repeated at least twice and representative images are shown.

Source data

Extended Data Fig. 2 Suppression of NK immunity specific to pancreas TME following T/P-induced senescence.

a-b, KPC2 PDAC or KP2 LUAD tumor cells expressing GFP were injected i.v. or orthotopically into the pancreas of 8-12 week old C57BL/6 female mice. Following tumor formation, mice were treated with vehicle (V) or combined trametinib (1 mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Flow cytometry analysis of NK cell numbers and degranulation in PDAC (PIP, PIL) (a) and LUAD-derived tumors (LIL, LIP) (b) are shown (PIP V, n = 3; PIP T/P, n = 4; PIL V and PIL T/P, n = 8; LIL V, n = 6; LIL T/P, n = 7; LIP V and T/P, n = 9 mice). c, Flow cytometry analysis of NK cell numbers and degranulation in spleens of mice with KPC1-derived PIP tumors treated as in (a) (n = 5 mice per group). d, Kaplan-Meier survival curve of mice with KPC2-derived PIP tumors treated with vehicle, combined trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or depleting antibodies against NK1.1 (PK136; 250 μg) or CD8 (2.43; 200 μg) (V, n = 5; T/P, T/P + αNK1.1 and T/P + αCD8, n = 8 mice). e, IVIS images showing luciferase signaling in KPC1-derived PIL tumors following treatment as in (a). Right, quantification of total luminescence in the thoracic region (V, n = 5; T/P and T/P + αNK1.1, n = 8 mice). f, Waterfall plot of the response of KPC1-derived PIP tumors following 2 week treatment with vehicle, combined trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or an NK1.1 depleting antibody (PK136; 250 μg) (V and T/P, n = 5; T/P + αNK1.1, n = 6 mice). g, Waterfall plot of the response of KPC2-derived PIP tumors following 2 week treatment with vehicle, combined trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or an NK1.1 (PK136; 250 μg) or CD8 (2.43; 200 μg) depleting antibody (V, n = 5; T/P, n = 7; T/P + αNK1.1 and T/P + αCD8, n = 8 mice). h, Kaplan-Meier survival curve of mice with KPC1-derived PIL tumors treated with vehicle, combined trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or depleting antibodies against CD8 (2.43; 200 μg) or CD4 (GK1.5; 200 μg) (V, n = 5; T/P, T/P + αCD4 and T/P + αCD8, n = 7 mice). i, Flow cytometry analysis of CD4+ and CD8+ T cell numbers and degranulation in KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD-derived tumors (LIL, LIP, LILiver) grown in different organs and treated as in (a) (PIP V and T/P, n = 5 or 10; PIL V, n = 5 or 9; PIL T/P, n = 5 or 10; LIL V, n = 3; LIL T/P, n = 5; LIP V, n = 5 or 13; LIP T/P, n = 6 or 15; PILiver V, n = 9; PILiver T/P, n = 10; LILiver V, n = 8; LILiver T/P, n = 10 mice). Data represents pool of 3 independent experiments. P values in a-c, e-g, and i were calculated using two-tailed, unpaired Student’s t-test, and those in d and h were calculated using log-rank test. Error bars, mean ± SEM.

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Extended Data Fig. 3 Repression of pro-inflammatory SASP gene expression specific to the pancreas TME following T/P treatment.

a, Heatmaps showing fold change in IFNα (left), IL-12 (middle), and TNFα pathway genes (right) following T/P treatment in indicated tumor settings from RNA-seq data in Fig. 2a (n = 2-4 independent samples per group). b, IHC staining of indicated KPC1 PDAC (PIP, PIL) and KP1 LUAD (LIL, LIP) derived-tumors grown in different organs and treated with vehicle (V) or combined trametinib (1 mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. H-score quantification of CCL2 and CXCL10 staining intensity is shown inset (n = 2-3 independent tumors per group). Scale bars, 50μm. Error bars, mean + SEM. c, Transcription factor enrichment analysis showing transcriptional regulators of targets differentially expressed in tumors in the lungs (LIL, PIL) following T/P treatment. IHC experiments were repeated at least twice and representative images are shown.

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Extended Data Fig. 4 Tumors in the pancreas TME are enriched for H3K27me3 repressive chromatin marks at SASP gene loci.

a, Heatmaps of normalized genome-wide H3K27me3 signaling intensities of consensus peaks from CUT&Tag analysis of KPC1 cells treated with vehicle or trametinib (25 nM) and palbociclib (500 nM) in vitro for 8 days, or KPC1 cells FACS sorted from transplanted PDAC tumors in C57BL/6 female mice treated with vehicle or trametinib (1 mg/kg) and palbociclib (100 mg/kg) for 2 weeks (n = 2-4 independent samples per group). b, Normalized H3K27me3 peak intensities of 87 SASP genes (see Table 47) from CUT&Tag analysis samples in (a) (n = 2-4 per independent samples group). c, Genome browser tracks showing H3K27me3 occupancy at pro-inflammatory SASP gene loci from CUT&Tag analysis samples in (a) (n = 2-4 independent samples per group).

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Extended Data Fig. 5 PDAC and LUAD tumor cells have a similar pro-inflammatory SASP response to T/P-induced senescence in vitro.

a, Cytokine array analysis of pro-inflammatory SASP factors in murine PDAC and LUAD cell lines treated with vehicle or combined trametinib (25 nM) and palbociclib (500 nM) for 8 days (n = 2 independent samples per group). #, outside the detectable limit. b, Normalized expression levels of pro-inflammatory SASP genes in human PDAC and LUAD cell lines following treatment as in (a) from analysis of RNA-seq data generated in Ruscetti et al. 201815 (n = 2 independent samples per group).

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Extended Data Fig. 6 Suppression of EZH2-mediated H3K27me3 induces pro-inflammatory SASP and immunomodulatory cell surface molecules following T/P treatment in PDAC cells.

a, Representative clonogenic assay images (from 3 biological replicates) of KPC1 cells harboring shRen or shEzh2 shRNAs replated in the absence of drugs after 8-day pre-treatment with vehicle or combined trametinib (25 nM) and palbociclib (500 nM) (top). Bottom, representative SA-β-gal staining (from 3 biological replicates) of KPC1 cells harboring shRen or shEzh2 shRNAs and treated as above for 8 days. Scale bar, 50μm. Experiments were repeated at least twice with similar results. b, RT-qPCR analysis of senescence and SASP gene expression in KPC1 cells harboring shRen or shEzh2 shRNAs treated as in (a) (n = 3 independent samples per group). A.U., arbitrary units. c, Representative histograms (top) and quantification of mean fluorescent intensity (MFI) of MHC-I (H-2kb) expression (bottom) on KPC1 cells harboring shRen or shEzh2 shRNAs (left) (n = 3 independent samples per group) or parental KPC1 cells (right) treated with vehicle, combined trametinib (25 nM) and palbociclib (500 nM), and/or tazemetostat (5 μM) for 8 days (n = 6 independent samples per group). Experiments were repeated at least twice with similar results. d, Representative clonogenic assay images (from 3 biological replicates) of KPC1 cells replated in the absence of drugs after 8-day pre-treatment with vehicle, combined trametinib (25 nM) and palbociclib (500 nM), and/or tazemetostat (5 μM) (top). Bottom, representative SA-β-gal staining (from 3 biological replicates) of KPC1 cells treated as above for 8 days. Scale bar, 50μm. Experiments were repeated at least twice with similar results. e, RT-qPCR analysis of SASP gene expression in human PANC-1 PDAC cells treated with vehicle, trametinib (25 nM), palbociclib (500 nM), and/or GSK126 (1 μM) for 8 days (n = 3 independent samples per group). A.U., arbitrary units. f, Heatmaps of normalized genome-wide H3K27me3 signaling intensities from CUT&Tag analysis of KPC1 cells harboring Ren or Ezh2 shRNAs treated with vehicle or trametinib (25 nM) and palbociclib (500 nM) for 8 days (n = 2-4 independent samples per group). g, Genome browser tracks showing H3K27me3 occupancy at pro-angiogenic SASP gene loci. P values in b, c, and e were calculated using two-tailed, unpaired Student’s t-test. Error bars, mean ± SEM.

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Extended Data Fig. 7 EZH2 knockdown in the KPC2 PDAC orthotopic transplant model potentiates anti-tumor NK and CD8+ T cell immunity and long-term tumor regressions following T/P treatment.

a, Ultrasound quantification of initial PDAC tumor volume 1-week post-transplantation of KPC1 or KPC2 cells harboring shRen or shEzh2 shRNAs into 8-12 week old C57BL/6 female mice prior to enrollment in treatment cohorts (KPC1 shRen, n = 18; KPC1 shEzh2, n = 75; KPC2 shRen, n = 21; KPC2 shEzh2, n = 20 mice). Data represents pool of 6 independent experiments. b, Western blots of shRen or shEzh2 KPC1 orthotopic PDAC tumors treated with vehicle or trametinib (1 mg/kg) and palbociclib (100 mg/kg) for 2 weeks. c, IHC staining of KPC1 and KPC2 orthotopic PDAC tumors harboring shRen or shEzh2 shRNAs treated as in (b). H-score quantification of EZH2 expression is shown inset (n = 2-3 independent tumors per group). Scale bars, 50μm. d-e, Flow cytometry analysis of NK (d) and T cell (e) numbers and activation markers in KPC2 orthotopic PDAC tumors harboring indicated shRNAs treated as in (b) (shRen V, n = 6; shRen T/P, n = 7; shEzh2 V, n = 5; shEzh2 T/P, n = 7 mice). f, Flow cytometry analysis of F4/80+ macrophages in KPC1 orthotopic PDAC tumors harboring indicated shRNAs treated as in (b) (shRen V, n = 9; shRen T/P, n = 8; shEzh2 V, n = 6; shEzh2 T/P, n = 9 mice). Data represents pool of 2 independent experiments. g, Waterfall plot of the response of KPC2 orthotopic PDAC tumors harboring indicated shRNAs to treatment as in (b) (shRen V, n = 9; shRen T/P, n = 12; shEzh2 V, n = 7; shEzh2 T/P, n = 13 mice). Data represents pool of 2 independent experiments. h, Kaplan-Meier survival curve of mice with shEzh2 KPC2 orthotopic PDAC tumors treated with vehicle, combined trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or depleting antibodies against NK1.1 (PK136; 250 μg) or CD8 (2.43; 200 μg) (V, n = 5; T/P, T/P + αNK1.1, and T/P + αCD8, n = 8 mice). Dotted line indicates timepoint when mice were taken off of treatment. P values in a were calculated using One-way ANOVA followed by Tukey’s multiple comparison test, d-g using two-tailed, unpaired Student’s t-test, and h using log-rank test. Error bars, mean ± SEM. IHC staining and western blots were repeated at least twice and representative images are shown.

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Extended Data Fig. 8 Combined EZH2 knockdown and T/P treatment reduces pancreatic metastasis growth and enhances NK and T cell immune surveillance in the lung.

a, KPC1 PDAC cells harboring shRen or shEzh2 shRNAs were injected i.v. into 8-12 week old C57BL/6 female mice. Following tumor formation in the lungs, mice were treated with vehicle (V) or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) (T/P) for 2 weeks. Quantification of lung tumor burden after 2 weeks of treatment is shown (shRen V and T/P, n = 4; shEzh2 V, n = 3; shEzh2 T/P, n = 5 mice). b-h, Flow cytometry analysis of total CD45+ immune cells (b), F4/80+ macrophages (c), NK cells (d), GZMB+ NK cells (e), CD4+ T cells (f), CD8+ T cells (g), and GZMB+ CD8+ T cells (h) in shRen or shEzh2 KPC1 PDAC tumors in the lung following treatment as in (a) (shRen V, n = 6; shRen T/P, n = 7; shEzh2 V, n = 8; shEzh2 T/P, n = 9 mice). P values in a-h were calculated using two-tailed, unpaired Student’s t-test. Error bars, mean ± SEM.

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Extended Data Fig. 9 EZH2 blockade reduces T/P-induced blood vessel formation and promotes CCL2 and CXCL9/10 secretion that increases NK and CD8+ T cell infiltration into PDAC.

a, IHC staining of KPC1 orthotopic PDAC tumors harboring shRen or shEzh2 shRNAs from mice treated with vehicle or combined trametinib (1 mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Quantification of blood vessels per field are shown in the inset (n = 2-4 independent tumors per group). Scale bar, 50μm. b-c, Flow cytometry analysis of NK cell activation markers (b) and CD4+ and CD8+ T cell numbers (c) in KPC1 orthotopic PDAC tumors expressing control Empty or Ccl2 vectors and treated as in (a) (Empty V, n = 2 or 4; Empty T/P, n = 4 or 9; Ccl2 O/E V, n = 6 or 11; Ccl2 O/E T/P, n = 7 or 12 mice). Data represents pool of 3 independent experiments d, Flow cytometry analysis of CD4+ and CD8+ T cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or a CCL2 depleting antibody (2H5; 200 μg) for 2 weeks (V and αCCL2, n = 3; T/P, n = 7; T/P + αCCL2, n = 9 mice). Data represents pool of 2 independent experiments. e, Flow cytometry analysis of NK cell numbers in shEzh2 KPC1 orthotopic PDAC tumors following treatment with vehicle, combined trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or a CXCR3 depleting antibody (CXCR3−173; 200 μg) for 2 weeks (V and T/P, n = 8; αCXCR3, n = 5; T/P + αCXCR3, n = 12 mice). Data represents pool of 2 independent experiments. P values in a-e were calculated using two-tailed, unpaired Student’s t-test. Error bars, mean ± SEM. IHC experiments were repeated at least twice and representative images are shown.

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Supplementary information

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Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Supplementary Tables 1–49.

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Source Data Figs. 1–8 and Extended Data Fig. 1–9

Statistical Source Data for Figs. 1–8 and Extended Data Figs. 1–9.

Source Data Figs. 4, 7 and Extended Data Fig. 7

Unprocessed western blots for Figs. 4 and 7 and Extended Data Fig. 7.

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Chibaya, L., Murphy, K.C., DeMarco, K.D. et al. EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance. Nat Cancer 4, 872–892 (2023). https://doi.org/10.1038/s43018-023-00553-8

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