. 2024 Feb 20;15(1):1532.

doi: 10.1038/s41467-024-46048-7.

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma

Il-Kyu Kim^#^{1

2}, Mark S Diamond^#^{1

2}, Salina Yuan^#^{1

2}, Samantha B Kemp^{1

2}, Benjamin M Kahn^{1

2

3}, Qinglan Li^{4

5}, Jeffrey H Lin^{1

2}, Jinyang Li^{1

2}, Robert J Norgard^{1

2}, Stacy K Thomas^{1

2}, Maria Merolle^{1

2}, Takeshi Katsuda^{1

2}, John W Tobias⁶, Timour Baslan⁷, Katerina Politi^{8

9

10}, Robert H Vonderheide^{11

12

13}, Ben Z Stanger^{14

15

16}

Affiliations

¹ Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
² Abramson Cancer Center and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
³ Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁴ Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁵ Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁶ Penn Genomic Analysis Core, University of Pennsylvania, Philadelphia, PA, USA.
⁷ Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.
⁹ Department of Pathology, Yale School of Medicine, New Haven, CT, USA.
¹⁰ Section of Medical Oncology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA.
¹¹ Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. rhv@upenn.edu.
¹² Abramson Cancer Center and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. rhv@upenn.edu.
¹³ Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA. rhv@upenn.edu.
¹⁴ Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. bstanger@upenn.edu.
¹⁵ Abramson Cancer Center and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. bstanger@upenn.edu.
¹⁶ Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. bstanger@upenn.edu.

^# Contributed equally.

PMID: 38378697
PMCID: PMC10879147
DOI: 10.1038/s41467-024-46048-7

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma

Il-Kyu Kim et al. Nat Commun. 2024.

. 2024 Feb 20;15(1):1532.

doi: 10.1038/s41467-024-46048-7.

Authors

Affiliations

¹ Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
² Abramson Cancer Center and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
³ Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁴ Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁵ Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁶ Penn Genomic Analysis Core, University of Pennsylvania, Philadelphia, PA, USA.
⁷ Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.
⁹ Department of Pathology, Yale School of Medicine, New Haven, CT, USA.
¹⁰ Section of Medical Oncology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA.
¹¹ Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. rhv@upenn.edu.
¹² Abramson Cancer Center and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. rhv@upenn.edu.
¹³ Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA. rhv@upenn.edu.
¹⁴ Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. bstanger@upenn.edu.
¹⁵ Abramson Cancer Center and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. bstanger@upenn.edu.
¹⁶ Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. bstanger@upenn.edu.

^# Contributed equally.

PMID: 38378697
PMCID: PMC10879147
DOI: 10.1038/s41467-024-46048-7

Abstract

Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (Irf6), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.

PubMed Disclaimer

Conflict of interest statement

Dr. Stanger receives research funding from Boehringer-Ingelheim and Revolution Medicines and previously served as a consultant to iTeos Therapeutics. Dr. Vonderheide has received consulting fees from BMS; research funding from Revolution Medicines; is an inventor on patients relating to cancer cellular immunotherapy, cancer vaccines, and KRAS immune epitopes; and receives royalties from Children’s Hospital Boston for a licensed research-only monoclonal antibody. The remaining authors declare no competing interests.

Figures

**Fig. 1. Recurrent PDAC acquires resistance to combination chemoimmunotherapy.**
a, b Mice were injected subcutaneously (s.c.) with 4662 PDAC cells and treated intraperitoneally (i.p.) with control IgG (n = 17) or chemoimmunotherapy (n = 34) (black arrow) consisting of gemcitabine (G), nab-paclitaxel (A), αCD40 agonistic Ab (F), αCTLA-4 Ab (C), and αPD-1 Ab (P). Tumor growth (a) and survival (b) were monitored. c The proportion of treated mice exhibiting no response/early progression, partial response, relapse after complete response (CR), or durable CR is depicted. d Mice with recurrent tumors after CR or near CR were re-treated with GFCP (blue arrows) and tumor size measured (n = 9). e, f Tumor cell lines were generated from s.c. tumors treated with control IgG (‘Ctrl’ lines, n = 4) or from tumors exhibiting early progression (‘EP’ lines, n = 2) or relapse after CR (‘Esc’ lines, n = 8) on chemoimmunotherapy (denoted by blue, green, and red lines in a, respectively). Naïve WT mice were challenged s.c. with these cell lines, and the resulting tumors were treated with control IgG (n = 3 or 4 per line) or GAFCP (n = 5 or 6 per line) (black arrow). Tumor growth (e) and survival (f) were monitored. g Response rates following treatment for each class of tumor cell line in e, f are shown. *** P < 0.0001 by log-rank (Mantel-Cox) test (b, f). Source data are provided as a Source Data file.

**Fig. 2. EMT induces immunotherapy resistance in PDAC.**
a PCA plot of RNA-seq data from parental, EP, and Esc cell lines (triplicates). b Representative bright field (top) and H&E (bottom) images of cultured cells and s.c. implanted tumors on day 18. Scale bars, 250 µm. c GSEA of the EMT Hallmark (Molecular Signature Database) in 4662 parental vs. Esc cell lines. Normalized enrichment score (NES) and false discovery rate (FDR) are shown. Individual tumor growth (d) and survival (e) of mice bearing s.c. implanted 4662 parental empty vector (EV, left) and *Zeb1*/*Snail* OE (right) tumors treated with either control IgG or FCP (arrow) (n = 10). Individual tumor growth (f) and survival (g) of mice bearing s.c. implanted 4662 Esc EV (left) and *Zeb1*^-/-*Snail*^-/- (right) tumors treated with either control IgG or FCP (arrow) (n = 10). SD, stable disease. Clonal 4662 (C7 and C10) and derived Esc (C7.e1 and C10.e1) lines were used for genetic modification, and both lines showed a similar phenotype. h Representative bright field (top) and H&E (bottom) images of in vitro and in vivo (orthotopic) control and Esc tumors following treatment with control IgG and immunotherapy (FCP), respectively. Scale bars, 250 µm. i qPCR of *E-cad*, *Zeb1*, and *Snail* in control and Esc tumor cell lines established from orthotopic tumors (n = 4). Mean tumor diameter over time by ultrasound imaging of orthotopic 4662 EV or *Zeb1/Snail* OE tumors treated with control IgG or FCP (arrow) (j). Bright field dissection images (k) and tumor weights (l) of orthotopic pancreas tumors 6 weeks post tumor implantation (n = 9 for controls and 10 for FCP). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 by log-rank (Mantel-Cox) test (e, g), Student’s t test (i), and one-way ANOVA (j, l). Data represent two independent experiments. Source data and exact P value are provided as a Source Data file.

**Fig. 3. EMT mediates cell-autonomous resistance to direct cytolytic T cell killing.**
Flow cytometric analysis of immune populations in s.c. implanted 4662 parental (P, n = 6) vs. Esc (E1 and E2, n = 4 per line) (a) and 4662 parental EV (n = 5) vs. *Zeb1/Snail* OE (n = 5) tumors (b) on day 18 post inoculation. c Representative αCD3 IHC images (left) and quantitation (right) from s.c. implanted parental and Esc tumors (n = 4 and 6 respectively). Scale bars, 250 µm. Mice that had been transplanted with 4662 parental cells and achieved CR or near CR following combination therapy (black arrows) were treated with control IgG (n = 11), depleting αNK1.1 Ab (n = 10), or αCD4 and αCD8 Abs (n = 13), starting from day 50 (blue arrows) post tumor inoculation, and monitored for tumor recurrence (d). The corresponding survival curves are shown in (e). n.s., non-significant. f Tumor growth of s.c. inoculated OVA-transduced 4662 parental and Esc tumors in NOD/SCID mice, with or without adoptive transfer of activated OVA-specific CD8⁺ T cells (OT-I) on day 14 (arrow) (n = 6 for parental and 8 for the other groups). g OVA-tdTomato⁺ 4662 parental and Esc tumors were co-cultured with non-activated or activated OT-I by αCD3 and αCD28 Abs overnight, at indicated tumor to effector (T:E) ratios. Each dot represents biological replicates. Two days later, AnnexinV and 7-AAD expression on tumor cells were determined by flow cytometry. h OVA⁺ 4662 parental and Esc tumors were additionally transduced with YFP or CFP expression constructs, plated separately or mixed, and co-cultured with activated OT-I. AnnexinV and 7-AAD on each tumor were measured 2 d after co-culture. The percentages of 7-AAD⁺ cells on co-cultured OVA⁺ 4662 parental EV vs. *Zeb1*/*Snail* OE tumors (i) and 4662 Esc EV vs. *Zeb1*^-/-*Snail*^-/- tumors (j) with or without activated OT-I for 2 d (n = 3). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 by Student’s t test (a, b, g–j), Mann-Whitney t test (c), log-rank (Mantel-Cox) test (e), and one-way ANOVA (f). Data represent two independent experiments. Source data and exact P value are provided as a Source Data file.

**Fig. 4. Transcriptional and chromatin profiling identifies Irf6 as a potential regulator of acquired immunotherapy resistance.**
a Venn diagram of HOMER de novo motifs identified in chromatin regions significantly enriched in EV vs. *Zeb1/Snail* OE (ZS) cell lines that have (right) or have not (left) been co-cultured with OT-1 cells (duplicates). Significantly enriched chromatin regions were defined as |log2fold change| > 1.4 and p value < 0.05 after DESeq2 analysis. b Genome browser track showing ATAC-seq reads along the *Irf6* gene, comparing EV vs. *Zeb1/Snail* OE cell lines. c Aggregate plots comparing the average ATAC signal of EV (blue) and *Zeb1/Snail* OE (red) tumors around putative *Irf6* promoter sequences. For details, see **Methods**. d Transcripts per million (TPMs) of *Irf6* in EV vs. *Zeb1/Snail* OE tumors that have (right) or have not (left) been co-cultured with OT-1 cells (duplicates). e Boxplots (log2fold) showing the expression of *Irf6*-associated genes with differentially open chromatin in parental EV (left, n = 470 genes) vs. *Zeb1/Snail* OE (right, n = 173 genes) tumors. Centre and whiskers represent means and 2.5-97.5 percentiles. f ChIP with control IgG or antibodies to ZEB1 and SNAIL in DNA from 4662 parental EV and *Zeb1/Snail* OE (ZS) tumor cells, followed by qPCR quantification in enriched DNA using primers for *Irf6* putative promoter and distal exon 6 regions. Data represent two independent experiments. g Representative IHC images of IRF6 in the original primary tumor tissues from control, EP, and Esc tumor-bearing mice. Scale bars, 250 µm. h GSEA plots of an *Irf6*-dependent gene signature (derived by comparing *Irf6*-expressing tumors to controls; provided in Supplementary Data 2) in parental vs. Esc (left) (triplicates) and parental EV vs. *Zeb1/Snail* OE (right) (duplicates) tumors. i GSEA of gene signatures derived from human PDAC cells that highly express *IRF6* in parental vs. Esc (left) and EV vs *Zeb1/Snail* OE tumors (right). Negative normalized enrichment scores (NES) demonstrate enrichment in parental and EV tumors compared to Esc and *Zeb1/Snail* OE tumors, respectively. Data are presented as mean ± SEM unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.0001 by Student’s t test (d, e) and one-way ANOVA (f). Source data and exact P value are provided as a Source Data file.

**Fig. 5. Irf6 loss contributes to EMT-induced immunotherapy resistance.**
a OVA-tdTomato⁺ 4662 parental EV, Esc EV, and Esc *Irf6* tumors were co-cultured with or without activated OT-I at the indicated 4662 tumor cell-to-OT-I ratios for 2 d. Tumor cell expression of 7-AAD expression was measured by flow cytometry. b OVA-tdTomato⁺ 4662 parental EV and *Irf6* KO tumors were used as target cells (1:5 ratio). For (a, b) n = 4 for tumor alone and 6 for co-cultures. Tumor growth (c) and survival (d) of mice bearing 4662 Esc EV and Esc *Irf6* tumors treated with control IgG or FCP (arrow) (n = 10). Data represent two independent experiments. Tumor growth (e) and survival (f) of mice bearing 4662 parental EV and parental *Irf6* KO tumors treated with control IgG or FCP (arrow) (n = 8). g TPMs of *IRF6* in 7 treatment-paired NSCLC patient samples (ref. ). All patients demonstrated initial response to combinatorial checkpoint blockade (anti-PD-1/PD-L1±anti-CTLA-4 Ab) before relapsing. Lines are drawn from patient-matched early treatment (Pre-ICB) to immunotherapy recurrence (IR). Red lines indicate 3 patient samples demonstrating a decrease in *IRF6* expression with recurrence. Blue lines indicate the others demonstrating unchanged or increased *IRF6* expression with recurrence. GSEA plots of an *Irf6*-dependent gene signature derived from *Irf6*-expressing 4662 tumors (h) and the EMT Hallmark (i) in patient-matched Pre-ICB vs. IR samples separately assorted based on *IRF6* expression as in (g). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 by Student’s t test (a, b) and log-rank (Mantel-Cox) test (d, f). Source data and exact P value are provided as a Source Data file.

**Fig. 6. Irf6 promotes susceptibility to T cell killing by enhancing TNF-induced apoptosis.**
a Normalized viability of 4662 parental EV, Esc EV, and Esc *Irf6* tumors with varying concentrations of TNF-α in the presence of IFN-γ (0.2 μg/ml) plus cycloheximide (1 μg/ml) for 48 h (n = 4). IC₅₀ values are 0.03472 ug/ml for parental EV, 0.6494 ug/ml for Esc *Irf6*, and not determined for Esc EV tumors. Mean fluorescence intensities (MFIs) or percentages of cleaved caspase-3 in 4662 parental EV, Esc EV, and Esc *Irf6* tumors treated with or without TNF-α (0.5 μg/ml) plus IFN-γ in the presence of cycloheximide by flow cytometry (n = 3) (b) or in s.c. implanted YFP⁺ those tumors with or without immunotherapy by IF staining. Each dot represents biological replicates (c). d Normalized viability of 4662 Esc EV and Esc *Irf6* tumors, treated with vehicle or z-VAD (20 μM), in response to TNF-α plus IFN-γ in the presence of cycloheximide (n = 3). e Left, immunoblots of IRF6 and TNF-related cell death mediators in 4662 Esc EV and Esc *Irf6* cells with or without ablation of each gene. Right, normalized viability of 4662 Esc EV and Esc *Irf6* tumors with or without indicated gene ablation in response to TNF-α plus IFN-γ in the presence of cycloheximide (n = 3). The percentages of 7-AAD⁺ cells among OVA-tdTomato⁺ 4662 Esc EV and Esc *Irf6* cells with or without the indicated gene ablation (f), or the same cells with or without TNF-α neutralizing Ab (5 μg/ml) (g), or OVA-tdTomato⁺ 4662 parental and Esc cells in the presence of vehicle or birinapant (5 μM) (h), co-cultured with or without activated OT-I for 2 d. n = 3 for tumor alone and 4 for co-cultures. Tumor growth (i) and survival (j) of mice bearing 4662 parental EV and *Casp8* KO tumors treated with control IgG or FCP (arrow) (n = 10). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 by Student’s t test (b–d), one-way ANOVA (e–h), and log-rank (Mantel-Cox) test (j). Data represent two independent experiments. Source data and exact P value are provided as a Source Data file.

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Update of

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy.
Kim IK, Diamond M, Yuan S, Kemp S, Li Q, Lin J, Li J, Norgard R, Thomas S, Merolle M, Katsuda T, Tobias J, Politi K, Vonderheide R, Stanger B. Kim IK, et al. Res Sq [Preprint]. 2023 Jun 1:rs.3.rs-2960521. doi: 10.21203/rs.3.rs-2960521/v1. Res Sq. 2023. Update in: Nat Commun. 2024 Feb 20;15(1):1532. doi: 10.1038/s41467-024-46048-7. PMID: 37398248 Free PMC article. Updated. Preprint.

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Musiu C, Lupo F, Agostini A, Lionetto G, Bevere M, Paiella S, Carbone C, Corbo V, Ugel S, De Sanctis F. Musiu C, et al. Front Immunol. 2024 May 16;15:1341079. doi: 10.3389/fimmu.2024.1341079. eCollection 2024. Front Immunol. 2024. PMID: 38817612 Free PMC article. Review.

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Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma

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Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma

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