Repression of the Type I Interferon Pathway Underlies MYC- and KRAS-Dependent Evasion of NK and B Cells in Pancreatic Ductal Adenocarcinoma

Abstract

MYC is implicated in the development and progression of pancreatic cancer, yet the precise level of MYC deregulation required to contribute to tumor development has been difficult to define. We used modestly elevated expression of human MYC, driven from the Rosa26 locus, to investigate the pancreatic phenotypes arising in mice from an approximation of MYC trisomy. We show that this level of MYC alone suffices to drive pancreatic neuroendocrine tumors, and to accelerate progression of KRAS-initiated precursor lesions to metastatic pancreatic ductal adenocarcinoma (PDAC). Our phenotype exposed suppression of the type I interferon (IFN) pathway by the combined actions of MYC and KRAS, and we present evidence of repressive MYC-MIZ1 complexes binding directly to the promoters of the genes encodiing the type I IFN regulators IRF5, IRF7, STAT1, and STAT2. Derepression of IFN regulator genes allows pancreatic tumor infiltration by B and natural killer (NK) cells, resulting in increased survival. SIGNIFICANCE: We define herein a novel mechanism of evasion of NK cell-mediated immunity through the combined actions of endogenously expressed mutant KRAS and modestly deregulated expression of MYC, via suppression of the type I IFN pathway. Restoration of IFN signaling may improve outcomes for patients with PDAC.This article is highlighted in the In This Issue feature, p. 747.

Figure 1. Rosa26-driven MYC is expressed at near physiological levels

A) RT-PCR comparison of Rosa26-driven MYC with endogenously expressed c-Myc in Rosa26^wt/wt, Rosa26^DM-lsl-MYC/+ and Rosa26^{DM-lsl-MYC/lsl-MYC} MEFs, 24hrs after infection with Adeno-Cre. N=3, * denotes p<0.05; ANOVA and post-hoc Tukey test. ND = not detected. B) FACS analysis of Annexin V (AV), propidium iodide (PI) labelling of MEFs, infected as per (A) with Adeno-Cre or Adeno-LacZ and cultured overnight in 0.2% FBS. N=3, ** denotes p<0.01; ANOVA with post-hoc Tukey test. C) Immunoblot of MYC expression in wild-type, Rosa26^DM-lsl-MYC/+, lsl-KRas^G12D and Rosa26^DM-lsl-MYC/+;lsl-KRas^G12D (double positive) MEFs 24 hrs after Adeno-Cre infection. Image is representative of >3 experiments.

Figure 2. Pancreatic cancer phenotypes induced by activation of Rosa26-driven MYC with and without KRas^G12D

A) Schematic of alleles used in panels B-D. B) Overall survival of MC (N=9) and M²C (N=11) mice. Mantel Cox logrank test (panels B, C, F). C) Overall survival of KC (N=65) versus MC (from (B)) and KMC (N=19) mice. Black hash marks indicate mice that were euthanised for reasons unrelated to pancreatic cancer. D) Representative images show tumour histology (H&E) and immunohistochemical detection of total MYC and KRas^G12D expression in end-stage tumours of mice of the indicated genotypes. Scale bar = 100μm. E) Schematic of alleles used in panels F-H. F) Overall survival of MC^ER (N=5), M²C^ER (N=6) and KMC^ER (N=10) mice measured in days post induction with Tamoxifen. G) Representative H&E images of ductal tumour progression in KMC^ER mice. Scale bars = 100μm. H) Top panels show Licor PEARL fluorescent imaging of IRFP-expressing metastases in KMC^ER mice. Left panels show liver metastases, right panels show diaphragm metastases. Lower panels show H&E and IHC for pan-cytokeratin and synaptophysin. Scale Bars = 100μm.

Figure 3. MYC expression levels modulate effector immune cell infiltration in PDAC

A) Genetic deletion of endogenous c-Myc in Rosa26^DM-lsl-MYC;lsl-KRas^G12D MEFs. Lysates were prepared 24hrs after Adeno-Cre infection. Representative of 2 independent experiments. B) Schematic of alleles used in panels C-G. C) Overall survival of KMC mice with 0 (N=19, from Fig. 2C), 1 (N=13) or 2 (N=8) copies of c-Myc^fl. Mantel Cox Logrank test. D) Normalised RNA-SEQ reads of indicated B cell markers in KC (N=6) and KMC (N=6) pancreatic tumours. Note that samples with zero reads are absent from log-scale graphics (pertains only to KMC samples). Adjusted P values were generated in R (panels D, E, H). E) Normalised RNA-SEQ reads of indicated NK cell markers in KC (N=6) and KMC (N=6) pancreatic tumours. F) Quantification of tumour infiltrating NK (NKp46⁺) and B (CD45R⁺) cells in KC (N=7), KMC (N=10) and KMC-cMyc^fl/fl (N=9 & 12, respectively) pancreatic tumours. Kruskal-Wallis and Dunn’s multiple comparison test. G) Representative images of IHC staining for NKp46⁺ NK cells (upper panels) and CD45R⁺ B cells (lower panels), as per (F). Scale bars = 100μm. H) RNA-SEQ reads of NK cell killer-type lectins in KMC (N=6) versus KMC-Myc^fl/+ (N=4) tumours. For all panels, ns = not significant; *** denotes p<0.001, **denotes p<0.01 and * denotes p<0.05.

Figure 4. MYC and KRas^G12D suppress the Type I Interferon pathway

A) RNA-SEQ analysis, of indicated gene expression in Rosa26^wt/wt (WT), Rosa26^DM-lsl-MYC/+(MYC), lsl-KRas^G12D (KRAS) and Rosa26^DM-lsl-MYC/+;lsl-KRas^G12D (MYC & KRAS) MEFs, 24hrs after infection with Adeno-Cre. Adjusted P values were generated in R (panels A, C). B) Heatmap of significant gene expression changes induced by depletion of MYC and KRas in KMC-derived cultured PDAC cells compared to non-targeting control, analysed by RNA-SEQ. N=4 biological replicates. C) RNA-SEQ analysis of the indicated genes upon depletion of MYC or KRas in KMC PDAC cells, as per (B). D) Reduced expression of MYC in human PDAC cells treated with Trametinib (T), compared with DMSO vehicle (D). Representative of 3 individual experiments. E) RT-PCR analysis of Interferon-related gene expression in human PDAC cell lines treated +/- Trametinib. Mean and SEM shown for N=3 biological replicates. For all panels, *** denotes p<0.001, **denotes p<0.01, * denotes p<0.05, ns = not significant.

Figure 5. Transcriptional repression of the Type I Interferon pathway by MYC/Miz1

A) Heatmap of gene expression changes induced by RNAi-mediated depletion of MYC or Miz1 in KMC-derived cultured PDAC cells compared to non-targeting (NT) control, analysed by RNA-SEQ. N=4 biological replicates. B) RT-PCR analysis of Interferon-related gene expression in KMC tumour cells upon siRNA mediated depletion of Miz1 compared to non-targeting control. Mean & SEM of 3 independent experiments shown. T test. C) Chromatin-IP analysis of MYC and Miz1 binding to the promoters of the indicated genes in human DAN-G PDAC cells compared with a known MYC/Miz1 target, VAMP4. Mean and SEM of technical replicates from 1 of 3 independent experiments shown. ANOVA and Tukey’s multiple comparison test. D) Re-CHIP analysis of Miz1 binding to anti-MYC-precipitated promoter regions. Mean and SEM of technical replicates from 1 of 3 independent experiments shown. T test. E) Schematic of alleles used in panels F-J. F) Overall survival of KMC (N=19, from Fig. 2C) and KMC-Miz1^fl/fl (N=12) mice. *** denotes p<0.001; Mantel Cox Logrank test (panels F, J). G) Quantification of tumour infiltrating NK (NKp46⁺) and B (CD45R⁺) cells in KMC and KMC Miz1^fl/fl pancreatic tumours. Mann-Whitney Test (panels G, I). H) IHC detection of tumour infiltrating NK and B cells after 3 weeks of IFNAR-1 blockade in KMC-Miz1^fl/fl PDAC. Scale bars = 100μm. I) Quantification of tumour infiltrating NK (NKp46⁺) and B (CD45R⁺) cells in untreated (N=11 & 10, respectively; from panel G) versus IFNAR1 antibody treated (3 weeks treatment, N=5) KMC-Miz1^fl/fl PDAC. J) Overall survival of KMC-Miz1^fl/fl mice treated with (N=7) or without (N=12, from panel F) anti-IFNAR1 blocking antibody. For all panels, *** denotes p<0.001, **denotes p<0.01, * denotes p<0.05, ns = not significant.

Figure 6. Cxcl13 mediates Interferon signalling to B and NK cells in PDAC

A) Normalised RNA-SEQ reads of Cxcl13 expression in KMC (N=6) and KMC Miz1^fl/fl (N=5) pancreatic tumours. B) In situ hybridization (ISH) analysis of Cxcl13-expressing cells in KMC and KMC-Miz1^fl/fl tumours. Lower panels show IHC for F4/80. Scale bar = 100μm. C) RT-PCR analysis of Cxcl13 gene expression in bone marrow-derived macrophages upon treatment (10ng/ml for 4hrs) with recombinant mouse IFNβ1. Mean and SEM of technical replicates from 1 of 3 independent experiments. T test; nd = none detected. D) RT-PCR analysis of Cxcl13 gene expression in bone marrow-derived macrophages after 24hrs treatment with conditioned media from MYC-depleted or control KMC tumour cells. Macrophages were pre-treated (20μg/ml, overnight) with IFNAR1 blocking antibody or isotype control, where indicated. Mean and SEM of technical replicates from 1 of 3 independent experiments. ANOVA and Tukey post-hoc test; nd = none detected. E) IHC detection of tumour infiltrating NK and B cells after 3 weeks blockade of Cxcl13 in KMC-Miz1^fl/fl PDAC. Scale bars = 100μm. F) Quantification of tumour infiltrating NK (NKp46⁺) and B (CD45R⁺) cells in untreated (N=11 & 10, respectively, from Figure 5G) and anti-Cxcl13-treated (N=6) KMC-Miz1^fl/fl PDAC. Mann Whitney test. G) Overall survival of KMC-Miz1^fl/fl mice treated with (N=6) or without (N=12, from Fig. 5F) anti-Cxcl13 blocking antibody for 3 weeks. Mantel Cox logrank test (G, H). H) Overall survival of KMC-Miz1^fl/fl mice treated with (N=6) or without (N=12, from Fig. 5F) anti-NK1.1 depleting antibody for 4 weeks. I) FACS analysis of Zombie NIR^™ labelling of target cells (KMC, Yac-1, Fibroblasts) co-cultured with IL-2 stimulated, NK cell enriched splenocytes for 4hrs. Mean and SEM of 3 independent experiments shown. 2-way ANOVA and Tukey’s multiple comparisons test. J) Model showing the mechanism of MYC/Miz1-dependent immune evasion in PDAC. For all panels, *** denotes p<0.001, **denotes p<0.01, * denotes p<0.05, ns = not significant.