MHC Class II is Induced by IFNγ and Follows Three Distinct Patterns of Expression in Colorectal Cancer Organoids

Abstract

Tumor-specific MHC class II (tsMHC-II) expression impacts tumor microenvironmental immunity. tsMHC-II positive cancer cells may act as surrogate antigen-presenting cells and targets for CD4⁺ T cell-mediated lysis. In colorectal cancer, tsMHC-II negativity is common, in cell lines due to CIITA promoter methylation. To clarify mechanisms of tsMHC-II repression in colorectal cancer, we analyzed colorectal cancer organoids which are epigenetically faithful to tissue of origin. 15 primary colorectal cancer organoids were treated with IFNγ ± epigenetic modifiers: flow cytometry was used for tsMHC-II expression. qRT-PCR, total RNA sequencing, nanopore sequencing, bisulfite conversion/pyrosequencing, and Western blotting was used to quantitate CIITA, STAT1, IRF1, and JAK1 expression, mutations and promoter methylation and chromatin immunoprecipitation to quantitate H3K9ac, H3K9Me2, and EZH2 occupancy at CIITA. We define three types of response to IFNγ in colorectal cancer: strong, weak, and noninducibility. Delayed and restricted expression even with prolonged IFNγ exposure was due to IFNγ-mediated EZH2 occupancy at CIITA. tsMHC-II expression was enhanced by EZH2 and histone deacetylase inhibition in the weakly inducible organoids. Noninducibility is seen in three consensus molecular subtype 1 (CMS1) organoids due to JAK1 mutation. No organoid demonstrates CIITA promoter methylation. Providing IFNγ signaling is intact, most colorectal cancer organoids are class II inducible. Upregulation of tsMHC-II through targeted epigenetic therapy is seen in one of fifteen organoids. Our approach can serve as a blueprint for investigating the heterogeneity of specific epigenetic mechanisms of immune suppression across individual patients in other cancers and how these might be targeted to inform the conduct of future trials of epigenetic therapies as immune adjuvants more strategically in cancer.

Significance: Cancer cell expression of MHC class II significantly impacts tumor microenvironmental immunity. Previous studies investigating mechanisms of repression of IFNγ-inducible class II expression using cell lines demonstrate epigenetic silencing of IFN pathway genes as a frequent immune evasion strategy. Unlike cell lines, patient-derived organoids maintain epigenetic fidelity to tissue of origin. In the first such study, we analyze patterns, dynamics, and epigenetic control of IFNγ-induced class II expression in a series of colorectal cancer organoids.

FIGURE 1

Class II inducibility across cell line and colorectal cancer organoids following IFNγ stimulation. A–C, Flow cytometry assessment of class I and II expression across lines reveal three groups of response to stimulation at 24 hours. Representative plots, with two lines displayed for each group, for noninducible (<10% class II expression; A), weakly inducible (10%–49%; B), and strongly inducible lines (>50%; C). Histogram overlay, right, for class II expression between control (red) and IFNγ (blue). Flow data from all 15 organoids available in Supplementary Fig. S1. D–F, Class II expression following 24 hours stimulation for all lines, performed in minimum of triplicate, unpaired t test (NS, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Lines grouped into non/weakly inducible (D), strongly inducible (E), and cell lines (F). G, A total of 72-hour stimulation of weakly and noninducible lines, results in triplicate and statisitcs as above. H, Flow cytometry gating strategy.

FIGURE 2

Western blot analysis for IFNγ signaling pathway component integretity. A–D, IFNγ responsiveness assessed with STAT1 and IRF1 following 24 hours stimulation demonstrating appropriate increases in protein in all cell lines (A), weakly (C), and strongly inducible (D) organoids. Loss of signaling response in noted in the noninducible lines (B). E, All organoid lines and A549, a control cell line with known JAK1 expression were assessed for JAK1 expression under basal conditions, with loss of protein expression confirmed in the noninducible lines. Representative blots following triplicate experiments displayed.

FIGURE 3

Genomic assessment of organoids. A, Tile plot of common mutations on WGS of organoid cultures, created with Maftools. B, Volcano plot for differential gene expression (RNA-seq, unstimulated organoids) between inducible and noninducible lines, most significant genes annotated including JAK1. Positive change upregulated in inducible lines.

FIGURE 4

CIITA accessibility and expression following IFNγ stimulation. A, qRT-PCR for change in CIITA mRNA expression following stimulation across the organoid lines. Results divided by response groups (dotted lines) Calculated mean expression change and SD displayed, with four technical replicates for each line. Note logarithmic scale on y-axis. Statistics applied across response groups (see main text) for biological replication. B, ChIP PCR for EZH2 occupancy of the CIITA promoter, mean change across the four regions of the CIITA-pIV, controlled for input DNA, given for the weakly inducible lines and two representative strongly inducible lines. Results performed with four replicates, with mean and SEM plotted. ChIP PCR for change in CIITA-pIV H3K9ac (C) and H3K9me2 (D), again displaying mean across the four regions of CIITA-pIV controlled for input DNA. All organoid lines and an example inducible (HT29) and noninducible (RKO) cell line treated. Horizontal line to demonstrate no change (relative change of 1). Results performed in triplicate with mean and SEM plotted. Statsitics (unpaired t test) displayed for significant results *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 5

Pharmaceutical enhancement of class II inducibility in weakly inducible organoids. Weakly inducible lines were stimulated for 24 and 72 hours and additionally treated with 2 μmol/L GSK126 (EZH2 inhibitor) for 6 days ± 5 μmol/L Entinostat (HDAC inhibitor) for 72 hours. Flow cytometry data (mean class II expression and SD) displayed from experiments performed in triplicate. Statistical analysis (one-way ANOVA followed by Sidak post hoc test) performed for the addition of GSK126. NS, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The addition of Entinostat was relatively toxic with relatively few viable events and therefore statistics have not been applied.

FIGURE 6

TCGA data on EZH2 and MHC class II expression. Inverse correlation (R = −0.314, P = 0.00017 Pearson correlation coefficient) of EZH2 and MHC class II expression derived from the pMMR colorectal cancer dataset. A total of 140 patients all derived from primary tumors. Characteristics: 43.6% female, 74.3% rectal/left-sided, 28.6% stage III/15% stage IV, 53.6% RAS mutant, 4.3% BRAF mutant.