NSD1 inactivation defines an immune cold, DNA hypomethylated subtype in squamous cell carcinoma

Abstract

Chromatin modifying enzymes are frequently mutated in cancer, resulting in widespread epigenetic deregulation. Recent reports indicate that inactivating mutations in the histone methyltransferase NSD1 define an intrinsic subtype of head and neck squamous cell carcinoma (HNSC) that features pronounced DNA hypomethylation. Here, we describe a similar hypomethylated subtype of lung squamous cell carcinoma (LUSC) that is enriched for both inactivating mutations and deletions in NSD1. The 'NSD1 subtypes' of HNSC and LUSC are highly correlated at the DNA methylation and gene expression levels, featuring ectopic expression of developmental transcription factors and genes that are also hypomethylated in Sotos syndrome, a congenital disorder caused by germline NSD1 mutations. Further, the NSD1 subtype of HNSC displays an 'immune cold' phenotype characterized by low infiltration of tumor-associated leukocytes, particularly macrophages and CD8⁺ T cells, as well as low expression of genes encoding the immunotherapy target PD-1 immune checkpoint receptor and its ligands. Using an in vivo model, we demonstrate that NSD1 inactivation results in reduced T cell infiltration into the tumor microenvironment, implicating NSD1 as a tumor cell-intrinsic driver of an immune cold phenotype. NSD1 inactivation therefore causes epigenetic deregulation across cancer sites, and has implications for immunotherapy.

Figure 1

Identification of NSD1 inactivated subtypes of squamous cell carcinomas featuring epigenetic de-repression of developmental oncogenes: (a) Heatmaps of differential methylation (DM) values illustrate five subtypes of head and neck squamous cell carcinoma (HNSC, n = 528 patients) and six subtypes of lung squamous cell carcinoma (LUSC, n = 502 patients) within TCGA studies, identified by consensus clustering of patients according to their profiles of abnormally methylated genes, subsequent to identification of these abnormally methylated genes by applying MethylMix to integrate DNA methylation and gene expression data. DM values represent the beta value difference in methylation between tumor and normal adjacent tissue for hypomethylated (<0) or hypermethylated (>0) methylation states for each gene, calculated by MethylMix. Red bars demarcate hypomethylated NSD1 subtypes, while light and dark grey bars demarcate other subtypes. (b) The average number of genes hypomethylated per patient (in tumor relative to normal tissue) was significantly higher in NSD1 subtypes (red) than each other subtype (grey) in both HNSC and LUSC. (c) Percentages of patients within each subtype that have NSD1 mutations (Striped bars) and NSD1 deletions (Solid bars) in HNSC and LUSC. Asterisks indicate the significance of enrichment of NSD1 mutations or deletions within the NSD1 subtype (red) compared with patients in all other subtypes (Pearson’s chi-squared test).

Figure 2

Concordant DNA methylation and gene profiles between HNSC and LUSC subtypes: (a) Heatmap of a correlation matrix illustrating pair-wise Pearson correlations of DNA methylation profiles between 528 HNSC patients (columns) and 503 LUSC patients (rows). Correlation coefficients indicate the correlation of each patient pair across 621 CpG sites, representing all CpG sites that were available for all patients (Measured on both Illumina 27 k and 450 k arrays), and which were abnormally methylated (hypermethylated in hypomethylated) in all or a subset of HNSCs. Patients are ordered according to DNA methylation subtypes (no clustering was performed), with sidebars indicate HNSC and LUSC subtypes (NSD1 subtypes illustrated in red, other subtypes grey). NSD1 mutation and deletion sidebars indicate patients with NSD1 mutations or deletions (black), absence of NSD1 mutations or deletions (grey), or missing data (white). The ‘NSD1 PAM class’ sidebar indicates predictions of PAM models for belonging to the NSD1 subtype, which were trained on one cancer type and used to classify patients of the other cancer type as either ‘NSD1 subtype’, or ‘Other subtype’ (e.g., ‘HNSC PAM class’ refers to predictions made by a model trained on HNSC subtypes and applied to predict subtypes of LUSC patients). (b) Scaled mean RNA expression in LUSC DNA methylation subtypes of genes that were upregulated (HNSC up) and downregulated (HNSC down) in the HNSC NSD1 subtype. Asterisks indicate the significance of differential mean expression between the NSD1 LUSC subtype (Red box) and each other subtype (Wilcoxon rank sum test): NS Not significant, *P < 0.05, **P < 0.01, ***P < 0.001. (c) DNA methylation of development-related transcription factor genes, in normal tumor-adjacent tissue (purple), and in tumor of patients within NSD1 subtypes (red) or other subtypes (grey), in HNSC and LUSC.

Figure 3

NSD1 inactivation is associated with immune cell exclusion from the tumor microenvironment in HNSC: (a) Compared with other HNSC subtypes, the NSD1 subtype (red box) displayed significantly lower mean signature levels of overall tumor associated leukocytes (TALs), and specific TAL types including M1 tumor associated macrophages (TAMs), CD8+ cytotoxic T cells, and CD4+ memory T cells (All inferred using CIBERSORT). The NSD1 subtype had the low mean RNA expression of immunotherapy-relevant genes, including CD274 (PD-L1), PDCD1 (PD-1) and PDCD1LG2 (PD-L2), and a lower mean level of T cell signature based on expression of 13 T cell transcripts. (b) Control and NSD1 shRNA knockdown HNSC cells (1 × 10⁶) were injected into the subcutaneous compartments of the flanks of NOD-scid IL2Rgamma^null (NSG) mice. In each mouse, one flank was injected with control cells (black) and the other with NSD1 knockdown cells (red). After tumors were established (5 mm diameter), 100 × 10⁶ Ficoll-purified human PBMCs per mouse were injected via tail vein. After 10 days, tumors were dissociated, and tumor-infiltrating T cells (CD45⁺CD3⁺) were quantified by FACS. Cohorts were n = 5 per set of control and knock-down cell line, as indicated. *P < 0.05; **P < 0.005 (paired two-tailed Students t-test, error bars represent S.D.). (c) NSD1 knockdown in HNSC results in the decreased expression of multiple chemokine genes. Control and NSD1 shRNA knockdown HNSC cells were assessed for the expression of chemokine and chemokine-related genes using a qRT-PCR array. Log2 fold expression of of 35 chemokine-related genes upon NSD1 knockdown (Relative expression NSD1-shRNA/Control) in three established HNSC cell lines (PCI13, FADU, SCC6). Log2 fold expression is indicated by a color gradient, with NA values indicated in grey. Asterisks indicate genes that were upregulated* or downregulated** in the NSD1 subtype (relative to other subtypes) in the TCGA study.