Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas

Abstract

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs) are deadly and common cancers. Recent genomic studies implicate multiple genetic pathways, including cell signaling, cell cycle and immune evasion, in their development. Here we analyze public data sets and uncover a previously unappreciated role of epigenome deregulation in the genesis of 13% of HPV-negative HNSCCs. Specifically, we identify novel recurrent mutations encoding p.Lys36Met (K36M) alterations in multiple H3 histone genes. histones. We further validate the presence of these alterations in multiple independent HNSCC data sets and show that, along with previously described NSD1 mutations, they correspond to a specific DNA methylation cluster. The K36M substitution and NSD1 defects converge on altering methylation of histone H3 at K36 (H3K36), subsequently blocking cellular differentiation and promoting oncogenesis. Our data further indicate limited redundancy for NSD family members in HPV-negative HNSCCs and suggest a potential role for impaired H3K36 methylation in their development. Further investigation of drugs targeting chromatin regulators is warranted in HPV-negative HNSCCs driven by aberrant H3K36 methylation.

Figure 1. H3K36M and NSD1 mutations define a specific HNSCC subgroup

Unsupervised hierarchical clustering of DNA methylation data identifies 5 HNSCC subgroups, including notably a subgroup comprised of NSD1 or H3K36M mutations (labeled the H3K36 cluster). The H3K36 cluster is enriched in HPV-, TP53 mutant, heavy smoking patients. Those tumors are found in the larynx and oral cavity. Another HPV+/TP53 wild-type subgroup is apparent (labeled the HPV+ cluster) as well as 3 other subgroups (labeled Groups A, B and C). Grey and white bars for clinical variables (HPV status, anatomical location) denote “No hit” or “Not available”, respectively.

Figure 2. Schematic representation of the anatomical locations of NSD1 and H3K36M mutations in HNSCCs

Tumors with H3K36M were mainly located in the oral cavity while NSD1 mutant tumors mainly occurred in the larynx and less frequently in other known locations of HNSCCs. The size of the dotted lines is proportional to the frequency of the mutation in a given anatomical location.

Figure 3. Representative immunohistochemical (IHC) staining of H3K36M, NSD1, H3K36me2 and H3K36me3 in H3K36M or NSD1 mutant HNSCCs and in a HNSCC sample wild-type for these genes

Representative IHC of HNSCC samples using anti-H3K36M, anti-NSD1, anti-H3K36me3, or anti-H3K36me2 antibodies and counterstained with hematoxylin. H3K36M was expressed in 3/85 HPV-HNSCCs. H3K36M shows strong nuclear positivity in tumor cells but no staining in adjacent nuclei of stromal/inflammatory cells. Tumors wild-type for H3K36M and/or NSD1 show no nuclear staining with the anti-H3K36M antibody. Corresponding H3K36me3 and H3K36me2 staining on the same samples shows global decrease in the levels of these histone marks in H3K36M mutant tumors compared to tumors wild-type for this mutation. Notably, strong positivity for H3K36me3 and H3K36me2 was seen in the adjacent stromal cells not carrying H3K36M mutation, while uniform strong staining in tumor and stroma was observed for both marks in HNSCCs wild-type for H3K36M. Loss of NSD1 was associated with a marked decrease in global levels of H3K36me2 but not in H3K36me3. Pictures were taken under a 40X magnification and collated using Photoshop.

Figure 4. H3K36M and NSD1 mutations decrease levels of H3K36me2 in HNSCC

(A) Immunoblots showing levels of NSD1/2/3, SETD2, H3K36me2 and H3K36me3 in NSD1 wild-type (Fadu and PCI-4B) and NSD1-deficient (SCC-4 and SKN-3) HNSCC cell lines. (B) Immunoblots showing levels of H3K36me2, H3K36me3 and H3K36M in NSD1 wild-type (Fadu and PCI-4B) and NSD1-deficient SCC-4 HNSCC cell lines, and SCC-4 cells stably expressing H3K36M mutant histone. (C) Immunoblots showing levels of NSD1, H3K36me2, H3K36me3 and H3K36M in 293T and NSD1 wild-type (Fadu and PCI-4B) HNSCC cell lines that were treated with NSD1 siRNA or stably express H3K36M mutant histone. (D) and (E) Mass spectrometry-based quantification of levels of H3K36 methylation in H3.1/2 (D) or H3.3 (E) in NSD1 wild-type (Fadu and PCI-4B) and NSD1-deficient (SCC-4 and SKN-3) HNSCC cell lines, and Fadu cells stably expressing H3K36M mutant histone. For (A) (B) and (C), blot images are cropped and representative of two independent experiments. Full-length blots are included in Supplementary Figure 14. For (D) and (E), error bars represent standard deviation from three independent cell cultures. Error bars represent standard deviation from biological triplicates. n.s., not significant; *, P<0.05; **, P<0.01 by Student’s t-test.