Disruption of NSD1 in Head and Neck Cancer Promotes Favorable Chemotherapeutic Responses Linked to Hypomethylation

Abstract

Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) represents a distinct classification of cancer with worse expected outcomes. Of the 11 genes recurrently mutated in HNSCC, we identify a singular and substantial survival advantage for mutations in the gene encoding Nuclear Set Domain Containing Protein 1 (NSD1), a histone methyltransferase altered in approximately 10% of patients. This effect, a 55% decrease in risk of death in NSD1-mutated versus non-mutated patients, can be validated in an independent cohort. NSD1 alterations are strongly associated with widespread genome hypomethylation in the same tumors, to a degree not observed for any other mutated gene. To address whether NSD1 plays a causal role in these associations, we use CRISPR-Cas9 to disrupt NSD1 in HNSCC cell lines and find that this leads to substantial CpG hypomethylation and sensitivity to cisplatin, a standard chemotherapy in head and neck cancer, with a 40% to 50% decrease in the IC₅₀ value. Such results are reinforced by a survey of 1,001 cancer cell lines, in which loss-of-function NSD1 mutations have an average 23% decrease in cisplatin IC₅₀ value compared with cell lines with wild-type NSD1Significance: This study identifies a favorable subtype of HPV-negative HNSCC linked to NSD1 mutation, hypomethylation, and cisplatin sensitivity. Mol Cancer Ther; 17(7); 1585-94. ©2018 AACR.

Figure 1

NSD1 mutations are associated with improved survival in the HPV(−) HNSCC cohort in TCGA. A, Forest plot of the prognostic influence of the 11 most recurrently mutated genes in the HPV(−) HNSCC cohort in TCGA. Hazard ratios derived from Cox proportional hazards model incorporating the clinical covariates age, stage, grade, gender, smoking status and anatomical location. B, Kaplan-Meier curve showing overall survival from the HPV(−) HNSCC cohort in TCGA. C, Head and neck cancer possess a high percentage of NSD1 mutations and a high percentage of relative truncating mutations. D, Loss-of-function NSD1 mutations and homozygous deletions, defined as a −2 copy number change by GISTIC (45), have significantly lower gene expression than wild-type or missense mutations. E, Lollipop plot of location of NSD1 mutations as generated by cBioPortal (46,47). The lines represent density plots of truncating (black) and missense (green) mutations.

Figure 2

CpG hypomethylation in patients with NSD1 loss-of-function mutations in the HPV(−) HNSCC cohort in TCGA. A, Unsupervised hierarchical clustering based on the methylation status of 500 selected CpG sites reveals a tight cluster of hypomethylation centered around NSD1 mutations. Analysis of alteration type (top color bar) reveals that missense mutations (orange) were more likely to be outliers while truncating (red) and homozygous deletions (purple) were associated with the hypomethylation signal. B, Gene level methylation analysis reveals that NSD1 is the only gene where mutations cause a significant change to the methylome (x-axis: ~13% of all CpG sites) with all other genes at <2%. The direction of methylation changes are strikingly in the hypomethylated direction with a full 98.9% of differentially methylated CpG probes being hypomethylated (y-axis).

Figure 3

CpG hypomethylation in cell lines with NSD1 disrupted. A–B, Methylation analysis of top 10,000 most differentially methylated CpG sites in CAL33 with and without NSD1 disrupted demonstrates that the cell lines with NSD1 disrupted have a much higher hypomethylation peak than their respective parents. C, Same as A and B except for UM-SCC47. D, Bar plot of the above three cell lines showing the increase in the hypomethylation peak in the NSD1 disrupted cell lines. NSD1 alleles from monoclonal populations are characterized as follows: wt, wild type; trunc, contains a truncating mutation

Figure 4

NSD1 loss-of-function mutations confer increased cisplatin sensitivity. A–B, Cisplatin sensitivity curves for cell lines with and without NSD1 disruption, showing greater sensitivity in the disrupted cell lines (blue and green lines). Pretreatment with the HMT inhibitor UNC0379 (HMTi) also increased sensitivity to cisplatin. NSD1 alleles from monoclonal populations are characterized as follows: wt, wild type; trunc, contains a truncating mutation. C, Barplot of cisplatin IC₅₀ in parental cell lines and cell lines with NSD1 disrupted. Asterisk (*) indicates f sum-of-squares p < 0.0001 when compared to parental cell line. D, Volcano plot showing differential effect of 143 drugs on NSD1 mutated versus NSD1 wild type cell lines. Cisplatin is highly effective (2nd most left point) and the most significant (most upward point). The drug classes “DNA replication” and “Genome integrity” are highly represented on the NSD1 sensitizing side. E, Violin plot showing increased sensitivity of NSD1 mutated cell lines to cisplatin.