Supplemental Tables 1-2, Supplemental Figures 1-6 from Disruption of <i>NSD1</i> in Head and Neck Cancer Promotes Favorable Chemotherapeutic Responses Linked to Hypomethylation
Authors: Bui Nam , Huang Justin K. , Bojorquez-Gomez Ana , Licon Katherine , Sanchez Kyle S. , Tang Sean N. , Beckett Alex N. , Wang Tina , Zhang Wei , Shen John Paul , Kreisberg Jason F. , Ideker Trey 
Published: April 2023

<p>Supplemental Table 1. Descriptions of the CpG-associated genes reveal a predominance of genes that function in MHC class or skin/connective tissue structure. Supplemental Table 2. Consistently hypomethylated genes in NSD1 disrupted cell lines. Supplemental Figure 1. Effect of various factors on NSD1 mutation survival effect in TCGA and validation of NSD1 survival effect in a second independent cohort from the University of Chicago (12). A, Distribution of smokers and non-smokers and B, Kaplan-Meier curve for overall survival of current and former smokers from the HPV(-) HNSCC cohort in TCGA (13). C, Kaplan-Meier curve for progression free survival and D, for overall survival for the HPV(-) HNSCC cohort from the University of Chicago. E, Kaplan-Meier curve for overall survival of patients with truncating (blue) or missense (orange) NSD1 mutations versus those with wild type NSD1 (green). Analysis is for the TCGA HPV(-) HNSCC cohort. All p-values for Kaplan-Meier curves in this figure were derived from the Log-Rank Test. Supplemental Figure 2. Regional analysis of differentially methylated CpG sites in TCGA patients. A strong cluster of differentially methylated CpG sites appear around chromosome 6 from 31 MB - 33 MB. Supplemental Figure 3. NSD1 protein expression levels in parental and monoclonal cell lines with NSD1 disrupted by CRISPR-Cas9. A, Immunoblots of NSD1 and TBP (TATA Binding Protein) for NSD1 wild type CAL33, two monoclonal and one polyclonal NSD1 disrupted cell lines. B, Relative NSD1 expression levels were calculated by taking the ratio of intensity of the NSD1 band relative to the TBP band and setting the wild type expression level to 100%. C, Immunoblots of NSD1 and TBP (TATA Binding Protein) for NSD1 wild type UM-SCC47 and one monoclonal NSD1 disrupted cell line. D, Same as B except with the NSD1 expression levels from the UM-SCC47 cell lines. NSD1 alleles from monoclonal populations are characterized as follows: wt, wild type; trunc, contains a truncating mutation. Supplemental Figure 4. Effect of NSD1 mutations on CpG site methylation and gene expression in HNSCC cell lines. A, Clustered bar chart describing the distribution of all differentially methylated CpG sites (DMRs) for Sotos Syndrome patients (14) (red), TCGA HNSCC NSD1 mutant tumors (gold), and the top 10,000 most differentially methylated probes from CAL33 (blue, green) and UM-SCC47 (purple) NSD1 knockout cell lines (Methods) compared to the standard distribution of CpG sites in the Illumina 450k chip for each site feature (light blue). B and C, Differential mRNA expression by RT-qPCR of selected genes in CAL33 (B) and UM-SCC47 (C) cell lines, with and without NSD1 disruption. Supplemental Figure 5. Effect of NSD1 mutations on cell line sensitivity to radiation and DNA damage repair. A, Relative gain in percentage of γH2AX positive CAL33 cells when treated with cisplatin compared to cells without cisplatin treatment for CAL33 wild-type and pooled NSD1-knockout cells. γH2AX gain is significantly increased at 2µM cisplatin (Holm-Sidak corrected p-value < 0.005, Student''s T-Test), consistent with the significant difference in cisplatin IC50 between the two groups. B, Clonogenic plating efficiency differences between CAL33 wild-type and pooled NSD1-knockout cells. C, Clonogenic survival assay of CAL33 wild-type and pooled NSD1-knockout cells with increasing doses of radiation. Two independent curves do not fit the data significantly better than a single curve (extra-sum-of-squares F-Test). Supplemental Figure 6. Distribution of cell line types in the Sanger dataset (blue) with number of NSD1 mutations (red) and truncating NSD1 mutations (orange).</p>