Fig 2: Immunohistochemical (IHC) staining of EGFRK721me1 in SCCHN tissues and correlation with WHSC1L1 staining.(a) (i) Representative examples of EGFRK721me1 IHC staining (+1, +2, +3 scores) in SCCHN tissue sections. Staining of EGFRK721me1 is mostly observed in the nucleus, with mild staining in the cytoplasm (40x). (ii) Representative example of EGFRK721me1 staining in normal squamous epithelium. Staining was mostly observed in the cells of the basal layer of the epithelium, corresponding to the staining of WHSC1L1 (20x). (b) Histogram of immunohistochemistry scores of EGFRK721me1 in normal, dysplastic epithelium and SCCHN tissue sections. The ratio of +2/+3 EGFRK721me1-positive staining samples was statistically significantly higher in SCCHN tissues compared to normal and dysplastic squamous epithelium (Cochran-Armitage test, *p < 0.001). (c) Representative examples of EGFRK721me1 and concordant WHSC1L1 staining in SCCHN sections from 3 different patients (40x). A table with the cumulative IHC score results of SCCHN sections (N = 127) staining for WHSC1L1 and EGFRK721me1 is also shown (Cochran-Armitage test, p = 0.0059).

Fig 3: A. Immunoblot for ERa in MCF-7 and SUM-44 cells. ß-actin was used to confirm equal loading. B. Immunoblot showing WHSC1L1 (left) and ERa (right) protein levels in whole cell lysates from SUM-44 cells lentivirally transduced with shRNA against LacZ or two shRNAs against both isoforms of WHSC1L1. ß-actin was used to confirm equal loading. C. (Left) Immunoblot showing WHSC1L1-long and WHSC1L1-short isoform levels in several breast cancer cell lines and MCF10A cells. Cell lines with increased WHSC1L1 copy number are denoted by an asterisk (*). (Right) Real-time PCR results showing transcript levels for WHSC1L1-long or WHSC1L1-short in several breast cancer cell lines relative to levels in normal breast epithelial cells, represented here by MCF10A cells. D. Transcript expression (RNAseqV2) of WHSC1L1-long and WHSC1L1-short isoforms from Cancer Genome Atlas (TCGA) breast cancer samples (N = 964). E. Immunoblot showing WHSC1L1 (left) and ERa (right) protein levels in whole cell lysates from SUM-44 cells lentivirally-transduced with shRNAs against LacZ or two shRNAs against WHSC1L1-short. ß-actin was used to confirm equal loading. F. (Left) Proliferation assay showing cell number after 12 days of culture in SFIH media for SUM-44 WH-total and WHSC1L1-short shRNA knock-downs compared to shLacZ control cells. The error bars represent 1.96 multiplied by the standard error of the mean (1.96*SEM). (Right) Boxplot showing the results of five replicates of the proliferation assay in SUM-44 shWHshort_1 vs. shLacZ control cells.

Fig 4: Clinical predictive value of NSD3 for immunotherapy outcome in LUSC. (A) Heatmap showing the Spearman correlation coefficients between the mRNA expression of NSD3 and a panel of inhibitory immune checkpoints in the TCGA LUSC cohort (n = 491). (B) Immunophenoscore (IPS) distribution plot in the non- and amplification groups of NSD3 across the TCGA LUSC cohort (n = 491). The Wilcoxon rank-sum test was used for comparison, and p < 0.05 was considered significant. (C) mRNA expression of NSD3 in the different clinical response groups (DCB: durable clinical benefit; non-durable clinical benefit) based on the transcriptomic dataset from the NSCLC immunotherapy cohort (n = 27; GSE135222). A two-tailed unpaired t-test was used for comparison, and p < 0.05 was considered significant. ns: none significant. (D) Kaplan–Meier analysis based on the mRNA level of NSD3 in the NSCLC immunotherapy cohort (n = 27; GSE135222) with high- (in red) or low- (in blue) expression was stratified by the optimal cut-off value of the individual expression across all patients using the surv_cutpoint function in the R “maxstat” package. The log-rank test was used for comparison, and p < 0.05 was considered significant.

Fig 5: The internal links between UPR and non-immunogenic features of NSD3-amplified LUSC. (A) The difference in the unfolded protein response (UPR) gene signature between the non- and amplification groups of NSD3 in the TCGA LUSC cohort (n = 491). The UPR gene signature was scored as the sum of the hallmark UPR gene sets. The Wilcoxon rank-sum test was used for comparison, and p < 0.05 was considered significant. (B) Kaplan–Meier analysis based on the UPR gene score in NSD3-amplified LUSC patients (upper panel; n = 207) or non-amplified group (lower panel; n = 236), with high (in red) or low (in blue) expression, was stratified by the optimal cut-off value of the individual expression across all patients using the surv_cutpoint function in the R “maxstat” package. The log-rank test was used for comparison, and p < 0.05 was considered significant. (C) Correlation analysis between the UPR gene score and several gene signatures was calculated using the “GSVA” R package in the TCGA LUSC cohort (n = 491). (D,E) Correlation analysis between the UPR gene score and a panel of immune cell-related gene markers (D) as well as inhibitory immune checkpoints (E) in the TCGA LUSC cohort (n = 491). (F,G) The expression levels of UPR gene signature in the indicated groups. Transcriptomic data were obtained from transgenic mice carrying a lung-specific active mutant (T1232A) or genetic knockout of Nsd3 (GSE149212 & GSE149482). A two-tailed unpaired t-test was used for comparison, and p < 0.05 was considered significant. * p < 0.05; **** p < 0.001. (H) The difference in the indicated immune cell types and the type II-IFN response was calculated using the “GSVA” R package between the wild-type and Nsd3 active mutant (T1232A) groups. A two-tailed unpaired t-test was used for comparison, and p < 0.05 was considered significant.