Extended Data Fig. 10: Proteomic changes resulting from ZCCHC7 alteration.
(a and b) Ranked gene set enrichment analysis (GSEA) of O-propargyl puromycin-mediated identification (OPP-ID) data using KEGG pathways collection. Genes (n = 5,176) corresponding to altered proteins were ranked in ascending order by two-way ANOVA test P-value of the genetic alteration factor on the x-axis in Fig. 7e. Pathways that show FDR adjusted P-value < 0.05 are shown in (a). The sign of normalized enrichment score (NES) suggests downregulation (negative NES) or upregulation (positive NES) in the SUDHL10 ZCCHC7-OE cells and SUDHL10 PAX5 TSS2Mut cells compared to SUDHL10 WT cells. GSEA shows downregulation of proteins in ribosome, DNA replication, mismatch repair, and non-homologous end joining pathways (b). (c) Fold changes of nascent proteins in ZCCHC7-OE or PAX5 TSS2Mut SUDHL10 cells compared to the wild type SUDHL10 cells. Each dot represents a protein with significantly higher abundance in OPP+ than OPP- in a two-way ANOVA (Fig. 7e). 1,690 proteins in total were captured via OPP and evaluated for nascent protein level. P-value and rho of Spearman’s correlation test between the x-axis and y-axis show a significant positive correlation between fold change of proteins in ZCCHC7-OE and PAX5 TSS2Mut. Tumor suppressor proteins including IKZF3, CHEK1, etc show decreasing translation in both ZCCHC7-OE cells and PAX5 TSS2Mut cells. (d) Independent whole cell proteomics for triplicate cultures of CL-01 (non-neoplastic B cells), SUDHL6 (which overexpress ZCCHC7 due to chromosomal translocation), SUDHL10 cells (no inherent alterations in PAX5-TSS2 or ZCCHC7) and ZCCHC7-OE (SUDHL10 cells overexpressing ZCCHC7). Of the 3428 proteins with different protein expression between SUDHL6 and CL-01, 215 showed alterations also observed in ZCCHC7-OE compared to SUDHL10. Some of these proteins are tumor suppressor proteins in B-NHL (for example IKZF3, TRAF3, etc.). (e and f) Polysome profiling of SUDHL10 WT and SUDHL10 ZCCHC7-OE (e) and analyses of IKZF3 mRNA distribution on the polysome (f). (g) ZCCHC7 transgene was introduced into SUDHL10, and stable lines isolated. ZCCHC7 protein expression is compared in ZCCHC7-OE and the SUDHL10 parental cell line. Three biological replicates were performed. (h) Evaluation of nascent levels of 20 proteins targeted by available therapeutics. The expression heatmap of the proteins in SUDHL10 and SUDHL10 PAX5-TSS2mut lymphoma cells is shown.