Additional file 1 of Multiomic analysis of papillary thyroid cancers identifies BAIAP2L1-BRAF fusion and requirement of TRIM25, PDE5A and PKCδ for tumorigenesis
Creators: name: Renaud, Emilie
name: Riegel, Kristina
name: Romero, Rossana
name: Suryamohan, Kushal
name: Distler, Ute
name: Tenzer, Stefan
name: Schad, Arno
name: Musholt, Thomas J.
name: Rajalingam, Krishnaraj
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Cancer
subject: Hematology

description: Additional file 1: Suppl. Fig. 1. RET fusions were identified in 3 patients. A. H&amp;E and α-thyroglobulin staining of indicated patients’ tumor tissues. B. Summary of patients’ features. C. Circos plots showing RET fusions detected in patients 14, 15 and 21. D./E. RET fusions detection by qPCR (E) and RT-PCR (F) in indicated samples. F. BAIAP2L1-BRAF detection by qPCR (upper panel) and RT-PCR (lower two panels) in the indicated patients’ samples. The upper graph shows three technical replicates. Suppl. Fig. 2. Numerous thyroid-related genes are dysregulated in tumor tissue compared to normal tissue. A. Heat map of RNA-seq analysis of normal vs tumor tissue of the 4 patients carrying the fusion proteins (adjusted p-value &lt; 0.05, log2 fold change ≠ 0). B. Comparison of RNA-seq data of 19 thyroid-related genes. Patient 17 is here compared to “all patients” representing the three pateints 14, 15 and 21. C. Individual normalized read counts of 2 thyroid-related genes, TPO and DIO1. D. Determination of the relative expression of TPO and DIO1 in tumor and metastasis vs normal samples of all four patients by qPCR. Three technical replicates of each patient are shown (error bars = SEM). E. Determination of the relative expression of TPO and DIO1 in normal patient 17 vs other patients’ normal samples by qPCR (error bars = SEM, three technical replicates). Suppl. Fig. 3. TRIM25 and PKCδ are not upregulated at the mRNA level. A. Heat map of mass spectrometry analysis of normal vs tumor vs metastasis. B. Relative protein intensities from mass spectrometric data of PKCδ, TRIM25 and IQGAP1 from the individual patients. C. Differential expression (log2(fold change)) of PKCδ, TRIM25 and IQGAP1 from mass spectrometry data in patients #14, #15, #17 and #21. Each dot represents the log2(fold change) of one patient. D. RNA-seq data showing PKCδ, TRIM25, and IQGAP1 expression in indicated samples. Suppl. Fig. 4. Combination of mass spectrometric vs RNA-seq data allowed identification of additional targetable factors. A. Table summarizing factors that are up- or downregulated on both the RNA and protein levels. B. Relative protein intensities from mass spectrometric data (left) or normalized read counts from RNA-seq data (right) of IGSF1 and PDE5A in the indicated samples. C. qPCR analysis of PDE5A and IGSF1 expression in indicated samples (error bars = SEM, three technical replicates shown). Suppl. Fig. 5. Combination of mass spectrometric vs RNA-seq data allowed identification of additional targetable factors. Scatter plots showing significantly differentially expressed RNA (blue), protein (green) and both (red) in patients 14, 15, 17 and 21 (healthy vs tumor). Suppl. Fig. 6. 3D growth and invasion of Nthy cells stable transfected with BAIAP2L1-BRAF mutants. A. Representative images of the colony formation assay that has been quantified in Fig. 1F. B. Schematic illustration of WT BAIAP2L1-BRAF fusion (WT aminoacids highlighted in green) and mutated BAIAP2L1-BRAF fusions (modified residues highlighted in red) used in this study. C. Same as in Fig. 1G, but representative images of BAIAP2L1-BRAF wildtype and mutants. Scale bar: 200 μm. D. Quantification of matrix invasion of spheroids (shown in C) from cells stably expressing BAIAP2L1-BRAF-FLAG WT after treatment with indicated compounds. E. Same as in D, but the mean invasion of four independent experiments is plotted relative to the WT control (error bars = SEM, Dunnett’s multiple comparisons test, P value: ** &lt; 0.002, **** &lt; 0.0001). Suppl. Fig. 7. MAPK activation depends on BAIAP2L1-BRAF DIF but not on CC domain or RAS activation status. A. MEK1/2 activation detected by Western blotting of proteins extracted from Nthy-ori 3–1 cells stable transfected with the indicated plasmids (B-BRAF = BAIAP2L1-BRAF). B. Western blot detection of a V5 pull-down experiment done with extracts from Nthy-ori 3–1 cells transiently transfected with the indicated constructs (B-BRAF = BAIAP2L1-BRAF). C. Nthy cells were transiently transfected with the indicated FLAG-tagged BAIAP2L1-BRAF constructs. The empty vector served as a control. A FLAG pull down was performed and the extracts were analzyed for the presence of endogenous RAF proteins by Western blot analysis (B-BRAF = BAIAP2L1-BRAF). D. Western blot detection of a FLAG pull-down experiment done with extracts from Nthy-ori 3–1 cells transiently transfected with the indicated constructs (B-BRAF = BAIAP2L1-BRAF). E. Nanobit experiment of HEK cells transiently transfected with LgBiT-N KRAS constructs and SmBiT-C RBD constructs (error bars = SEM, n = 3, paired t test, two-tailed, P value: ** &lt; 0.01, *** &lt; 0.0001, ns-not significant). F. NanoBit experiment of HEK cells transiently transfected with SmBiT-N BAIAP2L1-BRAF + LgBiT-C BAIAP2L1-BRAF constructs and treated for 1 hour with increasing doses of PLX839 (error bars = SEM, n = 3). Suppl. Fig. 8. Targeting PKCδ, PDE5A, or TRIM25 sensitizes 3D spheroids stably expressing BAIAP2L1-BRAF fusion. A. Nthy-ori 3–1 cells stably expressing EV, BAIAP2L1-BRAF-FLAG or BAIAP2L1-BRAF-V5 and B. Nthy-ori 3–1 cells stably expressing BAIAP2L1-BRAF-V5 along with indicated shRNAs were cultured as 3D spheroids for 3 days in the presence of 6 μg/mL collagen. C. Nthy-ori 3–1 cells stably expressing BAIAP2L1-BRAF-FLAG were cultured as 3D spheroids for 3 days, treated with indicated compounds and incubated for 3 more days. Suppl. Fig. 9. CCDC6-RET expressing cells are sensitive to PKCδ and PDE5A inhibitors. A. Quantification of matrix invasion of spheroids (shown in Fig. 2G) from cells stably expressing BAIAP2L1-BRAF-FLAG WT after treatment with indicated compounds. B. Same as in A, but the mean invasion of four independent experiments is plotted relative to the DMSO control (error bars = SEM, paired t test, two-tailed, P value: * &lt; 0.05, ** &lt; 0.01, ns-not significant). C. Colony counts of Nthy-ori 3–1 cells stably expressing the indicated constructs (error bars = SEM, n = 3, paired t test, two-tailed, P value: * &lt; 0.05). D. Kinase assay performed with extracts from Nthy-ori 3–1 cells transiently transfected with indicated constructs. Representative Western blot is shown. E. Representative Western blot analysis of protein extracts from Nthy ori cells transiently transfected with EV or CCDC6-RET-FLAG plasmids. F. Colonies counts (left) and 3D MTT (right) of cells stably expressing CCDC6-RET-FLAG WT analysed 5 days after indicated treatments (error bars = SEM, n = 3, paired t test, two-tailed, P value: * &lt; 0.05, ** &lt; 0.01). G. Western blot analysis of protein extracts from Nthy-ori 3–1 cells stably expressing CCDC6-RET-FLAG and treated with either DMSO or ponatinib for 6 hours. Representative Western blot is shown.