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2020-02-20 Supplemental Figures. Milosevic Et Al Copy Supplemental Figures outline 1. Fig S1A-F 2. Fig S2A-G 3. Fig S3A-I 4. Fig S4A-C 5. Fig S5A-B 6. Fig S6A-F 1 2 S1 400#NBL#cases#>#270#cases# Neuroblastoma tumors 11 Figure 22 33 44 55 66 77 number 88 99 1010 Chromosome 1111 1212 1313 1414 1515 1616 1717 1818 1919 2020 2121 2222 X MNA NumOnlyNumOnly 11q11q-del-del OtherSegmOtherSegm.. 17q-gain 17q-gain A 3 S1 MNA Pos Neg 11q-del Pos Neg 17q-gain Pos WCG17 Neg Figure Cell cycle & CDK4/6 CCND1 CDKN2A DNA repair G G G G G TP53 PPM1D Fanconi MDM2 ALIVE ALIVE DOD NA Outcome <5y >5y NA WES + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + WGS + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + T/N pair + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Abbreviations: MNA= MYCN-amplification; amp=amplification; WCG17= whole chromsome 17 gain; NA= not available; WES= whole exome sequencing, WGS= whole genome sequencing, G = germline variant, DOD = dead of disease E Disability Intellectual Neuroblastoma with with Disability mut mut mut - - C PPM1D PPM1D Intellectual Controls, Controls, Germline Germline patients ) months PPM1D PPM1D ( Survival for the extra plot SNE SNE - t probability survival Overal F D B Figure S1, related to Figure 1. Unfavorable chromosomal 17 q gain is common in neuroblastoma. A. Gain of chromosome 17q is the most common genetic aberration in neuroblastoma. Summary of SNP array data of gains and losses for neuroblastoma tumors from an unselected cohort of 271 Swedish patients included and analyzed during 25 years. Horizontal lines show segmental loss (clear blue) and gain (clear red) and whole chromosome loss (pale blue) and gain (pale red). Short vertical lines show amplification (red) or homozygous loss (dark blue). Green lines indicate chromothripsis. The genomic profile group is indicated to the right. The inclusion features for the three high-risk groups; MYCN amplification, 11q deletion and 17q gain-are indicated by ovals. For definition of the genomic groups see Carén et al., 2010. B. Gain of 17q correlates with poor survival in neuroblastoma. Neuroblastoma survival probability according to Kaplan-Meier analysis in a Swedish population-based patient material in relation to chromosome 17 status in the tumor tissue, shows significantly worse event-free survival (EFS) for children with 17q segmental gain. Abbreviations; MNA, MYCN amplification; 11q-del, 11q-deletion; NumOnly, numerical only (whole chromosome loss or gain); OS, Other segmental (no MNA, no 11q-del, no 17q-gain). C. Copy number profiling based on normalized sequencing coverage from two different metastatic sites, at diagnosis (Met 1) and relapse (Met 2), respectively, display different segmental aberrations except for the common 17q-gain and 11q-deletion resulting for one single unbalanced translocation being the first genetic event in neuroblastoma tumor development. D. Beside the primary unbalanced translocation t(11;17), all other mutations and structural aberrations showed sequence and/or breakpoint differences although multiple genomic alterations have emerged in parallel in the two different clones (same high-risk neuroblastoma patient as in Fig 1D and Fig S1C). E. Genetic landscape of sequenced Swedish neuroblastomas. Diagram of detected genetic defects of specific targets/pathways and clinical parameters (rows) in 73 whole-exome and/or whole-genome sequenced neuroblastoma samples (columns) including one with PPM1D mutation as described in detail in Fig 1D and three with TP53 mutations. F. t-SNE Plot of facial dysmorphic features showing the distribution of 12 individuals with germline variants in PPM1D (n=12, circles) eleven of which previously described (open circles) (Jansen et al., 2017). and including the patient with neuroblastoma (filled circle) versus controls with intellectual disability (ID, red crosses, p=0.0164). Figure S2 A Group comparison Fold change Student's t-test Significance Type 2A vs Type 1 2.10 0.0190 * Type 2B vs Type 1 2.26 0.0806 Type 2A & Type 2B vs Type 1 2.15 0.0070 ** B 17N 17+ 17q+ WIP1 H/E C D ANOVA (across all subgroups): P=9.5e-40 Expression PPM1D Expression Expression PPM1D Expression Infant MB Adult CB Adult MB Child MB Adult Fetal CB Fetal WNT SHH 4 Group Group 3 Group 5 Figure S2 E F G 6 Figure S2 Figure S2, related to Figure 2. PPM1D expression correlates with unfavorable neuroblastoma and medulloblastoma biological subgroups and is highly expressed in unfavorable chromosome 17q+ tumors. A. PPM1D as a prognostic marker for neuroblastoma. Principal Components Analysis (PCA) of the pre-processed McArdle/Wilzén microarray data sets (716 variables, 30 tumor samples/cases). PCA subgroups corresponding to molecular subtypes of NB; Type 1 (white), Type 2A (orange), Type 2B (red) and ganglioneuroma/ganglioneuroblastoma (GN/GNB; blue), and the expression of PPM1D in the four subgroups (Red = High expression; Green = Low expression). Samples/cases are represented as spheres joined by nearest Euclidean neighbor. The color scale of expression is based on standard deviations (SD) ranging from +2 SD (red) to -2 SD (green). Fold change and Student's t-test were calculated and compared between neuroblastoma subtypes; Type 1(n=10), Type 2A (=10), Type 2B (n=5). B. Representative images of H/E and Wip1 staining in neuroblastic tumors. Upper images from three neuroblastomas with normal chromosome 17 status (17N), whole chromosome 17 gain (17+), and chromosome 17q gain (17q+). C. High PPM1D expression correlates with aggressive medulloblastoma subgroups. PPM1D expression in medulloblastoma (MB) subgroups (WNT, SHH, Group 3 and Group 4) vs normal fetal or adult cerebellum (CB). PPM1D expression is significantly higher in Group 3 and Group 4 medulloblastoma compared to other MB subgroups and to normal adult and fetal cerebellum (Supplementary Table S2). D. PPM1D expression within SHH MB subgroup only, within the different age groups (infant, child and adult) shows three tumors with high PPM1D expression due to high level gene amplification in the SHH-child medulloblastoma subgroup harboring wild-type TP53. E. Gain-of function PPM1D mutation in medulloblastoma subtypes. Copy number profile (CN) of WNT medulloblastoma case MB124 with a PPM1D gain-of-function truncating mutation E525X (allele frequency 0.44). CN figure shows typical profile for a WNT medulloblastoma with loss of chromosome 6. F. Amplification of PPM1D in medulloblastoma. Heatmap of expression data for genes around PPM1D in SHH medulloblastomas. Gene amplifications including the PPM1D locus were detected in two SHH medulloblastoma samples belonging to the child SHH subgroup with wild-type TP53. G. Copy number (CN) profile of 2m21 SHH medulloblastoma shows several copy number aberrations, including amplifications of PPM1D, MDM4 and TERT and several small deletions across the genome. 7 Figure S3 A 3.5 3.0 Breast cancer cell lines 2.5 Neuroblastoma cell lines Medulloblastoma cell lines 2.0 1.5 1.0 0.5 Relative expression level (WIP1/18S) 0.0 MCF-7 IMR 32 SH-EP DAOY BT-474 MED 8A SK-N-FIPFSK-1 SK-N-DZ SH-SY5Y SK-N-SHSK-N-AS UW228-3 D384 MEDD458 MEDD283 MED SK-N-BE(2) B C D283-MED DAOY D458-MED PFSK-1 UW228-3 MEB-MED8A 8 Figure S3 D E F G H 9 Figure S3 I SK-N-BE(2) MEB-MED8A Wip1 24h Ctrl 24h Wip1 24h Ctrl 24h ShRNA ShRNA ShRNA ShRNA 73% 65,9% 59,3% 56% Ctrl Ctrl Cells Wip1 72h 72h Wip1 72h 72h # ShRNA ShRNA ShRNA Cells # ShRNA 52,1% 33,8% 42,3% 43,8% 20101812 D324 Workspace.joγH2AX γH2AXLayout: D342 MED 72h 250K 1.17 1.34 DAOY D283 MED 5 120 10 20 200K Ctrl Wip1 Ctrl 4 Wip1 24h 24h 24h 90 24h 10 14.8 6 15 ShRNA 150K ShRNA ShRNA ShRNA 17.9 Cells Cells 3 # SSC-A APC-A # 60 10 44,4% 10 40,3% 100K 30 2 5 50K 48.8 10 0 78.5 18.9 0 0 0 17,5% 10,2% 2 3 4 5 0 10 10 10 10 0 50K 100K 150K 200K 250K 2 3 4 5 2 3 4 5 20101812 D324 Workspace.jo 0 10 10 10 10 0Layout:10 10 D34210 10MED 72h APC-A FSC-A APC-A FITC-A FITC pos Ungated living cells living cells 011210_D324 F1 72h.fcs 011210_D324 F1 72h.fcs 011210_D324 F1 72h.fcs 011210_D324 F1 72h.fcs Event Count: 977 Event Count: 11172 Event Count: 5452 Event Count: 5452 250K 250K 120 1.17 1.34 2.57 1.94 5 5 20 Ctrl 120 Wip1 10Ctrl 10 Wip1 72h Cells 72h 72h 72h 20 ShRNA # ShRNA Cells # 200K 200K ShRNA ShRNA 90 72h 4 104 15 90 10 14.8 18.5 6 9.56 15 14,8% 18,5% 150K 150K 18.8 Cells Cells Cells 17.9 3 Cells 60 10 SSC-A APC-A # 3 10 # # SSC-A APC-A # 60 10 10 100K 100K 2 5 30 10 3050K 48 59,3% 2 56% 5 50K 48.8 10 0 0 76.5 19 0 0 78.5 18.9 0 0 0 0 2 3 4 5 2 3 4 5 0 50K 100K 150K 200K 250K 2 3 4 5 0 10 210 103 104 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 50K 100K 150K 200K 250K 2 3FSC-A 4 5 2 3 4 5 FITC-A APC-A 0 10 10 10 10 0 10 APC-A10 10 10 APC-A FSC-A Ungated living cells FITC pos APC-A living cells FITC-A FITC pos Ungated living011210_D324 cells NS 72h.fcs 011210_D324living cells NS 72h.fcs 011210_D324 NS 72h.fcs 011210_D324 NS 72h.fcs 011210_D324 F1 72h.fcs 011210_D324 F1 72h.fcs 011210_D324Event Count: F1 10949 72h.fcs Event011210_D324 Count: 5259 F1 72h.fcs Event Count: 5259 Event Count: 989 Event Count: 977 γH2AX Event Count: 11172 Event Count: 5452 γH2AXEvent Count: 5452 250K 3.49 43.9 100 5 250K 120 100 2.57 1.94 10 20 105 200K 80 80 4 200K 10 90 4 57 15 150K 10 61.4 18.5 60 9.56 60 150K 84.6 Cells SSC-A 3 Cells APC-A 10 # 10 18.8 # 100K Cells Cells 60 3 10 SSC-A 40 APC-A 10 # # 40 100K 42.3 50K 102 20 20 30 2 5 50K 48 10 0 0 9.16 43.4 00 0 0 50K 100K 150K 200K 250K 2 3 4 5 76.5 2 3 4 5 19 0 10 10 10 10 2 3 4 5 0 FSC-A 0 10 10 10 10 0 10 10 10 10 0 0 FITC-A
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