Lung Met 1 Lung Met 2 Lung Met Lung Met H3K4me1. Lung Met H3K27ac Primary H3K4me1

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1 a Gained Met-VELs Lung Met 1 Lung Met Lung Met 3 1. Lung Met H3K4me1 Lung Met H3K4me1 1 Lung Met H3K4me1 Lung Met H3K7ac 1.5 Lung Met H3K7ac Lung Met H3K7ac.8 Primary H3K4me1 Primary H3K7ac 1 Primary H3K4me1 Primary H3K7ac.6 Primary H3K4me1 Primary H3K7ac Lost Met-VELs Lung Met H3K4me1 Lung Met H3K7ac Primary H3K4me1 Primary H3K7ac Lung Met H3K4me1 Lung Met H3K7ac Primary H3K4me1 Primary H3K7ac Lung Met H3K4me1 Lung Met H3K7ac Primary H3K4me1 Primary H3K7ac Gained Met-VELs Lost Met-VELs Gained Met-VELs b 1.! 1!.8!.6!.4!.!! -.! -.4! Lung Met MNNG H3K4me1! MNNG H3K7ac! MNNG DNase! HOS H3K4me1! HOS H3K7ac! HOS DNase! 1.8! 1.6! 1.4! 1.! 1!.8!.6!.4!.!! -.! -.4! Lung Met H3K4me1 Lung Met H3K7ac Primary H3K4me1 Primary H3K7ac B H3K4me1! 143B H3K7ac! 143B DNase! HOS H3K4me1! HOS H3K7ac! HOS DNase! 1.6! 1.4! 1.! 1!.8!.6!.4!.!! -.! -.4! Lung Met H3K4me1 Lung Met H3K7ac Primary H3K4me1 Primary H3K7ac MNNG 143B LM7 LM7 H3K4me1! LM7 H3K7ac! LM7 DNase! SaOS H3K4me1! SaOS H3K7ac! SaOS DNase! Lost Met-VELs 1.! 1!.8!.6!.4!.!! -.! MNNG H3K4me1! MNNG H3K7ac! MNNG DNase! HOS H3K4me1! HOS H3K7ac! HOS DNase! 1.! 1!.8!.6!.4!.!! -.! 143B H3K4me1! 143B H3K7ac! 143B DNase! HOS H3K4me1! HOS H3K7ac! HOS DNase! 1.4! 1.! 1!.8!.6!.4!.!! -.! -.4! LM7 H3K4me1! LM7 H3K7ac! LM7 DNase! SaOS H3K4me1! SaOS H3K7ac! SaOS DNase! M Gained Met-VELs M11 H3K4me1 M11 H3K7ac M11 DNase Hu9 H3K4me1 Hu9 H3K7ac Hu9 DNase Lost Met-VELs M11 H3K4me1 M11 H3K7ac M11 DNase Hu9 H3K4me1 Hu9 H3K7ac Hu9 DNase Nature Medicine: doi:1.138/nm.4475

2 Supplementary Figure 1: Met-VEL profiles of osteosarcoma patient lung metastases and human osteosarcoma cell lines. a, Aggregate plots showing H3K4me1 ChIP-seq and H3K7ac ChIP-seq signal +/- 3Kb from midpoints of gained and lost Met-VELs in paired patient lung metastases and primary tumors. b, Aggregate plots showing H3K4me1 ChIP-seq, H3K7ac ChIP-seq and DNase-seq signal +/- 3Kb from mid-points of gained and lost Met-VELs in metastatic/parental human osteosarcoma cell lines. Nature Medicine: doi:1.138/nm.4475

3 a Gained Met-VEL Cluster Lost Met-VEL Cluster.5 1. H3K4me1 H3K4me1 GHR COL3A1 MG63 (Parental) MG63.3 (Metastatic) Gained Met-VEL 1kb MG63 (Parental) MG63.3 (Metastatic) Gained Met-VEL 1kb b MG63.3 Lost Met-VEL Clusters LHFP Scaled Coordinates WASF3 COL3A1 Chromosomes 16 Lost Met-VELs in kb window c Number of Clusters Lung Met 1 Lung Met Lung Met 3 Lung Met 4 Lung Met 5 MG63.3 LM7 M11 MNNG 143B Gained Met-VELs Lost Met-VELs d % Total Met-VELs 1 5 Gained Met-VELs outside clusters within clusters Lung Met 1 Lung Met Lung Met 3 Lung Met 4 Lung Met 5 MG63.3 LM7 M11 MNNG 143B % Total Met-VELs 1 5 Lung Met 1 Lung Met Lost Met-VELs outside clusters within clusters Lung Met 3 Lung Met 4 Lung Met 5 MG63.3 LM7 M11 MNNG 143B Nature Medicine: doi:1.138/nm.4475

4 Supplementary Figure : Met-VEL clusters occur across metastatic cancers. a. UCSC browser view of H3K4me1 profiles in MG63.3 (metastatic) and MG63 (parental) cell lines illustrating an example of a gained (left) and lost (right) Met-VEL cluster. Met-VELs identified by black bars. kb Met-VEL clusters highlighted in gray. b. Genome-wide lost Met-VEL landscape for MG63.3 cell line. Rows represent scaled chromosomal coordinates. Peaks represent maximum gained Met-VEL counts in kb sliding windows. Predicted target genes for selected peaks are labeled. c. Gained and lost Met-VEL cluster counts in patient lung metastases/primary tumors and metastatic/parental cell line pairs. d. Percentage of total gained (top) and lost (bottom) Met-VELs within and outside of clusters in patient lung metastases/primary tumors and metastatic/parental cell line pairs. Nature Medicine: doi:1.138/nm.4475

5 a Parental Metastatic b 1 MNNG Pair Gained Gained Met-VEL Met-VEL Genes Cluster Genes Lost Met-VEL Genes Lost Met-VEL Cluster Genes 4hrs 4hrs 14 days log(fpkm) *** *** *** *** ** *** * RNA-seq d Lost Met-VEL Motifs MG63.3 MNNG 143B FOSL1 JUNB JUND UBP1 FOS JUN JDP BACH1 MAF NFEL TFAPA NFIX ZIC1 CTCF TFAP4 ZBTB7B TFAPC EF6 EF1 ZIC SMAD4 TCF1 ATF4 NFIB MYC ZNF711 TFCP ATF MAFA MAFF log(fpkm) B Pair Gained Gained Met-VEL Met-VEL Genes Cluster Genes *** Lost Met-VEL Genes *** *** * Lost Met-VEL Cluster Genes Parental line 4hrs Metastatic line 4hrs Metastatic line day 14 *** * 3-1*log(p-value) G B Color Key e Lost Met-VEL Genes MG63.3 MNNG 143B embryonic morphogenesis extracellular matrix proteinaceous extracellular matrix transcription factor activity tube development ECM-receptor interaction cell motion -1*log p-value <3-7 <7-1 <1-13 <13-15 >15 enzyme linked receptor protein signaling pathway extracellular matrix part phosphoprotein nucleus embryonic skeletal system development acetylation 1 6 c MNNG 143B Early Constitutive Late Early Constitutive Late Normalized Expression -3 3 Met Day 1 Met Day 14 Parental Day 1 Met Day 1 Met Day 14 Parental Day 1 Nature Medicine: doi:1.138/nm.4475

6 Supplementary Figure 3: Assessment of Met-VEL associated gene expression during metastatic colonization of the lung. a. Schematic of experimental design for assessment of Met-VEL gene expression in parental and metastatic cell lines in ex vivo lung metastasis model. Image adapted from 9. b. Log quantile-normalized FPKM values for gained (left) and lost (right) Met-VEL and Met-VEL cluster genes in HOS/MNNG (top) and HOS/143B (bottom) cell line pairs. Asterisks indicate significant differences in FPKM distributions between parental and metastatic cell lines (* P<.5; ** P<1E-3; *** P<1E-4). P-values calculated by Mann-Whitney Test. c. Heatmap of up-regulated gained Met-VEL genes in HOS/MNNG (top) and HOS/143B (bottom) cell line pairs. d. Expressed transcription factors with enriched motifs in lost Met-VELs in three metastatic/parental cell line pairs and corresponding motif enrichment p-values. e. Gene Ontology (GO) terms for lost Met-VEL genes in three metastatic/parental cell line pairs and corresponding p-values. Nature Medicine: doi:1.138/nm.4475

7 a Gained Met-VEL Genes b Lost Met-VEL Genes MG63.3 MNNG 143B MG63.3 MNNG 143B 4 p=8.5e-4 4 p<.e-16 p<.e-16 p=.17 p=9.33e-5 p=4.1e-1 p=.76 p=.57 4 p=.15 p=1.74e-5 p=1.1e-4 p=1.1e-5 log(fold change expression) Met vs. Parental in vitro Met 4hrs lung vs. Parental 4hrs lung Met 14 days lung vs. Parental 4hrs lung Met vs. Parental in vitro Met 4hrs lung vs. Parental 4hrs lung Met 14 days lung vs. Parental 4hrs lung c log(fold-change expression met. vs. non-met) MG63.3 Pair MNNG Pair 143B Pair All Expressed Genes Gained Met-VEL Genes Lost Met-VEL Genes d Gained Met-VELs 5 5 Lost Met-VELs Number of Genes 15 1 Number of Genes MG63.3 MNNG 143B MG63.3 MNNG 143B Top up-regulated genes Overlap Gained Met-VEL Genes Top down-regulated genes Overlap Lost Met-VEL Genes Nature Medicine: doi:1.138/nm.4475

8 Supplementary Figure 4: Assessment of Met-VEL associated gene expression in vitro and in ex vivo lung culture. a. Log fold-change in quantile-normalized FPKM values of gained Met-VEL genes in metastatic cell lines versus parental cell lines in various conditions. P-values calculated by Mann-Whitney Test. b. Log fold-change in quantile-normalized FPKM values of lost Met-VEL genes in metastatic cell lines versus parental cell lines in various conditions. P-values calculated by Mann-Whitney Test. c. Violin plots of log(fold-change quantile-normalized FPKM values) distributions for gene sets in three metastatic cell lines relative to parental non-metastatic lines. Fold-change values for all expressed genes and gained Met-VEL gene sets represent comparisons of expression in metastatic lines at day 14 in ex vivo lung culture to non-metastatic lines at 4hrs. Fold-change values for lost Met-VEL gene set represent comparison of expression in metastatic lines at 4hrs in ex vivo lung culture to non-metastatic lines at 4hrs. d. Bar charts indicating overlap between gained (left) and lost (right) Met-VEL gene sets and top 1 up- and down-regulated genes in corresponding conditions, respectively. Nature Medicine: doi:1.138/nm.4475

9 Regulation of cell migration Angiogenesis and Endothelial cell migration Endothelial Developmental Programs Embyronic Epithelial tube Neg. regulation of cell migration Growth Urothelial Adherens junction Epithelial cell migration Actin cytoskeleton organization Epithelial cell proliferation Pathways in cancer Signaling Pathways Ossification Rap1 Regulation of cell shape WNT ERBB Hippo IL-6 response Node Color = FDR Protein dephosphorylation Protein localization Negative regulation to plasma membrane of protein phosphorylation.5.1 <.5 Nature Medicine: doi:1.138/nm.4475

10 Supplementary Figure 5: Assessment of enriched functions of gained Met-VEL genes in osteosarcoma patient metastases. Enriched Map representation of all Gene Ontology (GO) terms for gained Met-VEL genes calculated by aggregating gene lists from two patient metastases (Lung Met 4 and Lung Met 5). Nature Medicine: doi:1.138/nm.4475

11 a Normalized Metastatic Burden Vehicle 5nM JQ1 MNNG p=7.45e-8 b Mouse 1 Mouse Vehicle MNNG 5nM JQ1 c Day 15 Vehicle Time (days) 143B Vehicle 143B 5nM JQ1 Day 15 5nM JQ1 Normalized Metastatic Burden Vehicle 5nM JQ1 p=1.73e-5 Mouse 1 Mouse Time (days) d MNNG Activated Gained Met-VEL Genes. 143B Activated Gained Met-VEL Genes ES -.6 NES = FDR <.5 ES NES = -1.7 FDR < f 4 * * MNNG * 1 143B * * Vehicle Treated JQ1 Treated Vehicle Treated JQ1 Treated 3 5 e MNNG Activated Met-VEL Gene Expression Log(fold-change vs. parental line) p<.e Vehicle 5nM JQ1 Log(fold-change vs. parental line) 143B Activated Gained Met-VEL Genes 4 - p=5.78e-7 % Reduction in Expression 1-1 All Activated Genes Lost Met-VEL Genes Activated Gained Met-VEL Genes Activated Gained Met-VEL Genes no SEs SE Genes % Reduction in Expression All Activated Genes Lost Met-VEL Genes Activated Gained Met-VEL Genes Activated Gained Met-VEL Genes no SEs SE Genes Nature Medicine: doi:1.138/nm.4475

12 Supplementary Figure 6: Analysis of anti-metastatic and gene expression effects of BET inhibition with JQ1. a. Kinetics of metastatic outgrowth of MNNG (top) and 143B (bottom) metastatic cell lines in ex vivo lung culture with 5nM JQ1 or vehicle (DMSO) treatment. Metastatic burden measured as total GFP+ area per lung section normalized to GFP+ area on day. Values represent averages of 8 lung sections (4 sections per mouse x mice) +/-SEM. P-value calculated by Mann-Whitney Test. Dashed line indicates time points chosen for RNA-seq studies. b. Representative.5x images of vehicle (left) and 5nM JQ1 treated (right) lung sections at day 15. Lung sections outlined with dashed white line. Scale bar = 5µm. c. X image of hematoxylin and eosin stained section of lung slice after 15 days in ex vivo culture treated with DMSO (top) and 5nM JQ1 (bottom) illustrating viable lung cells and architecture. Scale bar = 1µm. d. GSEA plots of -fold up-regulated gained Met-VEL gene sets in vehicle versus JQ1-treated MNNG (left) and 143B (right) cells isolated from ex vivo lung culture. Cells isolated at time points indicated by dashed lines in Supplemental Figure -5a. e. Log fold-change expression -fold up-regulated gained Met-VEL gene sets in vehicle versus JQ1-treated MNNG (left) and 143B (right) cells isolated from ex vivo lung culture at time points indicated by dashed lines in Extended Data Figure 7a relative to parental cell line. f. Percent reduction in gene expression with 5nM JQ1 treatment of all genes up-regulated -fold in metastatic cell lines relative to parental cell lines at sorting time points, lost Met-VEL genes, gained Met-VEL genes -fold up-regulated relative to parental cell lines at sorting time points, upregulated gained Met-VEL genes without SE genes, and all SE genes in MNNG and 143B cells growing in ex vivo lung culture sorted at time points indicated in Extended Data Figure 7a. Nature Medicine: doi:1.138/nm.4475

13 Nature Medicine: doi:1.138/nm.4475

14 Supplementary Figure 7: Assessment of LT3REPIR shrna construct induction and leakiness. a. Schematic of doxycycline-inducible LT3REPIR shrna construct. Modified from (Fellmann et al., 13). b. Cytometric analysis of GFP and DsRed expression in GFP+ MG63.3 cells transduced with LT3REPIR construct at baseline (left) and 4hrs after exposure to 5ug/ml doxycycline (right). Nature Medicine: doi:1.138/nm.4475

15 Nature Medicine: doi:1.138/nm.4475

16 Supplementary Figure 8: Assessment of Tissue Factor (F3) dysregulation in metastatic osteosarcoma. a. IGV browser view of H3K7ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3 (metastatic) and MG63 (parental) cell lines. Top of figure shows local contact profile analysis of F3 locus in MG63.3. In the top panel (main trend), the contact intensity (black line) is calculated by using a running mean analysis of normalized read counts with a 1kb sliding window. The th and 8th percentile are visualized as a gray trend graph. In the bottom panel, contact intensities are computed using linearly increasing sliding windows (scaled -5 kb) and are displayed as a color-coded heatmap of positive 4C signals (maximum of interaction set to 1). Local color changes are log-scaled to indicate changes of statistical enrichment of captured sequences, corresponding to the enhancer-promoter interaction. Areas of significant contact highlighted. b. Fold-change quantile normalized F3 FPKM values in 3 metastatic cell lines at 4hrs and 14 days of metastatic outgrowth in ex vivo lung culture relative to parental line at 4hrs. c. Tissue Factor (F3) relative expression in human patient primary tumors and lung metastases normalized to expression in normal osteoblasts. d. IGV browser view of H3K7ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3, MNNG, and 143B metastatic cell lines. Nature Medicine: doi:1.138/nm.4475

17 Nature Medicine: doi:1.138/nm.4475

18 Supplementary Figure 9: F3 expression in human osteosarcoma tumors. Immunohistochemical staining of F3 in human osteosarcoma lung metastases and primary tumors. Scale bars = µm. Nature Medicine: doi:1.138/nm.4475

19 a. MG63.3 DHS chr1 p36.3 p36. p36.13 p36.11 p35.1 p34. p33 p3.3 p3.1 p31. p31.1 p.3 p.1 p1. p13.3 p13.1 p11.1 q1 q1.1 q1.3 q3.1 q3.3 q4. q5.1 q5.3 q31.1 q31.3 q3.1 q3. q41 q4.11 q4.mg63 q43h3k7ac q kb MG63.3 H3K7ac Patient Lung Mets F3 ESC TADs IMR9 TADs b. SEs lost in Mets SEs gained in Mets log1(wilcox p_val) F mean(met) mean(non_met) Nature Medicine: doi:1.138/nm.4475

20 Supplementary Figure 1: Assessment of Tissue Factor (F3) Met-VELs in patient lung metastases. a. IGV browser views of the F3 locus showing H3K7ac ChIP-seq tracks of lung metastases from 1 osteosarcoma patients. Blue bars below each track correspond to super enhancers defined using the ROSE script. Hi-C defined topologically associating domains (TADs) are displayed below. b. Volcano plot of 5571 total super enhancers detected in all osteosarcoma patient samples and cell lines used in this study. Points marked in red denote super enhancers meeting the threshold of significance (P <.5) for being gained or lost in metastatic samples. Points in grey denote super enhancers below the significance threshold. The F3 super enhancer is indicated by the arrow. Nature Medicine: doi:1.138/nm.4475

21 a GAPDH b F3 d.5 c Relative GAPDH Expression -Dox vs. +Dox MG63.3 shf3a MG63.3 shf3b Normalized F3 Expression Dox +Dox -Dox +Dox shf3-a shf3-b Nature Medicine: doi:1.138/nm.4475

22 Supplementary Figure 11: Assessment of F3 knockdown by RT-qPCR. a, Amplification plots and standard curves of GAPDH RT-qPCR using.ng, ng, ng, and ng of template cdna demonstrating efficiency value of 1% and R value of 1.. All Cq values used for quantification of GAPDH were confirmed to be within linear range of standards. b, Amplification plots and standard curves of F3 RT-qPCR using.ng, ng, ng, and ng of template cdna demonstrating efficiency value of 97.% and R value of.999. All Cq values used for quantification of F3 were confirmed to be within linear range of standards. c, Relative GAPDH expression in DsRed+ (induced) cells transduced with F3 shrna constructs after 4hrs treatment with 5ug/ml doxycycline relative to uninduced controls (average of 3 replicates +/-SEM). d, Relative F3 expression normalized to GAPDH in DsRed+ (induced) cells transduced with F3 shrna constructs after 4hrs treatment with 5ug/ml doxycycline relative to uninduced controls (average of 3 replicates +/-SEM). Nature Medicine: doi:1.138/nm.4475

23 +Dox (F3 kd) -Dox a 8 MG63.3 shf3a 1 MG63.3 shf3b Fold-Change Confluence 6 4 MG63.3 shf3a untreated MG63.3 shf3a 5ug/ml dox Fold-Change Confluence MG63.3 shf3b untreated MG63.3 shf3b 5ug/ml dox 5 1 Time (hrs) 5 1 Time (hrs) MNNG shf3a MNNG shf3b 5 5 Fold-Change Confluence MNNG shf3a untreated MNNG shf3a 5ug/ml dox Fold-Change Confluence MNNG shf3b untreated MNNG shf3b 5ug/ml dox 5 1 Time (hrs) d b Inducible shf3a MG63.3 Inducible shf3b Inducible shf3a MNNG Inducible shf3b Mouse 1 -Dox +Dox (F3 kd) Mouse 5 1 Time (hrs) c 1 MG63.3 MNNG 15 p =.3 p =. p =.13 p =.3 P=.3 P=. P=.13 P=.3 Mouse 3 Normalized Metastatic Burden Dox +Dox Inducible shf3a x x -Dox +Dox Inducible shf3b x x Burden Normalized Metastatic Burden 1 5 -Dox +Dox Inducible shf3a x x -Dox +Dox Inducible shf3b x x e log[total area per.5x field(pixels)] P<.1 6 n=5 mice per condition -Dox +Dox (F3 kd) Nature Medicine: doi:1.138/nm.4475

24 Supplementary Figure 1: Assessment of F3 knockdown on metastatic osteosarcoma cell in vitro growth and lung colonization. a. Change in confluence relative to day of MG63.3 (top) and MNNG (bottom) cells transduced with F3 shrna constructs grown in standard culture conditions over 18hrs in the presence or absence of 5ug/ml doxycycline. Values represent averages from 6 plates +/- SD. b. Representative.5x images of from day 1 of ex vivo lung culture sections of GFP+ MG63.3 (left) and MNNG (right) cells transduced with F3 shrna constructs untreated (top) or treated with 5ug/ml doxycycline (bottom). Lung sections outlined with dashed white line. Scale bar = 5µm. c. Quantification of metastatic burden at day 1 of ex vivo lung culture sections of GFP+ MG63.3 (left) and MNNG (right) cells transduced with F3 shrna constructs untreated (red) or treated (blue) with 5ug/ml doxycycline. Values represent averages +/-SEM from 8 sections per condition (4 sections per mouse x mice) normalized to the same section at day. P-Value calculated by Mann-Whitney Test. d. Representative.5x images of in vivo metastatic burden in untreated (left) or doxycyclinetreated (right) mice receiving tail vein injection of 5x1 5 GFP+ MG63.3 cells transduced with shf3b construct. Scale bar = 5µm. e. Quantification of in vivo metastatic burden in untreated (red) or doxycycline-treated (blue) mice receiving tail vein injection of 5x1 5 GFP+ MG63.3 cells transduced with shf3b construct. Values represent log of total GFP+ area per.5x field, black bars represent average +/- SEM (N= 5 mice per condition, 5 images per mouse). P-Value calculated by Mann-Whitney Test. Nature Medicine: doi:1.138/nm.4475

25 a Field 1 Field Field 3 Field 4 Field 5 Mouse 5 Mouse 4 Mouse 3 Mouse Mouse 1 Mouse 5 -Dox (Uninduced) Mouse 4 Mouse 3 Mouse Mouse 1 b Field 1 Field Field 3 Field 4 Field 5 +Dox (F3 knockdown) Nature Medicine: doi:1.138/nm.4475

26 Supplementary Figure 13: Assessment of lung metastatic burden at experimental end point of orthotopic spontaneous metastasis experiment with F3 knockdown. a. All images used for quantification of in vivo metastatic lesions in lungs 1 days after measureable tumor formation in untreated mice receiving orthotopic injection of 8x1 5 MG63.3 cells transduced with shf3b construct (5 mice per condition, 5 images per mouse). Scale bar = 5µm. b. All images used for quantification of in vivo metastatic lesions in lungs 1 days after measureable tumor formation in doxycycline-treated mice receiving orthotopic injection of 8x1 5 MG63.3 cells transduced with shf3b construct (5 mice per condition, 5 images per mouse). Scale bar = 5µm. Nature Medicine: doi:1.138/nm.4475

27 Gene Inclusion Criteria Metastasis Specific? BACH Enriched Motif N DOCK4 Common Gained Met-VEL N Gained Met-VEL Cluster (MG63.3) F3 Gained Met-VEL Cluster (MG63.3) Y FBXO4 Gained Met-VEL Cluster (MNNG/143B) Y FLNA Common Gained Met-VEL Y Gained Met-VEL Cluster (MNNG/143B) FOS Enriched Motif Y FOSL1 Enriched Motif Y FOXO3 Gained Met-VEL Cluster (MNNG/143B) Y GHR Gained Met-VEL Cluster (MG63.3) N JUN Enriched Motif N JUNB Enriched Motif N RASSF Common Gained Met-VEL N TMEM3 Common Gained Met-VEL N Nature Medicine: doi:1.138/nm.4475

28 Supplementary Table 1: Candidate metastasis dependency genes identified by in vivo highthroughput RNAi functional assay. Nature Medicine: doi:1.138/nm.4475