Matissek et al, Frequent Expressed Fusions in HR+ Breast Cancer

Inventory of Supplementary Data:

Supplementary Figures S1-S6 and their legends

Supplementary Tables S1-S8

Supplementary Figure S1

P A M Fusion P M FUSION AKT1 KRAS NEG PIK3CA BRAF IDH N.T.

B CTNNBL1 C ESR1 20q11.23 AF1 DBD HINGE LBD

Heat Arm PLEKGH1-ESR1 RhoGEF PH AF1 DBD HINGE LBD

3p25 RAF1 TNS3-ESR1 C1 C2 DBD HINGE LBD S6KC1 1q41 ESR1-CCDC170 AF1 SMC ATPase PX MIT PH Kinase Regulatory ESR1- CoA5 AF1 DBD 1q44 AKT3

ESR1 6q25.1

AF1 DBD

2q11.2 CoA5 Supplementary Figure S1. Identification of expressed intergenic fusions. A. Box plot summarizing somatic mutation (top) and fusion (bottom) analysis of the matched primary (P)/metastasis (M) cohort. NEG, none detected; NT, not tested. Not all patients testing negative who were not tested for both mutation and fusion are shown. B. Schematic diagrams of four predicted in-frame fusion . Chromosomal location and transcription direction (black arrows) of left (top) and right (bottom) fusion partner are shown. Dotted lines indicate contributions of left (red) and right (blue) partner genes. Arm: ARMadillo; PX: PhoX homology; PH: Pleckstrin Homology; MIT: Microtubule Interacting and Trafficking; AF1: Activation Function 1; DBD: DNA Binding Domain; DBL: DNase Binding Loop; RBD: Ras Binding Domain; C2: Calcium-dependent phospholipid binding. C. Schematic diagrams of ESR1 in-frame fusion proteins. LBD: Ligand Binding Domain; SMC: Structural Maintenance of ; C1: Phorbol esters/diacylglycerol binding domain; C2: C2 domain of PTEN tumor-suppressor . Supplementary Figure S2 A B ESR1 Primary

Different (11; 46%) Same Chromosome-Different Orientation (6; 25%) Same Chromosome-Same Orientation (7; 29%) C RAF1 Fusion Negative CTNNBL1-RAF1 Met 1 CTNNBL1-RAF1 Met 2

D AKT3 Fusion Negative RFWD2-AKT3 Primary

S6KC1-AKT3 Primary S6KC1-AKT3 Met 1 S6KC1-AKT3 Met 2

Supplementary Figure S2. FISH and IHC analysis of identified intergenic fusions. A. Chromosomal organization of identified intergenic fusions. The majority of fusions identified by AMP are amenable to detection by FISH. B. Representative photomicrograph showing FISH analysis of an ESR1-CCDC170 fusion detected by AMP. Adjacent red and green signals indicate an intact locus; white arrows indicate rearrangement (single signal). Scale bar, 10 μm. C&D. Representative images for IHC detecting RAF1 (C) and AKT3 (D) expression in indicated tumor tissues. For CTNNBL1-RAF1 and RPS6KC1-AKT3 fusions, all the same fusion- positive tissues were analyzed by FISH analysis, presented in Figure 2. Scale bar, 50 µm. Supplementary Figure S3

Phase EtBr Merged Vector CTNNBL1-RAF1 ACTL6A-PIK3CA

Supplementary Figure S3. Oncogenic phenotypes induced by kinase fusions in normal mammary epithelia. Decreased apoptosis conferred by kinase fusions, demonstrated in photomicrographs of representative ethidium bromide (EtBr)-stained 3D matrigel cultures of cells expressing the control vector, CTNNBL1-RAF1, or ACTL6A-PIK3CA fusion. Scale bar, 100 μm. Supplementary Figure S4 A B 3 4 S6KC1-AKT3Fusion p-FOXOa3 ** * AKT3WT *** 3 2 2

1 Fold 1 p-AKT p-AKT (fold)

0 0 -+ - + (serum) ------(serum) S6KC1-AKT3 AKT3 p-FOXO3A p-TSC2 p-S6

C D Whole cell lysate Plasma membrane

3 - + - + - + - + (serum) * * * S6KC1-AKT3 2 AKT3 1 Tubulin 0 - +- + (serum) Pan-Cadherin

Membrane Associated Associated AKT Membrane (fold) S6KC1-AKT3 AKT3 1 2 3 4 5 6 7 8

Supplementary Figure S4. A&B. Quantitation of Fig. 4B. The levels of phosphorylated proteins were determined and normalized against the respective total proteins, and the values shown are relative to the level in serum starved AKT3-expressing cells. (A) The average levels of p-AKT were determined from three independent experiments and error bars indicate SD; (B) The average levels of p-FOXO3A, p- TSC-2 and p-S6 were determined from two independent experiments and error bars indicate the range of the results. C. Quantitation of Fig. 4C. The levels of membrane-associated RPS6KC1-AKT3 and AKT3 were determined by normalizing against the pan-cadherin. The relative levels were determined by normalizing against the level in serum starved AKT3-expressing cells. The average levels were determined from three independent experiments and error bars indicate SD. P-value was calculated by Student’s t-test. *P<0.05, **P<0.01, ***P<0.001. D. Constitutive membrane localization of RPS6KC1- AKT3 in 293T cells. Whole cell or fractionated lysates expressing the fusion or wild-type AKT3 are shown. Compare lanes 5 vs. 6, 7 vs. 8. Tubulin and pan-cadherin serve as controls for cytosol and membrane fractions, respectively. Supplementary Figure S5 A T47D C

1.5 **

98 - 1 CTNNBL1-RAF1 64 - 0.5 36 - GAPDH 148 - 0 PIK3CA Relative Cell Number r 1 A 3 98 - ACTL6A-PIK3CA

36 - GAPDH 98 - S6KC1-AKT3 64 - GAPDH 36 -

B MK-2206 (AKTi) D 120

100 80 - + - + - + (serum) 60 Vector p-S6K CTNNBL1-RAF1 40 % Viable cells % Viable ACTL6A-PIK3CA * ** S6K 20 S6KC1-AKT3 0 p-S6 0

S6

p-S6KC1-AKT3 Trametinib (MEKi) 120 p-AKT1 E17K p-Akt 100 S6KC1-AKT3 80 AKT1 E17K 60 Vector Akt CTNNBL1-RAF1 *** 40 GAPDH

% Viable cells % Viable ** ACTL6A-PIK3CA 20 S6KC1-AKT3 0 0 1 0 10 0 0.1 10 Supplementary Figure S5

E CCND1 TFF1 12 6 10 * * 8 4 *** 6 * 4 2 * * 2 mRNA Relative Relative mRNA Relative * * ** 0 0

-E2 +E2 -E2 +E2

Supplementary Figure S5. Kinase fusions deregulate growth in hormone-dependent breast cancer models. A. Western blot showing lentiviral-mediated expression of the indicated fusion proteins in T47D cells. Endogenous PIK3CA co-migrates closes with ACLT6A-PIK3CA. B. Kinase fusions confer resistance to kinase-directed inhibitors. MCF7 cells expressing the indicated fusions were treated with an AKT inhibitor (left, AKTi) or MEK1/2 inhibitor (right, MEKi) at the indicated doses for 3 days. Values plotted represent the mean of quadruplicate wells in a representative experiment performed 3 times. Error bars indicate SD. P-value was calculated by two-sided multiple measures ANOVA. *P<0.05, **P<0.01, ***P<0.001. C. RPS6KC1- AKT3 fusion confers estrogen-independent proliferation in T47D cells propagated in charcoal-stripped serum for 5 days. Values plotted are relative cell numbers compared to day 0. Values represent the mean of quadruplicate wells in a representative experiment performed three times. Error bars indicate SD. P-value was determined by two-tailed Student’s t-test. **P < 0.01. D. Activation of mTORC1 signaling following RPS6KC1-AKT3 and AKT1 E17K expression in serum-starved MCF7PIK3CAWT cells, evidenced by increased pS6 Kinase (pS6K, T389) and pS6 (S235/236). Constitutive phosphorylation of RPS6KC1-AKT3 (AKT S473) is also apparent compared to endogenous AKT and AKT1 E17K. E. Estrogen-dependent transcription is deregulated following expression of kinase fusions. Canonical ER target genes CCND1 and TFF1 were assessed by qPCR in MCF7 cells expressing the indicated kinase fusions, following estrogen depletion (left, 3 days) then E2 stimulation (right, 100 pM, 12 hours). Values plotted represent the mean of quadruplicate wells in a representative experiment performed 4 times. P-value was determined by Student’s t-test compared to the respective vector controls. *P < 0.05; **P < 0.01; ***P < 0.001. Supplementary Figure S6 A 4 Vector S6KC1-AKT3 3 AKT1 E17K ** 2 *

1

Relative Fold Relative Change 0

0 5 10 15 20 25 30 Days B

20 10 0 -10

-20 ** Vector * -30 S6KC1-AKT3 -40 Delta Delta Tumor Volume (%) AKT1 E17K -50 0 5 10 15 Days C

Vector S6KC1 -AKT3 AKT1 E17K

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 (cm) Supplementary Figure S6. The RPS6KC1-AKT3 kinase fusion promotes tumor progression and confers hormonal therapy resistance in vivo. A. RPS6KC1-AKT3 and AKT1 E17K confer rapid growth in vivo. The ratio of tumor growth was determined by normalizing tumor size at each time point against the tumor size at Day 0, which is 15 days post injection and is the first day at which tumors are clearly palpable and measurable in all three experimental arms. Vector N=10; RPS6KC1-AKT3 N=10; AKT1 E17K N=10. B. Tumor-bearing mice underwent supplemental estrogen withdrawal (Day 0, corresponding to day 28 in Fig. S6A). Tumors expressing RPS6KC1-AKT3 and AKT1 E17K fail to regress relative to control tumors. C. The representative tumors from (B) 14 days post estrogen withdrawal. P-values were determined by two-way multiple-measures ANOVA. *P < 0.05; **P < 0.01. Supplementary Table S1 Genes and Exons Interrogated by AMP GENE Exons (Antisense Primer) Exons (Sense Primer) GENE Exons (Antisense Primer) Exons (Sense Primer) AKT3 1, 2, 3 NOTCH1 26, 27, 28, 29 2, 4, 29, 30, 31 ALK 19, (intron19), 20, 21, 22 NOTCH2 26, 27, 28 5, 6, 7 ARHGAP26 2, 10, 11, 12 NRG1 1, 2, 3, 6 BRAF 7, 8, 9, 10, 11, 12, 15 7, 8 NTRK1 8, 10, 11, 12, 13 BRD3 9, 10, 11, 12 NTRK2 11, 12, 13, 14, 15, 16, 17 BRD4 10, 11 NTRK3 13, 14, 15, 16 EGFR 7, 9, 16, 20 24, 25 NUMBL 3 ERG 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 NUTM1 3 ESR1 3, 4, 5, 6 PDGFRA 10, 11, 12, 13, 14, ETV1 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 PDGFRB 8, 9, 10, 11, 12, 13, 14 ETV4 2, 4, 5, 6, 7, 8, 9, 10 PIK3CA 2 ETV5 2, 3, 7, 8, 9 PKN1 10, 11, 12, 13 ETV6 2, 3, 5, 6, 7 1, 2, 3, 4, 5, 6 PPARG 1, 2, 3 EWSR1 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 PRKCA 4, 5, 6 FGFR1 2, 8, 9, 10, 17 PRKCB 3 FGFR2 2, 8, 9, 10 17 RAF1 4, 5, 6, 7, 9, 10, 11, 12 4, 5, 6, 7, 9 FGFR3 8, 9, 10 17, Intron 17 RELA 3, 4 FGR 2 RET 8, 9, 10, 11, 12, 13 INSR 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22 ROS1 31, 32, 33, 34, 35, 36, 37 MAML2 2, 3 RSPO2 1, 2 MAST1 7, 8, 9, 18, 19, 20, 21 RSPO3 2 MAST2 2, 3, 5, 6 TERT 2 MET 13 TFE3 2, 3, 4, 5, 6, 7, 8 2, 3, 4, 5, 6 MSMB 2, 3, 4 TFEB 1, 2 MUSK 7, 8, 9, 11, 12, 13, 14 THADA 28 MYB 7, 8, 9, 11, 12, 13, 14, 15, 16 TMPRSS2 1, 2, 3, 4, 5, 6 Supplementary Table S2 Summary of Advanced HR+ Breast Cancer Cohorts Clinical Genotyping Cohort Characteristic Total Fusion (+) Fusion (-) Age at diagnosis, years Median 49.5 53.3 48.6 Range 24-85 24-85 35-61 Stage at Diagnosis: I 15.5% (17/110) 14.3% (2/14) 15.6% (15/96) II 45.5% (50/110) 42.9% (6/14) 45.8% (44/96) III 14.5% (16/110) 21.4% (3/14) 13.5% (13/96) IV 21.8% (24/110) 21.4% (3/14) 21.9% (21/96) Unknown/Uncertain 2.7% (3/110) 0% (0/14) 3.1% (3/96) ER + 94.5% (104/110) 100% (14/14) 93.8% (90/96) PR + 72.7% (80/110) 71.4% (10/14) 72.9% (70/96) Adjuvant Tamoxifen 50.9% (56/110) 28.6% (4/14) 54.2% (52/96) Adjuvant AI 48.2% (53/110) 71.4% (10/14) 44.8% (43/96) Any Adjuvant Hormonal Therapy 87.3% (96/110)* 92.9% (13/14) 86.5% (83/96)* Adjuvant Chemotherapy 57.3% (63/110) 71.4% (10/14) 55.2% (53/96) * Includes patients initially thought to be curable but ultimately identified as de novo Stage IV. Matched Primary/Metastasis Cohort Characteristic Total Fusion (+) Fusion (-) Age at diagnosis, years Median 58.5 60 57 Range 31-91 47-89 31-91 Stage at Diagnosis: I 17.5% (11/63) 10% (1/10) 18.9% (10/53) II 54.0% (34/63) 50% (5/10) 54.7% (29/53) III 22.2% (14/63) 30% (3/10) 20.8% (11/53) IV 1.6% (1/63) 10% (1/10) 0% (0/53) Unknown/Uncertain 4.8% (3/63) 0% (0/10) 5.7% (3/53) ER + 95.2% (60/63) 80% (8/10) 98.1% (52/53) PR + 92.1% (58/63) 90% (9/10) 92.4% (49/53) Adjuvant Tamoxifen 74.6% (47/63) 90% (9/10) 71.7% (38/53) Adjuvant AI 33.3% (21/63) 40% (4/10) 32% (17/53) Any Adjuvant Hormonal Therapy 92.1% (58/63) 90% (9/10) 92.3% (49/53) Adjuvant Chemotherapy 60.3% (38/63) 70% (7/10) 54.7% (29/53) Supplementary Table S3 Summary of Detected Intergenic Fusions Case ID Primary or Metastasis Fusion (Left Partner-Right Partner) LP Exon Chromosome # (LP) RP Exon Chromosome # (RP) Mutation KM113 Primary PTCHD4-ESR1 Intron2-3 6 4 6 None KM166 Metastasis SND1-BRAF 18 7 8 7 None KM54 Metastasis FGFR1-IMMP2L 19 8 Intro3-4 7 None KM134 Metastasis VPS53-ESR1 Intron4-5 17 5 6 None KM14 Metastasis BCAS3-PRKCA 23 17 6 17 None KM23 Primary VPS53-ESR1 Intron4-5 17 5 6 None KM25 Primary TANC2-PRKCA Intron3-4 17 3 17 None KM33 Primary TIMP2-NOTCH1 2 17 2 9 PIK3CA-3140 KM42 Metastasis NBPF20-ESRRA Intron53-54 1 4 11 None KM90 Primary PLEKHG1-ESR1 3 6 2 6 None KM69 Metastasis TNS3-ESR1 16 7 5 6 PIK3CA-1633 KM115 Metastasis SEC16A-NOTCH1 5UTR 9 27 9 None KM81 Primary WDR45-TFE3 9 X 2 X None KM200 Metastasis ESR1-CCDC170 2 6 5 6 None MGH34 Primary ESR1-CoA5 4 6 2 2 None MGH31 Primary, Metastasis ESR1-ZBTB2 6 6 Intron 6 None CRO10 Primary, Metastasis CTNNBL1-RAF1 11 20 11 3 None CRO13 Metastasis ACTL6A-PIK3CA 1 3 3 3 None TRK3MII Metastasis RPS6KC1-AKT3 6 1 3 1 NT CRO05 Metastasis SND1-BRAF 17 7 9 7 None CRO08 Metastasis ITPR2-ETV6 36 12 9 12 PIK3CA-1633 CRO11 Primary LRGUK-PRKCA 4 7 4 17 None CRO18* Metastasis PRKAR2A-NOTCH2 Intron1-2 3 26 1 PIK3CA-1633 CRO18* Metastasis FGFR1-DNM1L 19 8 Intron9-10 12 PIK3CA-1633 CRO20 Primary RFWD2-AKT3 16 1 3 1 PIK3CA-1633 LN498 Primary ESR1-CCDC170 2 6 2 6 NT *Two Fusions Identified in One Sample Clinical Genotyping Cohort Matched Primary/Metastasis Cohort Primary Diagnosis Cohort Supplementary Table S4 Somatic Genotyping Genes And Alterations Interrogated Gene cDNA residues Gene Amino Acid cDNA residues AKT1 E17 49G KRAS G13 37G APC R1114 3340C KRAS G13 38G APC Q1338 4012C NOTCH1 L1575 4724T APC R1450 4348C NOTCH1 L1601 4802T APC T1556fs* 4666_4667insA NRAS G12 34G BRAF V600 1798G NRAS G12 35G BRAF V600 1799T NRAS G13 37G CTNNB1 D32 94G NRAS G13 38G CTNNB1 D32 95A NRAS Q61 181C CTNNB1 S33 98C NRAS Q61 182A CTNNB1 G34 101G NRAS Q61 183A CTNNB1 S37 109T PIK3CA R88 263G CTNNB1 S37 110C PIK3CA E542 1624G CTNNB1 T41 121A PIK3CA E545 1633G CTNNB1 T41 122C PIK3CA Q546 1636C CTNNB1 S45 133T PIK3CA Q546 1637A CTNNB1 S45 134C PIK3CA H1047 3139C EGFR G719 2155G PIK3CA H1047 3140A EGFR T790 2369C PIK3CA G1049 3145G EGFR L858 2573T PTEN R130 388C EGFR E746_A750 2235_2249del PTEN R173 517C EGFR E746_A750 2236_2250del PTEN R233 697C EGFR Exon 19 deletion PTEN K267fs* 800delA ERBB2 Exon 20 insertion TP53 R175 524G FLT3 D835 2503G TP53 G245 733G IDH1 R132 394G TP53 R248 742C IDH1 R132 395G TP53 R248 743G JAK2 V617 1849G TP53 R273 817C KIT D816 2447A TP53 R273 818G KRAS G12 34G TP53 R306 916C KRAS G12 35G Supplementary Table S5

Summary of Primary Diagnosis Cohort

Characteristic No. (N = 303) % Age at diagnosis, years Median 53 Range 30 - 84 Stage at Diagnosis: I 0 0 II 303 100 III 0 0 IV 0 0 ER + 299 98.7 PR + 275 90.7 Adjuvant Tamoxifen 242 79.9 Adjuvant AI 155 51 Any Adjuvant Hormonal Therapy 295 97.4 Adjuvant Chemotherapy 189 62.4 Mean Follow Up (Years) 12.51 Metastatic Recurrence 62 20.4 Supplementary Table S6

Primary Diagnosis Cohort FISH/AMP Summary Gene/Cases Case ID FISH Preliminary AMP Result LN283B-4 Rearrangement? Negative LN249B-4 Amplification Negative PIK3CA/303 LN267A Amplification Negative LN276A-5 Amplification Negative LN267A Rearrangement? Negative LN466 Amplification Negative LN357 Rearrangement? Negative LN289A Rearrangement? Negative ESR1/303 LN433 Rearrangement? Negative LN464 Amplification Negative LN164 Rearrangement? Negative LN498 Rearrangement? Positive LN393 Rearrangement? Negative LN42 Rearrangement? Negative LN185 Rearrangement? Negative LN364B4-4 Amplification Negative RAF1/303 LN476-5 Rearrangement? Negative LN273B Rearrangement? Negative LN164 Rearrangement? Negative AKT3/303* None None None * Not all cases exhibited optimal hybridization signal Supplementary Table S7 qPCR and Fusion Cloning Primers Gene Forward Primer Reverse Primer TFF1 GTACACGGAGGCCCAGACAGA AGGGCGTGACACCAGGAAA Cyclin D AGTGTTCAATGAAATCGTGCGGGGT AGCTCCTGTGCTGCGAAGTGGAAAC 18S GCTTAATTTGACTCAACACGGGA AGCTATCAATCTGTCAATCCTGTC RAF1 ACATGAAGGAGATGTTGCAGTAAAGATCCT CTAGAAGACAGGCAGCCTC PIK3CA CGGAGGAGGTTTTGCTATCGGCATGCCA TCAGTTCAATGCATGCTGTTTAA AKT3 TGTTCAAGGAGAATATATAAAAAACTGGAGGC TTATTCTCGTCCACTTGCAGAG CTNNBL1 CACCATGGGACGTCGGAAACAAACTGGTAC AACATCTCCTTCATGTTCCTTCTCAGTGGT S6KC1 CACCATGGGAACCTCTTACCGGGAGCGGA ATATTCTCCTTGAACACCTTCTAGGAGT ACTL6A CACCATGGGAAGCGGCGGCGTGTACGGGGGAGGTTTTGCT CTNNBL1-RAF1 CACCATGGGACGTCGGAAAC CTAGAAGACAGGCAGCCTC ACTL6A-PIK3CA CACCATGGGAAGTGGTGGT TCAGTTCAATGCATGCTGTTTAA S6KC1-AKT3 CACCATGGGAACCTCTTACC TTATTCTCGTCCACTTGCAGAG Supplementary Table S8 Antibodies Used Target Catalog Number (Provider) AKT 4685S (Cell Signaling Technologies) AKT-3 14982S (Cell Signaling Technologies) AKT-3 HPA002640 (Sigma) Alexa Fluor 488 A21121 (Molecular Probes) FoxO3a 12829S (Cell Signaling Technologies) GAPDH sc-25778 (Santa Cruz Biotechnology) p70 S6 Kinase 9202S (Cell Signaling Technologies) Pan-Cadherin 4068S (Cell Signaling Technologies) Phospho-AKT (S473) 4051S (Cell Signaling Technologies) Phospho-FoxO3a (Ser253) 9466S (Cell Signaling Technologies) Phospho-p70 S6 Kinase 9205S (Cell Signaling Technologies) Phospho-Rb(Ser780) 9307 (Cell Signaling Technologies) Phospho-S6 Ribosomal Protein (Ser235/236) 4858S (Cell Signaling Technologies) Phospho-Tuberin/TSC2 (Thr1462) 3617S (Cell Signaling Technologies) PI3 Kinase p110α 4249S (Cell Signaling Technologies) RAF1 sc-7267 (Santa Cruz Biotechnology) RAF1 HPA026441 (Sigma) Rb 9309 (Cell Signaling Technologies) S6 Ribosomal Protein 2217S (Cell Signaling Technologies) Tuberin/TSC2 3635S (Cell Signaling Technologies) Tubulin MAB3408 (Millipore) β-catenin 610153 (BD Transduction Laboratories) Fluorescein (FITC)-conjugated AffiniPure Donkey Anti-Rabbit IgG 711-095-152 (Jackson Immunoresearch) Biotin-conjugated Goat Anti-Rabbit IgG 111-066-047 (Jackson Immunoresearch)