Supplementary Figure 1 A B ER + Control PR + Control HER2 + Control

C

D E Change Effect Ppp2r5b c.578A>C p.Lys193Thr Raf1 c.1761C>G p.Asp587Glu Sf3b1 c.2838A>C p.Lys946Asn

Jak1 c.2870T>G p.Phe957Cys Jak1 c.2726T>C p.Leu909Pro Pten c.-834C>A Premature Start

Fancb c.1310A>C p.Lys437Thr Fancg c.1232A>T p.Gln411Leu Map3k2 c.1344A>C p.Gln448His

1.0 Brca1-deletion F 0.8

0.6

0.4 Cosine similarity

0.2

0.0

1.0 Signature 6 Signature 3 Signature 5 Signature 8 Signature 9 Signature 4 Signature 2 Signature 7 Signature 17 Signature 21 Signature 10 Signature 15 Signature 16 Signature 19 Signature 12 Signature 11 Signature 20 Signature 13 Signature 14 Signature 18 Signature 1A Signature 1B

0.8 Brca1-WT 0.6

0.4 Cosine similarity

0.2

0.0 Signature 5 Signature 9 Signature 3 Signature 8 Signature 4 Signature 6 Signature 7 Signature 2 Signature 17 Signature 16 Signature 12 Signature 19 Signature 20 Signature 14 Signature 11 Signature 21 Signature 15 Signature 18 Signature 13 Signature 10 Signature 1B Signature 1A G Brca 1 WT Brca 1 Del I Egfr (Amp) Fgfr2 (Amp) Tumor #1 Pten (Del) Fgfr2 (Gain) Tumor #6 ● ●● 150

●

● ●●

● 100

●● ●● ● ●● ● ●● ● ●

● ● ● 50 ● ●● ● ●● ● ● ● ●● ●●●●● ●● ● # of Chr Breakpoints # of Chr ● ● ●● ●●● ● ●●● ● ●●

●● ● ● ● ● ● 0

BRCA1 WT BRCA1 DEL Copy Number (log2) Copy Number H Position (Mb) Chr# Chr# Chr# Chr#

Met Amp Yap Amp Myc Amp Rb1 Del Figure S1. Establishment of mouse models that recapitulate human TNBCs. A) Schematics for mouse breeding to generate both cohorts of mice that developed breast tumors. B) Positive controls for ER and HER2 IHC staining. C) The majority of the mouse tumors are classified as basal-like with high probability using PAM50 classifier. D) Unsupervised hierarchical clustering groups mouse tumors with human TNBCs. E) Non-synonymous mutations identified in the

GEMM models. F) Mutation signatures of Brca1-deficient (top) and –WT (bottom) tumors show similarity between murine tumors and human breast cancer mutation signatures. G) Brca1- deficient tumors show increased genomic instability. H) Focal recurrent amplification of Met and

Yap1 in a subset of breast tumors. Yellow arrows point to the chromosomal localization of the

Met and Yap1 loci on 6 or 9, respectively. I) Focal amplification of Egfr and Fgfr2 or deletion of Pten in three different tumors.

Supplementary Figure 2

A Translocation Types Frequency C UTR-Intronic Region 16.6% Rpl32--Raf1 Kinase UTR-UTR 4.4% Junction point for Ffgr2-Dnm3 UTR-CDS (Truncated Coding Seq) 12.6% D

UTR-Exonic (No Known Coding) 2.5% CDS-Exonic 6.9%

CDS-Intronic 3.3% Intronic-Intronic 0.8% Exonic -Exonic 1.5%

Out-of-Frame Coding Sequence 21.6% E Junction point for Fgfr2-Tns1 In-frame Coding Sequence 29.6% B Gene 1 Chr Location Gene 2 Chr Location Fgfr2 7:130167703:- Dnm3 oral1:162019992:- Fgfr2 7:130167703:- Tns1 1:74016186:-

Tgfa 6:86229484:+ Unc119 11:78347218:+ Fgfr2 Tns1 Fancl 11:26434500:+ Wwox 8:115351638:+oral Shq1 6:100630883:- Ralgps2 1:156811536:-

Cyth3 5:143622673:+ Exoc4 6:33579982:+

Trim2 3:84177632:- Gabpb2 oral3:95200291:- Ank1 8:22975261:+ Lrrc16a 13:24025935:- F Cdc5l 17:45404594:- Cdk8 5:146268215:+ Junction point for Dhx9-Raf1 Anp32b 4:46469995:+ Ncl oral1:86357083:- Mapkapk2 1:131097289:- Pard3b 1:61767948:+ Fry 5:150372608:+ Stim2 5:54075280:+

Dlg1 16:31684294:+ Braf 6:39648486:-

Lamc1 1:153332128:- Emx2 19:59461662:+ Dhx9 Raf1 Fam65a 8:105617231:+ Farp1 14:121219377:+oral Dcaf11 14:55563019:+ Wdr45b 11:121341215:-

Moxd1 10:24223867:+ Letm2 8:25581786:- Plekha1 8:25088342:- Eya4 oral10:23226851:- Figure S2. Chromosomal translocation events identified in spontaneous tumors developed in

GEMM mice. A) Frequency of translocation events involving different genomic regions. B)

Examples of in-frame fusion events. C) Schematics of Rpl32-Raf fusion kinase. D) IGV plots showing RNAseq reads mapping to junction points for Fgfr2-Dnm3. E) IGV plots showing

RNAseq reads mapping to junction points for Fgfr2-Tns1 and Sanger sequencing traces for

Fgfr2-Tns1 fusion junction. F) IGV plot showing RNAseq reads mapping to junction point for

Dhx9-Raf1 and Sanger sequencing traces for Dhx9-Raf1 fusion junctions.

Supplementary Figure 3

A B Peptide Sequence Tumor# Fusion Fgfr2 DLVSCTYQLAR #1-1 Fgfr2-Dnm3 Fgfr2 DSGLYACTAAR #1-2 Fgfr2-Dnm3 Fgfr2 YQISQPEAYVVAPGESLELQCMLK #12 same genotype Fgfr2 W[oxM]APEALFDR #4-1 same genotype Fgfr2 NVLVTENNVMK #4-2 same genotype Fgfr2 IADFGLAR C Fgfr2 EAVTVAVK Fgfr2 QLVEDLDR Tumor# Fusion Fgfr2 QVTVSAESSSSMNSNTPLVR #5-1 Dhx9-Raf1 Fgfr2 APYWTNTEK #5-2 Dhx9-Raf1 Fgfr2 DAAVISWTK #5-3 Dhx9-Raf1 Fgfr2 GMEYLASQK #16 same genotype Fgfr2 VYSDAQPHIQWIK #12 same genotype Fgfr2 TVLIGEYLQIK #19 same genotype Fgfr2 DINNIDYYKK #1 Fgfr2-Dnm3 Fgfr2 MDKPTNCTNELYMMMR D Fgfr2 GMEYLASQK Tns1 VSAQGITLTDNQR Tumor# Fusion Tns1 GAYGLAMK #2-1 Met overexp. E #2-2 Met overexp. Tumor# Fusion

#2-3 Met overexp. #10 same genotype #2-4 Met overexp. #13 same genotype #2-5 Met overexp. #19 same genotype #20 same genotype #5 Dhx9-Raf1 #21 same genotype #12 same genotype #22 same genotype #28 same genotype #23 same genotype #16 same genotype #15 same genotype

#1 Fgfr2-Dnm3 #17 Pten deletion #18 same genotype #30 same genotype #29 same genotype Figure S3. Confirming the presence of fusion transcripts and fusion . A) Sample key for western lysates used in Figure 3A. B) Peptides identified by mass spectrometry that match mouse Fgfr2 and Tns1 from tumor #6 where RNAseq calls for Fgfr2-Tns1 fusion, but not from tumor #4 without out fusion call. C) Sample key for western lysates used in Figure 3B. D)

Sample key for western lysates used in Figure 3C. E) Sample key for western lysates used in

Figure 3D.

Supplementary Figure 4 Fusion Fusion Fusion Fusion Fusion Endo Endo * * FGFR2-CCDC6 C A pBabe-puro

Dhx9-Raf1 3T3 Dlg1-BRaf Fgfr2-Dnm3 0 HA BRaf Raf1 Fgfr2 Ig1 IG−like Ig2 IG−like Ig3 IG−like D E TM TM tERK pERK pAKT tAKT HMEC

Signal realtive to Day 1 FGFR2−CCDC6 Kinase HMEC Proliferation

pBabe Kinase Fgfr2-Dnm3 p=0.0003 *

pBabe Days Dhx9-Raf1 tERK pERK pAKT tAKT 1140 B

pBabe-puro 3T3 FGFR2-CCDC6 Fgfr2-Dnm3 tErk pErk (long) pErk (short) t Endo-Fgfr2 t Fusion-Fgfr2 p Endo-Fgfr2 p Fusion-Fgfr2 pS6 HA tS6 Figure S4. Effects of various fusion kinases on NIH3T3 cells and HMECs. A) Schematics of the domain structure of FGFR2-CCDC6 found in human breast cancer. B) and C NIH3T3 cells stably expressing various fusions confirmed by HA blotting and respective protein-specific antibody blotting. D) Fgfr2-Dnm3 or Dhx9-Raf1 increases AKT and ERK phosphorylation in immortalized human mammary epithelial cells (HMECs). E) Expression of cMet in HMECs increases cell proliferation.

Supplementary Figure 5

A GEMM Tumor Tumor End-Point B Spontaneous Tumors Implantation Treatment Responses IC50 In vivo Clinic FGFR-inhibitor (nM) (mg/Kg) Trial a BGJ398 1.4 10 oralII AZD4547 2.5 3 II/III

b CH5183284 7.6 100 I LY2874455 2.6 3 oralI c Tumor #1 (Fgfr2-Dnm3) Treatment C d

Tumor #1 (Fgfr2-Dnm3) Treatment D Tumor Size / Day 1 Tumor

Days F Tumor #6 (Fgfr2-Tns1): Brca1-Proficient

Tumor #6 Solvent Dose Route Tumor Size / Day 1 Tumor BKM120 0.5% Methylcellulose/0.5% Tween80 30 mg/Kg oralOral BGJ398 PEG300:D5W=2:1 30 mg/Kg Oral Days BKM120 0.5% Methylcellulose/0.5% Tween80 15 mg/Kg BKM Oral BGJ398 PEG300:D5W=2:1 30 mg/Kg BGJ Oral

Toxicity Profile E OS of Brca1-WT Tumors G Percent Body Weight Percent

Days Figure S5. In vivo treatment of tumors harboring Fgfr2-fusion proteins achieves complete response. A) Schematics of tumor implantation and treatment design. B) A list of commercially available FGFR-inhibitors. C) PI3K-inhibitor alone delays tumor progression without tumor recession. D) Combining BGJ398 and a different PARP-inhibitor, BMN673, induces sustained tumor remission. E) Overall survival of Brca1-WT tumors treated with a PARP-inhibitor

BMN673 and a PI3K-inhibitor BKM120 shows improved efficacy with BKM120. F) A list of

MEK-inhibitors currently in clinical trials. G) Toxicity profile combining BKM120 with

BGJ398.

Supplementary Figure 6 C A

Change From Baseline Day 14 Tumor #5(Dhx9-Raf1):Treatment Trametinib PD0326901 GDC-0623 GDC-0973 AZD8330 TAK-733 MEK-i Vehicle

Trametinib MEK1 0.13 0.92 3.2 4.2 7 Olaparib MEK2 0.33 100 1.8 Tram+Ola (mg/Kg) BGJ398 In vivo 25 40 10 10 1 1 D Clinic II/III Trial II I I I I cMET Inhibitor JNJ-38877605 PF-04217903 Capmatinib Crizotinib AMG208 MK2461 SGX523 B Percent Day 1 (nM) IC50 0.13 4.8 0.4 11 4 4 9 Tumor #5(Dhx9-Raf1):DrugToxicity In vivo (n/Kg) 134 45 40 60 30 25 2 Clinic II/III Trial NO I/II I/II I I I Days E Percent Day 1 Tumor #2(cMet):Drug Toxicity Days Figure S6. In vivo treatment of tumors harboring Raf-family fusion proteins. A) A list of commercially available MEK-inhibitors that are in clinical trials. B) Trametinib alone or in combination with PARP-inhibitor Olaparib does not cause general toxicity in nude mice at the doses used. C) Tumor #5 (Dhx9-Raf1-harboring) responses to different treatments on day 14.

D) A list of commercially available MET-inhibitors that are in clinical trials. E) Toxicity profile of Crizotinib alone or in combination with BKM120.

Supplementary Figure 7 A Oncoprint of TNBC cases from TCGA B Oncoprint of all BC cases from TCGA

F Frequency of Fusion Events in Different Human Tumors C FGF3 Applification 15% (TCGA) FGF4 Applification 15% (TCGA)

D FGF4 Expression FGF19 Expression FGF4 expression FGF19 expression

●

● 15 p=0.012 10 p=1.7e-08 P−value = 0.0012 P−value = 1.7e−08 8 % Tumor with fusions % Tumor 10 6 FPKM FPKM

●

FPKM 4 5 ● FPKM

● 2

● ●

● ● ●

●

● ● ● ●

● ●● ●●● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ● ● ● ● ●● ● ●●● ●● ● ●● ●● ● ● ● ●●●● ● ●●●●●● ●●●●●●● ●●●●●●● ●●●●● ●●●●● ●●●●●●●●●●● ●●●●● ●●●●●●●●●●●● ●● ●●●●●●●●●●●●● ●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● 0 0 ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

Amplified Copy number neutral Amplified Copy number neutral Amp Neutral Amp Neutral E METABRIC TCGA Frequncy Frequncy FGFR1 Amp and EXP>2 14% 17%

FGFR2 Amp and EXP>2 4% 6% # of fusions/ Tumor FGF3 Amp and EXP>2 14% 16% Overall Frequency 26% 34% G 5‘- fusion partner 3‘-fusion partner ANK1, BAG4, BCR, CEP110, CPSF6, ERLIN2, FGFR1op, FN1, FPXP1, HERV-k, LRRFIP1, MYO18a, NVP98, RANBP2, RHOT1, SQTM1, THOT1, TPR, TRIM24, FGFR1 WHSC1l1, ZNF198, ZNF577 CCDC6, LCCAR2, AFF3, AHCYL, BICC1, C10ORF68, CASP7, CIT, FGFR2 FAM76a, KIAA1598, KIAA1967, NCALD, NOL4, OFD1, PPAPDC1a, PPHLN1, SCL45a3, TACC2, TACC3, USP10 FGFR3 TACC3, ELAVL3, BAIAP2l1, AES, TNIP2, TPRG1, WHSC1

H 5‘- fusion partner 3‘-fusion partner

KIAA1549, AGK, AGTRAP, AP3B1, ATG7, BAIAP2l1, BCL2l11, C7ORF73, CCNY, CD27, EML4, FAM114a2, FAM131b, FAM73a, BRAF HIBADH, JHDAM10, KDM7a, MACF1, MKRN1, PARP12, PICALM, PPF1BP2, SCL45A3, SND1, TAX1BP1, TRIM14, ZC3HAV

AGGF1, CLCN6, CMTM8, RSRP1, LMNA, MPRIP, NFIA, PAPD7, SRGAP1, TRAK1 RAF1 Figure S7. Genomics reveal potential mechanisms for frequent MAPK and PI3K pathway activation in TNBC. A) Oncoprint showing the prevalence of different genetic alterations in

TNBC patients in TCGA. B) Oncoprint showing the prevalence of different genetic alterations in all breast cancer patients in TCGA. C) Frequency of FGF3 and FGF4 amplification in breast cancer patients. D) FGF4, and FGF19 mRNA levels are higher in amplified populations compared to non-amplified. E) METABRIC dataset confirms the prevalence of FGFR family member amplification and overexpression. F) TCGA fusion portal data analysis reveals human breast tumors harbor high incidences of chromosomal translocations among all tumors analyzed.

G) Fusions involving the FGFR-family members identified in different human cancer types. H)

Fusions involving the RAF-family members identified in different human cancer types.