Experimental Procedures

Immunoblotting

Cells were washed twice with cold PBS and lysed in lysis buffer (containing 125mM HEPES, pH7.5, 750mM NaCl, 5% Igepal CA-630, 50mM MgCl2, 5mM EDTA and 10% glycerol) supplemented with proteinase and phosphatase inhibitors (Roche). concentration was determined using the BCA (Thermo Scientific). Western blots were conducted on 20 μg protein separated by 4-12% Bis-Tris SDS-PAGE gels (Invitrogen), transferred to PVDF membranes, and immunoblotted after blocking with 5% skim milk with the corresponding primary antibodies (listed below) in 5% BSA (SIGMA). This was followed by incubation for 1h with secondary antibodies conjugated with goat anti-rabbit HRP-conjugated antibody (1:5,000; Santa Cruz sc-2054) or goat anti-mouse HRP-conjugated antibody (1:5,000; Santa Cruz sc-2005). Bound antibodies were detected by chemiluminescence with the ECL detection system (GE Healthcare Biosciences). In Figures 2C, 3C and 4C we show the same representative Merlin and blots as aliquots of the same shNF2 clone M2-transduced C643 cell lysate were used for multiple different blots. Supplementary Figure 11 shows the individual blots and the original films corresponding to Figures 2C, 3C, and 4A. Aliquots of the same shNF2 clone M4-transduced C643 cell lysates were used for all blots in Figure 2C and 3C.

RAS-GTP or RAC1-GTP immunoprecipitation was conducted using the RAS or RAC1 activation assay kits, respectively, from Millipore, according to the manufacturer's protocol and subjected to Western blotting with the indicated antibodies. Cells were treated during 72h with dox in media with 1% of FBS.

Subcellular fractions were prepared using the Subcellular Protein Fractionation Kit for cultured cells following the manufacturer’s instructions (Thermo Fisher Scientific). Fractions were subjected to Western blotting with the indicated antibodies. Copy number alterations, chr 22, IMPACT Copy number alterations, chr 22, CGH-array

A. CRKL MAPK1 SMARCB1 CHEK2 NF2 EP300 B. CRKL MAPK1 SMARCB1 CHEK2 NF2 EP300

JF_thy_005_PDTC -0.86 JF_thy_005_PDTC -0.45 JF_thy_019_PDTC -0.80 JF_thy_019_PDTC -0.45 JF_thy_020_PDTC -0.74 JF_thy_020_PDTC -0.37 JF_thy_021_PDTC -0.63 JF_thy_021_PDTC -0.36 JF_thy_009_PDTC -0.55 JF_thy_009_PDTC -0.32 JF_thy_018_PDTC -0.52 JF_thy_018_PDTC -0.31 JF_thy_007_PDTC -0.39 JF_thy_007_PDTC -0.25 JF_thy_034_ATC -0.17 JF_thy_034_ATC -0.13

JF_thy_008_ATC -0.17 JF_thy_008_ATC 9 NF2 NF2 9loss

9 NF2 NF2 9loss -0.10

37 frozen tumors frozen 37 37 frozen tumors frozen 37

C. D. NF2 CNA values CRKL MAPK1 SMARCB1 CHEK2 NF2 EP300 log ratio log ratio Tumor ID Tumor type Preservation Driver alteration JJ_thy_027_PDTC -0.73 IMPACT CGH-array JJ_thy_053_PDTC -0.53 JJ_thy_007_PDTC -0.47 JF_thy_005 PDTC frozen NRAS Q61R -0.86 0.45 JJ_thy_004_PDTC -0.43 JJ_thy_026_PDTC -0.42 JF_thy_019 PDTC frozen NRAS Q61R -0.80 -0.45 JJ_thy_017_PDTC -0.36 JF_thy_020 PDTC frozen unknownRET/PTC -0.64 -0.37 JJ_thy_001_PDTC -0.28

7 NF2 NF2 7loss JF_thy_021 PDTC frozen HRAS Q61R -0.63 -0.36 JF_thy_009 PDTC frozen unknownRET/PTC -0.55 -0.32 JF_thy_018 PDTC frozen unknown -0.52 -0.31 JF_thy_007 PDTC frozen unknownRET/PTC -0.39 -0.25 JF_thy_034 ATC frozen unknown -0.17 -0.13 JF_thy_008 ATC frozen NRAS Q61R -0.17 -0.10 JJ_thy_027 PDTC FFPE NRAS Q61R -0.73 N/A JJ_thy_053 PDTC FFPE NRAS Q61R -0.53 N/A JJ_thy_007 PDTC FFPE NRAS Q61R -0.47 N/A JJ_thy_004 PDTC FFPE NRAS Q61R -0.43 N/A

46 FFPE tumors 46 JJ_thy_026 PDTC FFPE unknown -0.42 N/A JJ_thy_017 PDTC FFPE HRAS Q61R -0.36 N/A JJ_thy_001 PDTC FFPE unknown -0.28 N/A

Supplementary Figure S1: Loss of heterozygosity of Ch22q genes in poorly differentiated (PDTC) and anaplastic thyroid cancers (ATC). Copy number alterations (CNA) were investigated in frozen (A,B) or formalin-fixed paraffin embedded (FFPE) (C) tissue samples. Frozen tissues were studied with two different approaches: A) CNA- IMPACT, an -capture, next generation sequencing panel of 341 cancer genes, 6 of which mapped to 22q (CRKL, MAPK1, SMARCB1, CHEK2, NF2, EP300), from which copy number information was derived by comparing sequencing read counts to those of a diploid control. B) SNP-CGH, using an Agilent 500K SNP array. Ch22q loss was present in 9/37 tumors, all of which had NF2 LOH. This was confirmed with both strategies, including the relative quantification of allelic loss in the individual tumors (D), thus confirming the ability of CNA-IMPACT to detect copy number losses. In most cases the Ch22q arm was lost in its entirety, with the exception of one PDTC that lost only the distal arm of the , encompassing CHEK2, NF2 and EP300. C) CNA of 46 FFPE tumors by IMPACT, 7 of which had Ch22q and NF2 loss. One tumor had LOH of the distal arm only. D) NF2 CNA log ratios in tumors analyzed by IMPACT or CGH. 9/16 tumors with NF2 LOH had RAS . line with wild analysis by IMPACT, shows selective copy number loss of exon 4 of Top panel: Supplementary Figure S2:

NF2 normal IMPACT exon-level IMPACT exon-level copy array-CGH

cell line aligned reads number copy number Focal Focal deletion within NF2 gene as detected by array - type NF2 merlin log ratio [log2 (CN/2)] - - 12 10 - - - - 8 6 4 2 0 2

0 , demonstrating adequate sequencing coverage at that site. 1

Focal NF2 exon2 2

3 - exon 4 homozygous deletion in KHM 4 5 6 exon3 7 NF2 exon exon # 8 -

CGH. CGH. 9 NF2 Middle panels 10 . Bottom panel: 11 - 5Manaplastic thyroid cancer cells 12

: NF2 exon 13

Exon 4 read counts in a cell 14 exon4 - level copy number 15 16

.

A B

Supplementary Figure S3: FISH for NF2 in anaplastic thyroid cancers. FISH analysis for NF2 was performed by hybridizing a tissue microarray consisting of 16 different ATC (anaplastic) specimens with NF2 probe (BAC clone RP11-551L12, 22q12.2, red) and BCR probe (22q11.2, green), as control. Deletion of NF2 was defined as tumors with > 25% of cells with a single NF2 hybridization signal (a minimum of 200 cells were scored for each tumor). This threshold was selected because of the high admixture of stromal cells, particularly tumor associated macrophages, in these cancers (Ryder et al., 2008; Caillou et al., 2011). A) A total of 10/16 ATCs had NF2 LOH or homozygous deletion, with the remainder being polyploid at this site (note that the FISH analysis alone is not sufficient to determine loss of NF2 locus in a polyploid population) B) Representative FISH image to show nuclei with either LOH (yellow arrows) or homozygous loss (red arrows) of NF2 locus in an ATC sample. A

MERLIN protein

BRAF RAS WT PTEN B MERLIN protein

C D

1

-

8505c Cal62 TCO KMH5 Hth74 BCPAP Hth83 TTA1 Hom Exon Del Decreased mRNA Stability Decreased mRNA

Supplementary Figure S4: NF2/Merlin defects in thyroid cancer cell lines. A) Western blots of thyroid cancer cell lines for merlin. The driver mutation of each line is indicated. Cell lines were defined as merlin normal (black), low (grey) or null (white) based on protein abundance. B) Merlin mRNA by quantitative RT-PCR in thyroid cancer lines categorized based on merlin protein levels. C) Decreased merlin mRNA half-life in HTh74 cells. Merlin mRNA levels were measured by quantitative RT-PCR at various times after addition of actinomycin D. D) Events associated with merlin loss in thyroid cancer cell lines. A B

C643 (HRASG13R)

Cal62 (KRASG12R) C

*

** **

C643(HRASG13R) D

Supplementary Figure S5: Merlin inhibits RAC1-PAK activity: A) Top: RAC1-GTP levels in Cal62 (KRASG12R) cells treated with dox for 72h. Bottom: Westerns of input lysates for the indicated are shown below. pMEK-S298 is a PAK substrate. B) Western blots of: Left: Cal62 cells and Hth83 cells following dox-induction of merlin. Right: C643 cells transfected with five NF2 siRNAs. C) Growth of Cal62 cells expressing control (pEBG) or a dominant negative PAK (dnPAK) vector treated with or without dox for 4 days. ***p ≤ E x 10-3; ** p ≤ E x 10-2 ; n=3, two independent experiments). Bars represent mean +/- SD. D) Left: Growth effects of FRAX597 in C643 (HRASG13R, NF2-WT) cells stably expressing scrambled or shNF2.M2. Cells were counted at 6days. *p < 4 x E-2; **p < 1 x E -2. Right: Western blots of C643 cells expressing scrambled or shNF2.M2 after incubation with FRAX 597 for 48h. Supplementary Figure S6: Merlin effects on EGFR signaling and growth of RAS mutant thyroid cancer cell lines. Left: Western blots of Cal62 (KRASG12R) cells treated with dox for 72h in 1% serum, and then with EGF (5nM) for 10 min as shown. Right: hEGF (5ng/ml) effects on growth of Cal62 cells treated with or without dox in 1% serum (n=3). HrasG12V+/- wt HrasG12V+/- HrasG12V+/+ Nf2 1 2 3 4 1 2 3 1 2 3 1 2 3 4 5

M WT

Supplementary Figure S7: PCR of genomic DNA from mouse thyroid tissues of the indicated genotypes with primers that distinguish wild-type from mutant Hras alleles. M: 668 bp; WT: 622 bp.

A TEAD expression in TCGA-PTC B Hth83(HRASQ61R) C643(HRASG13R)

seq seq

-

RNA expression

C Hth83(HRASQ61R) D Cal62 (KRASG12R)

G13R E C643(HRAS )

Supplementary Figure S8: A) Data represent TEAD isoform mRNA levels derived from RNASeq of PTCs reported by the TCGA program (Cancer Genome Atlas Research Network., 2014). B) ChIP-PCR with antibodies to YAP and TEAD1 in Hth83 cells treated with or without dox for 72h and in C643 cells transfected with plko.1 or shNF2 (*p < 1 x E-2) C) Left: Verteporfin (VP) dose- dependent decrease of YAP, pYAP and TEAD at 72h in Hth83 cells. This is associated with lower HRAS and KRAS, and decreased pMEK and pERK. Control cells were treated with dox for 72h. Right: VP decreases growth of Hth83 cells in a dose-dependent manner. Cells were counted 3 days after incubation with the indicated concentration of the compound (*p < 2x E-2; ** p< 2 x E-3). D) Growth of Cal62 cells after 4d with or without dox in the presence of the indicated concentration of VP. (*p <2 x E-2; ** p < 1 x E- 2; *** p < 1 X E 3). E) Growth inhibitory effects of VP in C643 (HRASG13R, NF2-WT) cells stably expressing scrambled or 2 different NF2 shRNAs (M2 and M4). Cells were counted at 4days (* p < 5 X E -2; ** p < 2 X E -2). A

B C643(HRASG13R)

Supplementary Figure S9: A) Left: Concentration-dependent growth inhibitory effects of the MEK inhibitor selumetinib (AZD6244) in RAS mutant thyroid cancer cell lines cells. Black: null/low NF2; Red: wt NF2. The IC50 of each cell line is shown in the boxed legend. Right: Western blots for merlin, pMEK and pERK in the cell lines studied. B) C643 cells expressing scrambled (pLKO.1) or shNF2 were treated with FRAX, AZD or their combination at the indicated concentrations (nM) in 1% of serum. Cells were counted at 6 days (*p<5 x E -3). p=0.002

A p=0.002 )

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i 200

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u 0 T x x x lo lo lo f F F 3 5 f2 n p N te , , P s s , a a s r r a H H r - - H R R - F F R F

B

Supplementary Figure S10: A) Tumor doubling time of the indicated mouse models of PDTC.B) Kaplan-Meier survival curves of TPO-Cre, TPO-Cre/FR-HrasG12V/NF2flox2, TPO-Cre/FR-HrasG12V/PTENflox2 and TPO-Cre/FR- HrasG12V/p53flox2 mice. Left: HrasG12V heterozygotes. Right: HrasG12V homozygotes. (*p < 3 x E-2; **p < 1 x E -4).

Supplementary Figure 11 A

B

Supplementary Figure 11: Individual blots with corresponding Actin and Merlin controls (A), and original films (B) for all blots shown in Figures 2C, 3C, and 4A (shNF2 clone M2-transducted C643 cells).

Red boxes show different blots from the same membranes. The black boxes show the corresponding Western blots used in the paper. Blue boxes show blots or films corresponding to these experiments but not shown in the paper. As they are all Western blots from aliquots of the same lysate, the same representative actin and merlin blots were illustrated in the final figure manuscript. Supplementary Table S1: 22q loss is associated with RAS mutations in PTC, PDTC and ATC. NF2 status loss Tumor type Driver gene n, total n % BRAF 249 32 12.9 Papillary thyroid cancers RAS 49 22 44.9 (PTC), TCGA non-BRAF/RAS 198 32 16.2 total 496 86 17.3 BRAF 26 0 0 Poorly-differentiated RAS 16 8 50 thyroid cancers (PDTC), non-BRAF/RAS 21 6 28.6 MSKCC total 63 14 22.2 BRAF 11 0 0 Anaplastic thyroid cancers RAS 5 1 20 (ATC), MSKCC non-BRAF/RAS 4 1 25 total 20 2 10

Chromosome 22q loss significantly co-occurs with RAS-mutated PTC tumors in the TCGA study (chi-square p <0.0001) and in MSKCC analysis of poorly-differentiated thyroid cancers (PDTC, p=0.0005). Supplementary Table S2: Penetrance of thyroid cancer in HRASG12V/NF2-null mice. Supplementary Table 3: PCR primer sequences

Gene Fw:5’-3’ Rw:5’-3’ hNF2 AGATCAGCTGAAGCAGGACC GCTGTCACCAATGAGGTTGA hYAP1 CAACTCCAACCAGCAGCAAC TTGGTAACTGGCTACGCAGG hHRAS GCTGACCATCCAGCTGATCC CCCATCAATGACCACCTGCT hKRAS TAGGCAAGAGTGCCTTGACG TTGACCTGCTGTGTCGAGAA hNRAS AGCAGGTGGTGTTGGGAAAA TGTCCAACAAACAGGTTTCACC hCTGF CACCCGGGTTACCAATGACA GGATGCACTTTTTGCCCTTCTTA hGADPH CATGGCCTTCCAACTTGACT CATCTGCCGTCACATTGTTC hACTIN ATGATGATATCGCCGCGCTC TCGATGGGGTACTTCAGGGT hHRAS promoter-ChIP GCTGGGATTATAGGCGTGAG AAACCGTACCCACAAGTTGC hKRAS promoter-ChIP GCCCGTGTAAGAGTAGAAAGGA GCATACCCGGAGTCTGTGTT hNRAS promoter-ChIP CGCCTGGTTACTGTGTCCTG AAAGCCTTCAGTCCACTGGC mHras allelic imbalance GCCATCCCTCGCGTTCCTGTAGT CCTGCCCCACCTGCCAATGAGAA h: ; m: mouse C G