Supporting online material

MATERIALS AND METHODS

Cell lines

All T-ALL cell lines were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA)

supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 1mM sodium

pyruvate, 2mM L-glutamine, 100U/ml penicillin G, and 100 µg/ml streptomycin at

37o C under 5% CO2. Human U2OS osteosarcoma cells were grown in

Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal

bovine serum (Hyclone), 2mM L-glutamine, 100U/ml penicillin G, and 100 µg/ml

streptomycin at 37o C under 5% CO2.

Gamma-secretase inhibitor treatment of cell lines

Gamma-secretase activity was inhibited by addition of compound E to growing

cells to a final concentration of 1µM. Mock-treated cultures were exposed to

vehicle only (DMSO) at a final concentration of 0.01%. The following human T-

ALL cell lines were tested for sensitivity to compound E: TALL-1, ALL-SIL,

KOPT-K1, HPB-ALL, DND-41, CCRF-HSB2, RPMI8402, THP-6, BE-13, JM-

Jurkat, Jurkat-E6, REX, CEM, LOUCY, DU-528, KE-37, SKW-3, MKB-1, MOLT-

3, MOLT-4, MOLT-13, MOLT-15, MOLT-16, SUPT-7, SUPT-11, SUPT-13, P12-

Ichikawa, Karpas-45, DSMZ-PEER, and PF-382. The murine cell line T6E,

derived from a murine T-ALL expressing a membrane-tethered form of truncated

NOTCH1 that requires gamma-secretase cleavage for activation (1), was used

as a positive control.

Retroviruses

Construction of retroviral expression plasmids (Murine stem cell virus (MSCV)-

GFP-ICN1 and MSCV-dominant negative Mastermind-like-1-GFP), production of

high-titer, pseudotyped, amphotropic, replication-defective retroviruses, and

spinoculation procedure were performed in (S1).

Cell cycle analysis

Cells were stained with propidium iodide after overnight fixation in cold 70%

ethanol, or DRAQ5 (Biostatus, Leicester, UK) in complete medium in instances

where GFP fluorescence and DNA content measurement were measured

simultaneously. DNA content was ascertained by flow cytometry and cell cycle

fractions determined using Multicycle software (Phoenix Flow Systems, San

Diego, CA). Intact cells were gated by forward/side scatter criteria. A minimum of

15,000 events was collected to create each DNA content histogram.

NOTCH1 expression plasmids

NOTCH1 expression plasmids in pcDNA3 have been described previously. To

create a generic PEST deletion, the insertion mutation in the PEST domain of the

T-ALL cell line ALL-SIL was amplified by PCR, identified by sequencing, and

then sub-cloned into pcDNA3-NOTCH1. Site-directed mutagenesis of the HD

domain was conducted using the QuikChange kit (Stratagene, La Jolla, CA).

Mutagenic PCR primers were designed using the Stratagene web tool.

Reporter gene assays

NOTCH1 expression plasmids (pcDNA3) were transiently transfected into U2OS

cells together with (i) an artificial luciferase reporter gene under the control of a

promoter containing CSL binding sites, and (ii) an internal Renilla luciferase

control gene, as described previously ( S2), using Lipofectamine Plus

(Invitrogen). Total DNA was kept constant by adding empty vector as needed. All

transfections were carried out in triplicate, and each experiment was repeated at

least three times. Cell lysates were harvested 44-48 hours post-transfection and

luciferase assays were carried out using the Dual Luciferase Assay System

(Promega, Madison, WI) on a Turner Systems luminometer.

PCR amplification of NOTCH1 cDNAs

RNA isolated from T-ALL cell lines (1 µg) was reverse transcribed into cDNA

using oligo-dT priming and Superscript reverse transcriptase (Invitrogen) in 20 µl

reactions. PCRs contained 1 µl cDNA template, 400 µM forward

(TTTGAATTCGGGCTGGACTGTGCGGAGCATGTACCCGA) and reverse

(TTTGGATCCTCCGGAATGCGGGCGATCTGGGACTGCA) NOTCH1-specific

primers, 0.25 µl Ex-Taq (TAKARA BIO, Shiga, Japan), 1X reaction buffer, 1X

dNTPs, and 7.5% DMSO in a 50 µl reaction volume. Cycle parameters were: 94o

C X 3 min; 30 cycles of 94o C x 1 min, 60o C x 30 sec, 72o C x 2 min; 72o C x 8

min. PCR products were purified on Qiagen columns, digested with Bam HI and

Eco RI, ligated into pSP72, and used to transform DH5α E. coli cells. Miniprep

DNA from individual colonies was sequenced using T7 and SP6 primers.

Patient Material

Samples of cryopreserved lymphoblasts from 96 children and young adults with

T-ALL treated at St. Jude Children’s Research Hospital and Dana-Farber Cancer

Institute were obtained with informed consent at the time of diagnosis. Genomic

DNA from each sample was extracted with a commercial kit (GENTRA,

Minneapolis, MN). RNA was prepared from cryopreserved lymphoblasts with

RNAaqueous reagents (Ambion, Austin, TX).

Quantitative RT-PCR

Quantitative RT-PCR analysis of HOX11, HOX11L2, TAL1, LYL1, TAL2, and

BHLHB1 was performed as previously described ( S3, S4) using an ABI PRISM

7700 Sequence Detection System instrument (Perkin Elmer Applied

Biosystems, Foster City, CA). RT-PCR-based detection of MLL-ENL and CALM-

AF10 fusion transcripts was performed as previously described ( S4, S5).

Genomic DNA preparation

Genomic DNA was prepared from primary T-ALLs by extraction with a

commercial kit (GENTRA). DNA was prepared from cell lines by SDS

lysis/proteinase K digestion, followed by phenol:chloroform extraction and

ethanol precipitation. DNAs were resuspended in TE buffer, quantified

spectrophotometrically, and stored at -20o C.

Mutation Detection

Mutation detection in exons 26, 27, and 34 of NOTCH1 was performed via PCR-

based denaturing HPLC using a WAVE DNA fragment analysis system

(Transgenomic, Omaha, NE) equipped with a DNASep HT cartridge. Amplicons

for mutation analysis were prepared with the following primer combinations. The

N terminal region of the HD domain of NOTCH 1, encoded in exon 26, was

divided in two amplicons. Amplicon HD-N1 was amplified by PCR using primers

HD-N1FW1: AGCCCCCTGTACGACCAGTA, HD-N1RV1:

CTTGCGCAGCTCCTCCTC; and by nested PCR using primers HD-N-1FW2:

GACCAGTACTGCAAGGACCA, HD-N1RV2 TCCTCGCGGCCGTAGTAG.

Amplicon HD-N2 was amplified by PCR using primers HD-N2FW1:

GTGCTGCACACCAACGTG, HD-N2RV1: GAGGGCCCAGGAGAGTTG, and by

nested PCR using primers HD-N2FW2: GCACACGGCCAGCAGATGAT and HD-

N2RV2: CGCCGGGTCTCACTCAC. The C terminal region of the HD domain of

NOTCH 1, encoded in exon 27, was amplified by PCR using primers HD-CFW1:

GTGGCGTCATGGGCCTCA HD-CRV1: TAGCAACTGGCACAAACAGC, and by

nested PCR using primers HD-CFW2: CATGGGCCTCAGTGTCCT and HD-

CRV2: GCACAAACAGCCAGCGTGTC Sequences of exon 34 encoding the

PEST domain and the continuous N-region containing the TAD of NOTCH1 were

divided in three amplicons. Amplicon PEST1 and PEST2 were amplified by PCR

using primers PESTFW1: GCAGCATGGCATGGTAGG and PESTRV1:

AACATGTGTTTTAAAAAGGCTCC TC; and by nested PCR using primers

PEST1FW: AAACATCCAGCAGCAGCAAA, PEST1RV:

CACAGGCGAGGAGTAGCTGTG and PEST2FW: GTGACCGCAGCCCAGTTC,

PEST2RV: AAAGGAAGCCGGGGTCTC. Amplicon TAD1 was amplified by PCR

using primers TAD1FW1: AGACTGGCCCACCTCGTCTCT, TAD1RV1:

GCTCTCCACTCAGGAAGCTC and by nested PCR using primers TAD1FW2:

CGTCTCTCCCACCTGCCTGT and TAD1RV2: CTGAGCTCACGCCAAGGT.

Before DHPLC analysis, nested PCR products were denatured at 95o C for 5

minutes and slowly ramped to 40o C using a PTC-225 Peltier Thermal Cycler (MJ

Research, Waltham, MA) in order to allow the formation of homo- and

heteroduplexes in samples heterozygous for a mutant allele.

Annealed PCR fragments (15-30 µl per sample) were injected onto the

DNASep HT cartridge for analysis. Products were eluted at a constant flow rate

of 1.5 ml/min with a linear acetonitrile gradient determined by Navigator software

(Transgenomic) based on the size and GC-content of the amplicon. The gradient

was produced by combining 0.1M triethylammonium acetate (TEAA) buffer (pH

7) (Transgenomic) and Buffer B (0.1M TEAA with 25% acetonitrile)

(Transgenomic). The elution profiles of DNA fragments, monitored by the

system’s UV detector, were used to produce chromatographs. The analysis

sequence for each sample was as follows: a 1.9-min time lapse for the detector,

a 0.1-min loading stage with a 3% decrease in Buffer B, a 2.0-min linear gradient

with an increasing slope of 2% Buffer B per min, a 0.1-min cleaning stage using

75% acetonitrile, and a 0.1-min equilibration before the next injection. Homo-

and hetero-duplex peaks were detected between the initial injection peak,

produced by residual nucleotides and primers in the reaction, and the wash

stage.

Melting profiles for the nested PCR products, ranging from 276 to 452

base pairs, were constructed using the Navigator software. Appropriate partial

denaturing temperatures for mutation detection, which produced percent

helicities between 30% and 98% at each base contained in regions of interest,

were predicted for all amplicons: PEST1, 66.5o C; PEST2 65.0 o C; HD1, 67.0 o C

and 64.5 o C; HD2, 67.0 o C and 64.0 o C; and TAD1, 64.5 o C and 63.9 o C. Less

stable heteroduplexes denature earlier than homoduplexes and, thus, appear

first in elution profiles. Therefore, direct sequencing was performed on samples

with chromatographs displaying species that eluted before normal

homoduplexes.

Fig. S1. Induction of cell cycle arrest by dominant negative MSCV-GFP-Mastermind-like-1 retrovirus. T-ALL cell line cultures were transduced with empty control virus of Mastermind-like-1 virus. Cultures were harvested on day 7 post-transduction and stained with the DNA dye DRAQ5, which pemits flow cytometric measurement of DNA content in GFP- and GFP+Sub-populations. A minimum of 15,000 events was were collected to create each DNA histogram.

GFP

DN-MAML1

ALL-SIL DND-41 HPB-ALL KOPT-K1 TALL-1

1571 1618 human NOTCH1 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFrat notch1 AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFmouse notch1 AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAHGQQMIFchicken notch1 ADGTLVVVVLITPENLKNNSFNFLRELSRVLHTNVVFKKNAHGQQMIFfrog notch1 AEGTLVLVVLMPPERLKNNSVNFLRELSRVLHTNVVFKKDAHGQQMIFfish notch1 AVGLLVVVVHIHPDQLRNNSFGFLRELSRVLHTNVVFRRDAHGQQMIFCONSENSUS A-G-LV-VV---P--L-N-S--FLRELS-VLHTNVVF--DA-GQQMIF

1571 1618NOTCH1 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFKOPTK1 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVPHTNVVFKRDAHGQQMIFTALL 07 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVPHTNVVFKRDAHGQQMIFTALL 40 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVPHTNVVFKRDAHGQQMIFTALL 41 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVPHTNVVFKRDAHGQQMIFTALL 47 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVPHTNVVFKRDAHGQQMIFTALL 68 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVPHTNVVFKRDAHGQQMIFTALL 83 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVPHTNVVFKRDAHGQQMIFALL-SIL AAGTLVVVVLMPPEQLRNSSFHFPRELSRVLHTNVVFKRDAHGQQMIFDND-41 AAGTLVVVVLMPPEQLRNSSFHFPRELSRVLHTNVVFKRVAHGQQMIFTALL 12 AAGTLVVVVLMPPEQLRNSSFHFPRELSRVLHTNVVFKRDAHGQQMIFTALL 18 AAGTLVVVVLMPPEQLRNSSFHFPRELSRVLHTNVVFKRDAHGQQMIFTALL 44 AAGTLVVVVLMPPEQLRNSSFHFPRELSRVLHTNVVFKRDAHGQQMIFTALL 45 AAGTLVVVVLMPPEQLRNSSFHFPRELSRVLHTNVVFKRDAHGQQMIFTALL 55 AAGTLVVVVLMPPEQLRNSSFHFLRELSPVLHTNVVFKRDAHGQQMIFTALL 64 AAGTLVVVVLMPPEQLRNSSFHFLRELSPVLHTNVVFKRDAHGQQMIFTALL 87 AAGTLVVVVLMPPEQLRNSSFHFLRELSPVLHTNVVFKRDAHGQQMIFTALL 89 AAGTLVVVVLMPPEQLRNSSFHFLRELSPVLHTNVVFKRDAHGQQMIFTALL 91 AAGTLVVVVLMPPEQLRNSSFHFLRELSPVLHTNVVFKRDAHGQQMIFHPB-ALL AAGTPVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 03 AAGTPVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 79 AAGTPVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 96 AAGTPVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 05 AAGTLVVV-LMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 21 AAGTLVVV-LMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 23 AAGTLVVV-LMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 93 AAGTLVVV-LMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 30 AAGTLVVVVLMPPEQLRNSSFHSLRELSRVLHTNVVFKRDAHGQQMIFTALL 04 AAGTLVVVVLMPPEQPRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFTALL 76 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKSDAHGQQMIFTALL 02 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNV-FKRDAHGQQMIFTALL 42 AAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKT--HGQQMIF

1571 1622NOTCH1 AAGTLVVVVLMPPE.QLRNSSFHF.LRE...LSRVLHTNVVFK....RDAHGQQMIFPYY.GTALL 09 AAGTLVVVVLMPPE.QLRNSSFHF.LRE...LSRVLHTNVVFK....RDAHGQQMIFPPGGGTALL 19 AAGTLVVVVLMPPAQQLRNSSFHF.LRE...LSRVLHTNVVFK....RDAHGQQMIFPYY.GTALL 24 AAGTLVVVVLMPPE.QLRNSSFHF.LRE...LSRVLHTNVVLGR...RDAHGQQMIFPYY.GTALL 46 AAGTLVVVVLMPPE.QLRNSSFHLALRE...LSRVLHTNVVFK....RDAHGQQMIFPYY.GTALL 53 AAGTLVVVVLMPPE.QLRNSSFHF.LREKEDLSRVLHTNVVFK....RDAHGQQMIFPYY.GTALL 86 AAGTLVVVVLMPPE.QLRNSSFHF.LRE...LSRVLHTNVVFKSMPPRDAHGQQMIFPYY.GTALL 95 AAGTLVVVVLMPPE.QLRNSSFHTALRE...LSRVLHTNVVFK....RDAHGQQMIFPYY.G

Fig. S2. NOTCH1 HD-N domainmutations. Upper panel: Alignment of HD-N sequences from diverse vertebrateNOTCH1 proteins. Residues mutated inone or more T-ALL cell lines or primarytumors are highlighted in yellow. Middlepanel: list of HD-N missense and deletionmutations identified in primary T-ALLsamples and NOTCH-inhibitor sensitive T-ALL cell lines (underlined). Lower panel:insertional mutations identified in primaryT-ALL samples. Numbers correspond toposition of residues in human pro-NOTCH1, including the signal peptide.

Human NOTCH1 GSIVYLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVQSEFish notch1 GSIVYLEIDNRLCSQGSDDCFRNADSAAEYLGALSAREMLR-FPYPIKEVTSEMouse notch1 GSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVKSERat notch1 GSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVKSEChicken notch1 GSIVYLEIDNRQCIQSSSQCFQSATDVAAFLGALASLGNLN-IPYKIEAVKSEFrog notch1 GSIVYLEIDNRQCYKSSSQCFNSATDVAAFLGALASLGSLDTLSYKIEAVKSEConsensus GSIVYLEIDNR-C------CF--A---A--LGALAS--------Y-IE-V--E

1674 1722NOTCH1 GSIVYLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVTALL 1 GSIDYLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVTALL 11 GSIVYPEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVTALL 37 GSIVYPEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVTALL 43 GSIVYPEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVTALL 80 GSIVYPEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVTALL 15 GSIVYLENDNRQCVQASSQCFQSATDVAAFLGALASLGSLN-IPYKIEAVTALL 16 GSIVYLEIDNRQCVQASSQCFQSATDVAPFLGALASLGSLN-IPYKIEAVTALL 26 GSIVYLEIDNRQCVQASSQCFQSATDVAPFLGALASLGSLN-IPYKIEAV

Fig. S3. NOTCH1 HD-C domain mutations. Upper panel: Alignment of HD-C sequences from diversevertebrate NOTCH1 proteins. Residues mutated in one or more T-ALL cell lines are highlighted inyellow. Lower panel: HD-C mutations identified in primary T-ALL samples. Numbers correspond toposition of residues in human pro-NOTCH1, including the signal peptide.

2311NOTCH1 WLSRLQSGMVPNQYNPLRGSVAPGPLSTQAPSLQHGMVGPLHSSLAASALSQMMSYQGLPSTRLATQPHLVQTQQVQPQNLQMQQQNLQPTALL 05 ........................................................................................*TALL 13 ....................................................................................*TALL 13 ...................................................................................*TALL 44 ...............................*

2401NOTCH1 ANIQQQQSLQPPPPPPQPHLGVSSAASGHLGRSFLSGEPSQADVQPLGPSSLAVHTILPQESPALPTSLPSSLVPPVTAAQFLTPPSQHSTALL 35 ............................................................................*ALL-SIL ..........................................................................AHP*TALL 53 ....................................................................GCHPRWSHP*TALL 96 ..................................................................*TALL 40 ...............................................................PCPRRCHPRWSHP*TALL 08 ............................................................*TALL 37 ...........................................LSRDLCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSH*DND41 ...........................................CCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*HPB-ALL ..........................................EGRGRCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*TALL 23 .........................................GGGGMGSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*TALL 91 ......................................ERARQTCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*TALL 84 ......................................RVRARQTCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*TALL 07 ......................................*TALL 01 .................................APS*TALL 36 ....................AA*TALL 32 ...........RHHHHHSRTLVA*TALL 68 ......*TALL 57 ...*

2491 2556NOTCH1 YSSPVDNTPSHQLQVPEHPFLTPSPESPDQWSSSSPHSNVSDWSEGVSSPPTSMQSQIARIPEAFK*TALL 41 .........................DSGPARPRIPTSPTGPRASPALPPACSPRSPAFRRPSSKRRAPRDPGFLSQAFGRLCALCGCQGRPEEPF*TALL 30 ........................SVP*KOPTK1 ........................RVP*TALL 83 ........................RVP*TALL 94 ........................RVP*TALL 80 ........................RVP*TALL 81 ........................RVP*TALL 47 .......................VP*TALL 52 .......................VP*TALL 41 .................Y.*TALL 93 ................*TALL 63 ...GRQHPQPPATGA*TALL 92 ..*

Fig. S4. PEST domain mutations in T-ALL cell lines and primary T-ALL samples. Predicted consequences of point mutations,insertions, and deletions on the PEST domain are shown. Asterisks indicate the position of stop codons. Numbers correspond toposition of residues in human pro-NOTCH1, including the signal peptide.

KOPT-K1 cDNA 4

HPB-ALL cDNA 4

DND-41 cDNA 1

HD-N Domain PEST Domain

ALL-SIL cDNA 3

ΔCT

insert

insert-1 bp

insert

Fig. S5. HD and PEST domain mutations in gamma-secretase-sensitiveT-ALL cell lines lie in cis. Sequencing traces are shown for individual cDNAclones, demonstrating the presence of HD-N domain and PEST domainmutations within single cDNAs.

Figure S6. Model for functional consequences of NOTCH1 HD domain and PEST domainmutations. HD domain mutations are posited to destabilize NEC/NTM subunit interaction,rendering the NTM subunit susceptible to activating proteolytic cleavages that generateICN1. Once ICN1 is created, the absence of PEST sequences increases ICN1 stability.

NC

NEC

EGF-like LNR HD

NTM

TM

AnkyrinX X

Increased ICN1

Production

Increased ICN1

Stability

X

Table S1. NOTCH1 Heterodimerization domain and PEST domain mutationalstatus of gamma-secretase-resistant T-ALL cell lines.Key: HD = heterodimerization domain; blue = HD mutation only; green = PESTmutation only; red = both HD and PEST mutations present; black = wild typesequence. Nucleotide and amino acid positions correspond to pro-NOTCH1 withthe signal peptide sequence included.

Cell Line SEQ HETERODIMERIZATION DOMAIN

MUTHETERODIMERIZATION

DOMAINMOLT4 4802 T/C 1601 L/PMOLT3 4802 T/C 1601 L/P

CCRF CEM 4784 INS<CGCGCCTTCCCCACAACAGCTCCTTCCACTTCCTGC> 1595 INS PRLPHNSSFHFLSKW3 - -

KARPAS45 - -PF382 4724 T/C, 5097 C/T (SNP) 1575 L/P

MOLT15 4678 C/T(SNP), 5097 C/T (SNP) -CCRFHSB2 4678 C/T(SNP), 5097 C/T (SNP) -

SUPT13 5097 C/T (SNP) -SUPT11 - -JURKAT 5097 C/T (SNP) -LOUCY - -

REX 4746 G/A(SNP), 5097 C/T (SNP) -P12

ICHIKAWA5165

INS<CCCGGTTGGGCAGCCTCAACATCCCCTACAAGATCGAGGCCG> -DU528 - -CMLT1 - -

MOLT16 - -JM

JURKAT 4883 G/A 1628 R/QRPMI 8402 4754 INS<CCGTGGAGCTGATGCCGCCGGAGC> 1584 INS PVELMPPE

Cell LineSEQ PESTDOMAIN MUT PEST DOMAIN

MOLT4 7544 DEL(CT) 2515 RVP*STOPMOLT3 7544 DEL(CT) 2515 RVP*STOP

CCRF CEM 7237 C/A 2413 P/SSKW3 7378 C/T 2460 *STOP

KARPAS45 7378 C/T 2460 *STOP

PF3827480

INS<GCCTCTTGCAT>

2494ASCILWTTPPATSYRCLSTPSSPRPLSPLTSGPARPRIPTSPTGPRASPALPPACSPRSPAFRRPSSKRRAPRDPGFLSQAFGRLCALCGCQ

GRPEEPF*STOPMOLT15 7571 C/A 2524 *STOP

CCRFHSB2 - -

SUPT13 - -SUPT11 - -

JURKAT - -LOUCY - -

REX 7400 C/T 2467 T/MP12

ICHIKAWA WT WTDU528 ND WTCMLT1 ND WT

MOLT16 6856 G/A 2286 V/I (SNP)JM

JURKAT ND WTRPMI 8402 ND WT

Table S2. NOTCH1 Heterodimerization domain and PEST domain mutational status, karyotype, and RT-PCR analysis ofprimary T-ALLs and gamma-secretase-sensitive T-ALL cell lines.Key: HD = heterodimerization domain; blue = HD mutation only; green = PEST mutation only; red = both HD and PESTmutations present; black = wild type sequence. Nucleotide and amino acid positions correspond to pro-NOTCH1 with thesignal peptide sequence included.

ID HD SEQ HETERODIMERIZATION DOMAIN MUTATION HETERODIMERIZATION DOMAIN1 MUT 5030 T/A 1677 V/D2 MUT 4815 DEL(GGT) 1606 DEL V3 MUT 4724 T/C, 5097 C/T (SNP) 1575 L/P4 MUT 4757 T/C, 5097 C/T (SNP) 1586 L/P5 MUT 4735 DEL(GTG), 5097 C/T (SNP) 1579 DEL V6 WT - -7 MUT 4802 T/C 1601 L/P8 WT 5097 C/T (SNP)9 MUT 4857 DEL (TACTAC) INS<CCTGGGGGA> 1620 Y/P, 1621 Y/G, 1622 INS G10 WT - -11 MUT 5036 T/C, 3127 G/T (SNP) 1679 L/P12 MUT 4781 T/C 1594 L/P13 WT - -14 WT 5097 C/T (SNP) -15 MUT 5042 T/A 1681 I/N16 MUT 5104 G/C 1702 A/P17 WT - -18 MUT 4781 T/C 1594 L/P19 MUT 4751 INS<CCC>, 5097 C/T (SNP) 1584 E/A, 1585 INS Q20 WT - -21 MUT 4735 DEL(GTG), 4890 C/T (SNP) 1579 DEL V22 WT - -23 MUT 4735 DEL(GTG) 1579 DEL V24 MUT 4821 DEL(CAAGC) INS<GG>, 5097 C/T (SNP) 1607 F/L, 1608 K/G, 1609 INS R

25 WT - -26 MUT 5104 G/C 1702 A/P27 WT - -28 WT - -29 WT - -30 MUT 4778 T/C 1593 F/S31 WT - -32 WT - -33 WT - -34 WT 5097 C/T (SNP) -35 WT - -36 WT 5097 C/T (SNP) -37 MUT 5036 T/C 1679 L/P38 WT 5097 C/T (SNP) -39 WT 5097 C/T (SNP) -40 MUT 4802 T/C 1601 L/P41 MUT 4802 T/C, 5097 C/T (SNP) 1601 L/P42 MUT 4825 DEL (CGTGACGCA) INS<ACG>, 5097 C/T (SNP) 1609 R/T, 1610 DEL D, 1611 DEL A43 MUT 5036 T/C 1679 L/P44 MUT 4781 T/C, 5097 C/T (SNP) 1594 L/P45 MUT 4781 T/C 1594 L/P46 MUT 4779 INS<AGC> 1593 F/L, 1594 INS A47 MUT 4802 T/C 1601 L/P48 WT 5097 C/T (SNP) -49 WT - -50 WT 5097 C/T (SNP) -51 WT - -52 WT 5097 C/T (SNP) -53 MUT 4779 INS<TAAGGAGGA>, 5097 C/T (SNP) 1594 INS K, 1595 INS E, 1596 INS D54 WT - -

55 MUT 4796 G/C 1599 R/P56 WT 5097 C/T (SNP) -57 WT 5097 C/T (SNP) -58 WT - -59 WT - -60 WT - -61 WT - -62 WT - -63 WT - -64 MUT 4796 G/C, 5097 C/T (SNP) 1599 R/P65 WT - -66 SNP 4621 T/C 1541 F/L67 WT - -68 MUT 4802 T/C 1601 L/P69 SNP 5014 G/A (SNP) 1672 V/I (SNP)70 WT 5097 C/T (SNP) -71 WT -72 WT - -73 WT - -74 WT - -75 WT - -76 MUT 4825 C/A 1609 R/S77 WT 5097 C/T (SNP) -78 WT - -79 MUT 4724 T/C, 5097 C/T (SNP) 1575 L/P80 MUT 5036 T/C 1679 L/P81 WT - -82 WT 5043 T/A (SNP), 5097 C/T (SNP) -83 MUT 4802 T/C 1601 L/P84 WT - -

85 WT 5097 C/T (SNP) -86 MUT 4821 INS<ATCAATGCCCCC>, 5097 C/T (SNP) 1608 INS S, 1609 INS M, 1610 INS P, 1611 INS P87 MUT 4796 G/C 1599 R/P88 WT - -89 MUT 4796 G/C 1599 R/P90 WT - -91 MUT 4796 G/C, 5014 G/A (SNP), 5097 C/T (SNP) 1599 R/P, 1672 V/I (SNP)92 WT 5097 C/T (SNP) -93 MUT 4735 DEL(GTG) 1579 DEL V94 WT - -95 MUT 4777 DEL(TTCC) INS<ACTGCG> 1593 F/T, 1594 INS A96 MUT 4724 T/C 1575 L/P

HPB-ALL MUT 4724 T/C 1575 L/PDND-41 MUT 4781 T/C, 4829 A/T 1594 L/P, 1610 D/VALL-SIL MUT 4724 T/C 1575 L/PKOP-TK1 MUT 4802 T/C 1601 L/PTALL-1 WT - -

ID PEST SEQ PEST DOMAIN MUTATION PEST DOMAIN1 MUT 7399 INS <TGCCC> 2434 APS*STOP2 WT - -3 WT - -4 SNP 6856 G/A (SNP) 2286 V/I (SNP)5 MUT 7195 C/T 2399 STOP6 WT - -7 MUT 7321 C/T 2441 *STOP8 MUT 6856 G/A (SNP), 7378 C/T 2286 V/I (SNP), 2460 *STOP9 WT - -10 WT - -11 WT - -12 WT

13 MUT 7110 C/T, 7113 C/T 2394 STOP, 2395 STOP14 WT - -15 WT - -16 WT - -17 WT - -18 WT - -19 WT - -20 WT - -21 WT - -22 WT - -23 MUT 7331 DEL(T) INS<GTGGAGGGGGGATGG> 2444 GGGGMGSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*STOP24 WT - -25 WT - -26 WT - -27 WT - -28 WT - -29 SNP 6839 C/T 2280 A/V30 MUT 7542 INS<GAGCGTC> 2515 SVP*STOP31 WT - -32 MUT 7233 INS<CC> 2412 RHHHHHSRTLVA*STOP33 WT - -34 WT - -35 MUT 7428 DEL(C ) 2477 *STOP36 MUT 7262 INS <CC>, 2421 AA*STOP37 MUT 7330 INS<TTAAGTAGAGACTT> 2444 LSRDLCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSH*STOP38 WT - -39 WT - -40 MUT 7389 DEL (C ) 2464 PCPRRCHPRWSHP*STOP

41 MUT7522 C/T, 7529 DEL(TCCTCACCCC)INS<GA>, 7548 DEL(GTCCCCTGAC)

2286 V/I (SNP), 2508 H/Y, 2510 *STOP, 2516DSGPARPRIPTSPTGPRASPALPPACSPRSPAFRRPSSKRRAPRDPGFLSQAFGRLCALCGCQGRPEEPF*

42 WT - -

43 WT - -44 MUT 7025 INS<GAGCACACAGGCCCCCT> 2342 STOP45 WT - -46 WT - -47 MUT 7539 INS <C> 2514 VP*STOP48 WT - -49 WT - -50 WT - -51 WT - -52 MUT 7539 INS <C> 2514 VP*STOP53 MUT 7405 INS<GG> 2469 GCHPRWSHP*STOP54 WT - -55 SNP 6856 G/A (SNP) 2286 V/I (SNP)56 WT - -57 MUT 7110 C/T 2404 STOP58 WT - -59 WT - -60 WT - -61 WT - -62 WT - -63 MUT 7480 DEL (CCTGTG) INS<GGGC> 2494 GRQHPQPPATGA *STOP64 WT - -65 WT - -66 WT ND -67 WT - -68 MUT 7116 C/T 2396 STOP69 SNP 7518 T/C (SNP), 7236 A/G (SNP) -70 WT - -

71 SNP7236 A/G(SNP), 7233 G/A (SNP), 7518

T/C (SNP), 7677 A/G (SNP) -

72 WT - -73 SNP 7661 C/A 25554 A/D74 WT - -75 WT - -76 WT - -77 WT - -78 SNP 6856 G/A (SNP) 2286 V/I (SNP)79 SNP 7538 C/T (SNP) 2499 P/L80 MUT 7544 DEL(CT) 2515 RVP*STOP81 MUT 7544 DEL(CT) 2515 RVP*STOP82 WT - -83 MUT 7544 DEL(CT) 2515 RVP*STOP84 MUT 7068 T/C (SNP),7316 INS<GGGTT> 2439 RVRARQTCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*STOP85 WT - -86 WT - -87 WT - -88 WT - -89 WT - -90 WT - -91 MUT 7317 INS<AGAAC>, 7518 T/G 2440 ERARQTCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*STOP92 MUT 7478 C/A 2493 STOP93 MUT 7519 G/T 2507 *STOP94 MUT 7544 DEL(CT) 2515 RVP*STOP95 WT - -96 MUT 7403 C/A 2468 *STOP

HPB-ALL MUT 7329 DEL (C) INS<GGGCCGTGGACG> 2443 EGRGRCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*STOPDND-41 MUT 7330 DEL (G) INS<TGT> 2444 CCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*ALL-SIL MUT 7422 INS<GG> 2475 AHP*STOPKOP-TK1 MUT 7544 DEL(CT) 2515 RVP*STOPTALL-1 WT - -

ID Group Classification Cytogenetics1 HOX11L2 -2 CALM-AF10 -3 HOX11L2 -4 HOX11L2 -5 HOX11L2 -67 Other -8 TAL1 TAL1/SIL deletion9 HOX11L2 -

10 LYL1 -111213 TAL1 TAL1/SIL deletion1415 HOX11L2 -16 HOX11L2 -17 TAL1 TAL1/SIL deletion18 TAL1 -19 LYL1 -20 TAL1 -21 HOX11L2 -2223 HOX11L2 -24 TAL1 -25 LYL (TAL1+, LYL+) -2627 TAL1 TAL1/SIL deletion

28 Other (low levels of HOX11 expression) -29 TAL1 TAL1/SIL deletion30313233343536 TAL1 TAL1/SIL deletion37 TAL1 -38 LYL (TAL1+, LYL+) -39 Other -40 Other -41 TAL1 -42 Not classified del7(q35), del(9)(p13)43 TAL1 del(1)(p22p32),del(6)(q15q21),i(9)(q10),t(10;14)(q24;q11),TAL1d+44 Not classified t(7;7)(p15;q35)45 HOX11 t(5;11)(q33;q13),+846 HOX11 t(10;14)(q24;q11)47 HOX11L2 Normal48 TAL1 Normal49 TAL1 Normal50 LYL1 del(11)(q23), FLT3 ITD+51 LYL1 47,t(3;11)(q12;p15),+1952 LYL1 Normal53 MLL-ENL t(11,19)(q23;p13.3)/MLL-ENL+ by RT-PCR54 MLL-ENL add(10)(q24), t(11;19)(q23;p13.3)/MLL-ENL+ RT PCR55 TAL1 add(14)q22,TAL1d+56 TAL1 add(19)(p13);TAL1d+

57 TAL1 Normal58 TAL1 Normal, TAL1d+59 TAL1 del(6)(q21q23);TAL1d+60 TAL1 add(4)(q31);TAL1d+61 TAL1 del(16)(q13);TAL1d+62 TAL1 dup(4)(q28-32),del(6)(q13q21);TAL1d+63 Not classified Normal64 Not classified near-tetraploid: del(11)(q22-23)65 TAL1 Normal66 HOX11 -5,-8,-19,+3mar67 HOX11L2 t(2;16)(q35-37;q13-21),t(9;11)(p21;p14)68 NA NA69 NA NA70 Not classified del(6)(q13q21),del(9)(p22),add(9)(q34)71 CALM-AF10 add(7)(p22), t(10;11)(p14-15;q22), del(18)(q12)72 TAL1 Normal73 LYL (TAL1+, LYL+) +7/der(1) t(1;7)(q32;q11)74 TAL1 del(9)(p21)75 TAL2 del(6)(q16q25),t(6;8)(p21;q24)76 TAL1 add(9)(p13),?I(9)(p10)77 MLL-ENL Normal/MLL-ENL+ by RT-PCR78 TAL1 del(6)(q15q21)79 Not classified Normal80 HOX11L2 52,+8,del(15)(q15q22),+21,+22,+3mar81 TAL1 ins(5;?)(q31), t(8;14)(q24;q11)82 TAL1 Normal83 TAL1 del(9)(p21)84 LYL1 del(9)(p13)85 NA NA

86 LYL (TAL1+, LYL+) del(11)(q21q25), del(13)(q22,q34)87 HOX11L2 (HOX11L2+, TAL1+, BHLHB1+) del(5)(q13q22),+2188 LYL (TAL1+, LYL+) del(16)(p12)89 LYL (TAL1+, LYL+) inv(11)(p13q23)90 TAL1 Normal, Tal1d+91 LYL (TAL1+, LYL+) add(15)(p11.1), del(5)(q22q33), del(13)(q12q14), del(17)(p11.2),-1992 LYL1 ?inv(12)(p11.2q24.1), del(5)(q13q22), del(13)(q12q22)93949596

HPB-ALL HOX11L2DND-41 HOX11L2ALL-SIL HOX11KOP-TK1 TAL1TALL-1 Not classified

References for Supplemental Materials

S1. A. P. Weng et al., Mol. Cell. Biol. 23, 655 (2003).

S2. J. C. Aster et al., Mol. Cell. Biol. 20, 7505 (2000).

S3. A. A. Ferrando et al., Cancer Cell 1, 75 (2002).

S4. A. A. Ferrando et al., Lancet 363, 535 (2004).

S5. V. Asnafi et al., Blood 102, 1000 (2003).