Supplementary methods: Patient material & consents Bone marrow (BM) and peripheral blood (PB) collection from all living patients was performed following a written informed consent, according to the protocols approved by local Institutional Review Boards and in accordance with the Declaration of Helsinki. Approval to use archival samples from deceased patients was obtained from the National Supervisory Authority for Welfare and Health operating under the Ministry of Social Affairs and Health in Finland. Mononuclear cell preparation Mononuclear cells (MNC) were prepared from BM aspirates or PB using standard methods including Ficoll gradient separation (GE Healthcare, Little Chalfont, UK) or using Vacutainer CPT cell preparation tubes (Becton Dickinson, Franklin Lakes, NJ, USA). For drug sensitivity testing, the cells were cultured in mononuclear cell medium (PromoCell, Heidelberg, Germany). CD3 positive cell selection CD3+ cells were selected from the PB MNC fraction of a healthy donor using the Easy Sep Human CD3 Positive Selection kit (StemCell Technologies, Vancouver, Canada) following the manufacturer’s protocol. DNA sequencing Genomic DNA was isolated from the MNC fraction of BM samples and a skin biopsy using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). Exome capture was performed on 3 μg DNA using the Nimblegen SeqCap EZ v2 capture kit (Roche NimbleGen, Madison, WI, USA). Exomes were sequenced using the HiSeq2000 instrument (Illumina, Inc., San Diego, CA, USA). For the germline control 4×107 and for tumor samples 10×107 2×100 bp paired-end reads were sequenced per sample. Somatic mutation calling and annotation from exome sequencing data Sequence reads were processed and aligned to GRCh37.66 reference genome primary assembly as described previously1,2. Somatic mutation calls were made for exome capture target regions of the NimbleGen SeqCap EZ v2 capture kit (Roche NimbleGen, Madison, WI, USA) and the flanking 500 bps. High confidence somatic mutations were called for each tumor sample using the VarScan2 somatic algorithm3 with the following parameters: strand-filter 1, min-coverage-normal 8, min-coverage-tumor 6, somatic-pvalue 0.01, normal-purity 1, min-var-freq 0.20. Mutations were annotated with SnpEff4 using the Ensembl v68 annotation database. To filter out misclassified germline variants, population variants included in dbSNP version 130 were removed. Remaining non-synonymous mutations were visually validated using the Integrated Genomics Viewer (Broad Institute). Library preparation, sequencing and data analysis of transcriptomes 5 µg of total RNA was used for depletion of ribosomal RNA (Ribo-Zero™ rRNA Removal Kit, Epicentre, Madison, WI, USA) and reverse transcribed to double-stranded cDNA (NEBNext® mRNA Library Prep Master Mix Set 1, New England BioLabs, Ipswich, MA, USA). Random hexamers (New England BioLabs) were used for priming the first strand synthesis reaction and SPRI beads (Agencourt AMPure XP, Beckman Coulter, Brea, CA, USA) for purification of ribodepleted RNA and cDNA. RNAseq libraries were prepared using Illumina compatible Nextera™ Technology (Illumina, Inc., San Diego, CA, USA) and 60 ng of cDNA. In this technology DNA fragmentation and tagging is performed by in vitro cut-and-paste transposition. After the tagmentation reaction the fragmented cDNA was purified with SPRI beads. The RNAseq libraries were amplified according to the manufacturer´s instructions and size selected (350-700 bp) with the Caliper LabChip XT Instrument (PerkinElmer, Waltham, MA, USA). Each transcriptome was loaded to occupy 1/3 of the lane capacity in the flow cell. C-Bot (TruSeq PE Cluster Kit v3, Illumina) was used for cluster generation and Illumina HiSeq2000 platform (TruSeq SBS Kit v3-HS reagent kit, Illumina) for paired end sequencing with 93 bp read length. RNAseq data analysis, such as fusion gene identification, mutation calling and gene expression quantitation (Tophat and Cufflinks) was done as described previously. 5 The STAT5B haplotype of the index patient’s relapse sample regarding mutations N642H, T648S, I704L was reconstructed by extracting from Tophat alignment the pairs (38 in total) properly aligned and overlapping with the affected exons (ENSE00002394532 and ENSE00001118418). The pairs supporting each of the possible haplotypes were counted manually using the Integrated Genomics Viewer (Broad Institute). Pairs supporting the triple mutation (N642H, T648S, I704L) = 23; pairs supporting triple wt = 15, pairs supporting other combinations = 0. Targeted PCR amplification and amplicon sequencing Sample preparation was performed according to an in-house targeted PCR amplification protocol. Each amplicon was generated using locus specific PCR primers carrying Illumina compatible adapter sequences (primer sequences in Table 1). All oligonucleotides were synthesized by Sigma-Aldrich (St. Louis, MO, USA). The PCR reaction was performed in a volume of 20 µl containing 5-10 ng of sample DNA, 10 µl of 2x Phusion High-Fidelity PCR Master Mix (Thermo Scientific Inc., Waltham, MA, USA), 0.025 or 0.05 µM of each locus specific primers, 0.5 or 0.375 µM of an adapter primer carrying Illumina grafting P7 sequence, 0.5 or 0.375 µM of an adapter primer carrying Illumina grafting P5 sequence (primer sequences in Table 2)** and the reaction mix was brought to a final volume with water (Sigma-Aldrich, St. Louis, MO, USA). Samples were cycled according to Phusion High-Fidelity PCR Master Mix cycling instructions (annealing temperature 59 °C). Following PCR amplification, samples were purified using Performa® V3 96-Well Short Plate (EdgeBio, Gaithersburg, MD, USA) and QuickStep™2 SOPE™ Resin (EdgeBio, Gaithersburg, MD, USA) or with Agencourt® AMPure® XP beads (Beckman Coulter Inc., Beverly, MA, USA) and then pooled together without exact quantification. Purified sample pools were analyzed on Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) to quantify amplification performance and yield. Sequencing of PCR amplicons was performed using the Illumina MiSeq instrument with MiSeq Control Software v2.2.29 or later versions (Illumina, Inc., San Diego, CA, USA). Samples were sequenced as 151 or 251 bp pairedend reads and two 8 bp index reads. Data obtained from sequencing were processed with an in-house amplicon pipeline. STAT5B mutations were validated from the index patient with capillary sequencing as described previously7. STAT5B mutagenesis An expression plasmid pCMV6-XL6 containing the wild-type coding sequence of STAT5B (OriGene, Rockville, MD, USA) was modified to include the N642H, T648S and I704L mutations. After generating the single mutated constructs the double (N642H+T648S, N642H+I704L, T648S+I704L) and triple (N642H+T648S+I704L) mutated constructs were prepared. The mutant constructs were created using the GENEART Site-Directed Mutagenesis System (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The primer sequences used for the mutagenesis are listed in Table 3. The entire STAT5B cDNA sequence with mutations was confirmed by capillary sequencing (primer sequences in Table 4). Cell culture and transfection HeLa cells were cultured in high glucose DMEM (Life Technologies, Carlsbad, CA, USA) containing 10% FBS, 2mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. For transfection, HeLa cells were plated in 6-well plates (400 000 cells/well) for Western blot analysis and in 96-well plates (15 000 cells/well) for the STAT5B reporter assay one day before the transfection. Transfection was carried out with STAT5B expression plasmids mixed with the luciferase reporter plasmid pGL4.52 (Luc2P/STAT5RE/Hygro, Promega, Madison, WI, USA) using Fugene HD transfection reagent (Promega); Fugene HD:DNA ratio 3.5:1. A master mix was prepared with each mutant construct + luciferase plasmid and appropriate amounts were added to each well; 6-well plate 3μg + 1μg and 96-well plate 0.1μg + 0.033μg. Western blot analysis One day after transfection the HeLa cells were lysed in RIPA buffer (0.1% SDS, 150mM NaCl, Tris 50mM, 1% Triton-X100, 0.5% sodium deoxycholate) containing 1mM sodium orthovanadate and Roche's Complete Protease Inhibitor Cocktail Tablet. Lysates were then sonicated for 4×1s and after addition of 2×Laemmli buffer, 50 μg of protein was loaded per sample to 10% SDS-PAGE gels. Detection of phopho-STAT5 and total STAT5 was done using two separate gels run in parallel with the same protein lysates. The proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), after which the membranes were blocked with 5% bovine serum albumin (BSA) for 1 hour. Primary antibodies for STAT5 were obtained from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, US): STAT5 (rabbit polyclonal antibody, cat. 9363) dilution 1:1000 and phospho-STAT5 (Tyr694/699, rabbit mAb, cat. 9359) dilution 1:1000. Anti-α-Tubulin was obtained from Sigma Aldrich (T9026 mouse mAb) dilution 1:500. Membranes were incubated with primary antibodies diluted in phosphate-buffered saline and 0.1% Tween 20 (PBS-T) + 5% BSA 1h at room temperature (RT). The membranes were then incubated with a secondary infrared antibodies: goat anti-rabbit IRDye 800CW and donkey anti-mouse IRDye 800CW (LICOR Biosciences, Lincoln, NE, USA) diluted 1:15 000 in 5% BSA PBS-T for 1 hour at RT. The proteins were visualized with the Odyssey imaging system (LI-COR Biosciences) and its application software version 3.0 was used to quantify band intensity levels. STAT5B reporter assay After 24 h incubation at 37 °C, transfected HeLa cells were lysed and luciferase activity was assessed using the One-Glo luciferase detection reagent (Promega, Madison, WI, USA). The assay was repeated 3 times and the mean fold change in transcriptional activity induced by the different mutant constructs compared to the activity of the WT STAT5B construct was calculated (±SD). qRT-PCR Total RNA was prepared from peripheral blood CD3+ cells from a healthy donor and from BM MNCs of the index patient and two T-ALL controls using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was prepared from 224 ng of total RNA using SuperScript III reverse transcriptase and random primers (Life Technologies, Carlsbad, CA, USA) in a 20 μl reaction, including 40 U RiboLock RNase inhibitor (Thermo Scientific, Waltham, MA, USA). For primer efficiency reactions, 5 μg of the index patient RNA was used in the reverse transcription reaction. Primer efficiencies were determined using ten fold dilutions of RNA: 125 ng, 12.5 ng, 1.25 ng, 0.125 ng per reaction. Reference genes were chosen based on uniform expression in all samples with POLR1B and GUSB determined to be the best reference candidates. qPCR was performed for 4 target transcripts: BCL2, BCLXL, BCL-XS and MCL. Primer sequences are listed in Table 5. BCL-XL and BCL-XS are alternative transcripts of the BCL2L1 gene, so the primers were designed to be transcript specific. Other targets were non-specific for transcript variants. qPCR reactions were performed using iQ SYBR Green Supermix (Bio Rad, Hercules, CA, USA), and the specificities of the amplification products verified by melting curve analysis. Gene expression was quantified using the Pfaffl method based on calculated primer efficiencies.6 Protein structure analysis The human STAT5B dimer homology model was prepared as described previously.7 Protein structure visualization was prepared with Swiss PDB viewer.8 Table 1. Locus specific primer sequences carrying tails corresponding to the Illumina adapter sequences. Locus specific primer sequences are underlined.** Primer name Primer sequence STAT3_exon21-F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCCAAAAATTAAATGCCAGGA-3’ STAT3_exon21-R 5’-AGACGTGTGCTCTTCCGATCTGGTTCCATGATCTTTCCTTCC-3’ STAT5A_exon17-F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTGCTGCTGGTGGATTAT-3’ STAT5A_exon17-R 5’-AGACGTGTGCTCTTCCGATCTAGCCCAAGGCTTTGTCTATG-3’ STAT5B_exon14-15-F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGACCTCAACAAATAGTAAGTACCC-3’ STAT5B_exon14-15-R 5’-AGACGTGTGCTCTTCCGATCTTTCCAAGTGTTACTCTGGTGTTC-3’ STAT5B_exon16-F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTTGGGGTTTTAAGATTTCC-3’ STAT5B_exon16-R 5’-AGACGTGTGCTCTTCCGATCTCAAATCAGAATGCGAACATTG-3’ STAT5B_exon17-F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCCAGGGCTGAGACAGTTT-3’ STAT5B_exon17-R 5’-AGACGTGTGCTCTTCCGATCTAGATTGCACCACCGTACTCC-3’ STAT5B_exon18-F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGATTCCTTTGACCCCAGC-3’ STAT5B_exon18-R 5’-AGACGTGTGCTCTTCCGATCTAGATACCCCTTGGTCCCCTC-3’ STAT5B_exon19-F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGGTGGCCTGTGGGGCTTG-3’ STAT5B_exon19-R 5’-AGACGTGTGCTCTTCCGATCTTCTGTCTGTGGCCCCTCTGCT-3’ Table 2. Adapter primer sequences carrying P7/P5 grafting sequences. TruSeq Adapter index primers are adapted from Illumina and contain the P7 grafting sequence. FIMM P5 index primers partly contain Illumina indices and carry the P5 grafting sequence. Index sequences are underlined. ** Primer name Primer sequence TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 4 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 5 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 6 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 7 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 8 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 9 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 10 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 11 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 12 TruSeq Adapter index 13 5'-CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 14 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 15 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATCTCTACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 17 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 18 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 19 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATCGAAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 21 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATGTATAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 27 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATCGATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 30 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATGCTGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 31 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATATTATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 32 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATGAATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 33 TruSeq Adapter 5'-CAAGCAGAAGACGGCATACGAGATCTTCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' index 35 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACAGTTGGTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 9 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACGTACCGGACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 10 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACCGGAGTTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 11 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACTGAACCTTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 14 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACTGCTAAGTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 15 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACTGTTCTCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 16 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACTAAGACACACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 17 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACCTAATCGAACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 18 FIMM P5 index 5-‘AATGATACGGCGACCACCGAGATCTACACCTAGAACAACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 19 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACTAAGTTCCACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 20 FIMM P5 index 5’-AATGATACGGCGACCACCGAGATCTACACTAGACCTAACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ 21 * denotes a phosphorothioate-modified bond **Oligonucleotide sequences © 2007-2012 Illumina, Inc. All rights reserved. Derivative works created by Illumina customers are authorized for use with Illumina instruments and products only. All other uses are strictly prohibited Table 3: STAT5B mutagenesis primers N642H: F: 5'- GAA AGA ATG TTT TGG CAT CTG ATG CCT TTT AC -3' R: 5'- GTA AAA GGC ATC AGA TGC CAA AAC ATT CTT TC -3' T648S: F: 5'- CTG ATG CCT TTT ACC TCC AGA GAC TTC TCC -3' R: 5'- GGA GAA GTC TCT GGA GGT AAA AGG CAT CAG -3' I704L: F: 5'- CGT GAA GCC ACA GCT CAA GCA AGT GGT CCC -3' R: 5'- GGG ACC ACT TGC TTG AGC TGT GGC TTC ACG -3' Table 4: Sequencing primers of the STAT5B constructs F_1: GGACTTTCCAAAATGTCG F_2: GAACAGAGGTTGGTCCGAGA F_3: GAACACCCGCAATGATTACA F_4: CAACAGGCCCATGACCTACT R_1: CCACCAGCCTTGTCCTAAT Table 5: Primer sequences for qPCR POLR1B: GCATACCCCAGACGGGGAGC TCGGTGGGGAGCTCCATCAAT GUSB: TGGAGCTTCCGCGCCGACTT GCATGTCCACGGTGGGGCCT BCL2: AGATTGATGGGATCGTTGCCT AGTCTACTTCCTCTGTGATGTTGT bcl-xL: ATTGGTGAGTCGGATCGCAG GCTGCTGCATTGTTCCCATAG bcl-xS: TCCCCATGGCAGCAGTAAAG TCCACAAAAGTATCCTGTTCAAAGC MCL1: TTTGGCACTGTGAAACCCCT GTGTCACAGCACCCATGGTA References: 1. 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