Mutagenesis and generation of expression vectors Gene expression profiling Lentiviral production and infection of TF1 cells

Transcription

1 Mutagenesis and generation of expression vectors Human c-kit wild-type cdna encoding the short c-kit isoform was excised from vector pbshkitwt (kind gift from Dr. L Gros, France) by Sal I-Acc65 I digestion. The cdna fragment was subcloned into pentr1a vector (Invitrogen, France). c-kit mutations identified in patients were introduced into pentr1a-hkitwt vector with QuickChange site-directed mutagenesis Kit (Stratagene, Netherlands) according to the manufacturer s instructions. All the mutations were confirmed by bidirectional sequencing. The plxsn-gw vector was constructed with the plxsn retroviral vector (Clontech, Germany) by inserting a Gateway cassette (Invitrogen, France). The Gateway reaction was performed between pentr-kit and plxsn-gw vectors following the manufacturer s recommendations (Invitrogen, France) to obtain the c-kit expression vector. To generate cdnas encoding the hybrid protein EGFP-AKT or MyrEGFP-AKT separated by a 6 Glycines linker, fragments EGFP-6Gly, MyrEGFP-6Gly, and AKT were respectively cloned from three vectors (kindly provided by Dr. P Lecine, France) by using primers containing the Gateway recombination sequences AttB1 or AttB2 (Invitrogen, France). A second PCR was then performed to fuse EGFP-6Gly or MyrEGFP-6Gly to AKT. The resulting amplicons were cloned into the pdonr21 vector (Invitrogen, France) by Gateway technique. Finally, the chimeric cdnas were introduced into pmscvpuro expression vector (Clontech, Germany) which had been previously modified by inserting a Gateway cassette. Gene expression profiling RNA expression profiling of cell lines was done with Affymetrix M43 2. mouse oligonucleotide microarrays containing probe sets, representing transcripts and variants including well-characterized mouse genes. Preparation of crna, hybridizations, washes, detection, and quantification were done as recommended by the supplier. For each sample, synthesis of the first-strand cdna was done from 3 µg total RNA by T7-oligo(dT) priming, followed by second-strand cdna synthesis. After purification, in vitro transcription associated with amplification generated crna-containing biotinylated pseudouridine. Biotinylated crna was purified, quantified, and chemically fragmented (95 C for 35 min), then hybridized to microarrays in 2 µl hybridization buffer at 45 C for 16 h. Automated washes and staining with streptavidin-phycoerythrin were done as recommended. Signal amplification was done by biotinylated antistreptavidin antibody with goat-igg blocking antibody. Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GCOS software. Validation study using cdna-spotted arrays was done with Ipsogen Nylon microarrays containing 8, genes/expressed sequence tags (EST). Expression data were analyzed by the Robust Multichip Average method in R using Bioconductor and associated packages. Filtered and log 2 -transformed data were analyzed by hierarchical clustering using the Cluster program. Results were displayed using TreeView. Lentiviral production and infection of TF1 cells Human Kit cdnas were cloned in prrl lentiviral vector (*). For infection of TF-1 cells, 1 6 cells were incubated for 1 hour in 1 ml of media supplemented with GM-CSF and 8 µg/ml of polybrene (hexadimethrine bromide). Cells were then incubated for 3 hours with infectious particles, spun, and diluted in fresh media. GFP-positive infected cells were sorted 4 days later and immediately cultured in media without GM-CSF.

2 (*) Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery. Romain Zufferey, Thomas Dull, Ronald J. Mandel, Anatoly Bukovsky, Dulce Quiroz, Luigi Naldini, and Didier Trono. J Virol December; 72(12): Figure S1. Growth curves of mutant c-kit expressed in Ba/F3 cells Ba/F3 cells expressing the different mutants were seeded in culture in the absence of SCF and counted every day by Trypan blue exclusion. Control native Ba/F3 cells were incubated in the presence of IL3 and counted with the same procedures. Figure S2. Effect of ECD and PTD mutant ectopic expression in human TF1 cells (A) TF1 cells expressing ECD and PTD mutants were seeded in culture in the absence of SCF and counted every day by Trypan blue exclusion. Control parental TF1 cells were incubated in the presence of GM-CSF (5 ng/ml) or SCF (25ng/mL) and were counted with the same procedures. (B) TF1 cells expressing WT or KIT mutants were introduced in a proliferation assay and incubated for 48 h with 5 ng/ml GM-CSF, 25ng/mL SCF or without growth factor and in the presence of 1 µm Imatinib or Dasatinib. Values are presented relative to cell proliferation in the absence of inhibitors.data represent mean values ± standard deviation (SD) of triplicates. Results shown are representative of two independent experiments. (C) Parental and KIT variants expressing TF1 cells were serum starved in RPMI.5% FBS for 3 h and stimulated with or without mscf for 5 min. Total lysates were analysed by SDS-PAGE followed by anti P- KIT (Y719) and anti phospho-akt (S473). The blot was stripped and reprobed with anti-akt antibodies to illustrate equal loading. Figure S3. ECD- or PTD-mutants display very different whole-genome transcriptional profiles Each EML cell line (WT-KIT, Del insy mutant and mutant) was cultured with or without 25 ng/ml SCF for 48 or 72h. Gene expression profile analysis was performed using whole-genome microarrays. Hierarchical clustering was applied to the four samples (2 for Del insy; 2 for ) and to the probe sets most variable across all samples. Samples were sorted into two large groups, which correlated with the type of mutation: Del insy in the right group and in the left group. The two groups appeared homogeneous (correlation coefficient.85 for mutants and.8 for Del insy mutants) and very distinct from each other (correlation coefficient.17). Figure S4. Differential activation of AKT pathway by ECD- and PTD-mutants in EML cells Parental and KIT variants expressing EML cells were serum starved in RPMI.5% FBS for 3 h and stimulated with or without mscf for 5 min. Total lysates were analysed by SDS-PAGE followed by anti phospho-akt (S473), anti-p STAT3, and anti-p ERK antibody blotting. The blot was stripped and reprobed with anti-akt, STAT3, and ERK2 antibody to illustrate equal loading. Figure S5. AKTVIII inhibits ECD-mutants dependent proliferation of EML cells and clonogenicity of Rat2 cells

3 (A) Differential sensitivity of ECD- and PTD-mutants towards AKTVIII in EML cells. EML cells expressing WT-KIT or mutants and Ba/F3 cells were seeded in 96-well plates and cultured for 48 h with or without mscf or mil-3 and in the presence of 5 µm AKTVIII. Cell growth was assessed using WST-1. Values are presented relative to cell proliferation in the absence of AKTVIII and represent mean values ± SD of triplicates. (B) Effect of AKTVIII on colony formation of Rat2 cells. Rat2 cells expressing WT-KIT, Del insy KIT mutant and RasV12 were plated at cells per dish in soft-agar medium with or without mscf and in the presence of 5 µm AKTVIII. Colonies were counted at day 1. Values are shown relative to colony number in the absence of AKTVIII. Figure S6. Analysis of surface expression of KIT by FACS Ba/F3 cells were transfected with WT or mutant forms of KIT. Surface expression of KIT was assessed by FACS using anti-kit monoclonal antibody (solid lines). Untransfected Ba/F3 cells (dotted lines) were used as a negative control.

4 Figure S1 6 5 Number of cells x BaF3 IL3 D Y D816I D816V D816Y K59I Ins 53AY D D1 D2 D3 Growth curves in Ba/F3 cells

5 Figure S2 A B Cell count x1-6 2, 1,8 1,6 1,4 1,2 1,,8,6,4,2, D1 D2 D3 Days DMSO Imatinib Dasatinib TF-1 without GF TF-1 + GM-CSF TF-1 + SCF TF-1 Del InsY TF-1 TF-1 Cell proliferation in OD TF-1 +GM-CSF TF-1 +SCF D InsY TF-1 +GM-CSF TF-1 +SCF D InsY C TF1 cells WT WT D insy SCF P-KIT Y719 P-S 473 AKT AKT

6 Figure S3 r =.17 r =.85 r =.8-72h - 48h Del insy - 72h Del insy - 48h log 2-1 1

7 Figure S4 EML Cells EML Del insy D 816 I SCF P-S 473 AKT AKT P-STAT3 STAT3 P-ERK1/2 ERK1/2

8 Figure S5 A Proliferation (%DMSO control) AKTVIII (µm) EML+SCF Del insy D 816 I BaF3+IL-3 B % colonies of DMSO control AKTVIII (µm) WT+SCF Del insy RasV12

9 Figure S6 WT Del insy S 476 I Cell count K 59 I D 816 Y D 816 I KIT